Background Hürthle cell carcinoma is a variant of follicular cell carcinoma of thyroid. It may present as a low-grade tumour or as a more aggressive type. Prognosis depends upon the age of the patient, tumour size, extent of invasion and initial nodal or distant metastasis.
Background
Hürthle cell carcinoma is a variant of follicular cell carcinoma of thyroid. It may present as a low-grade tumour or as a more aggressive type. Prognosis depends upon the age of the patient, tumour size, extent of invasion and initial nodal or distant metastasis.Patient and methods The case of Hürthle cell carcinoma is reported in a 79-year-old man who presented with a rapidly increasing lump on the left side of his neck, having had a right hemithyroidectomy for colloid goitre 24-years-ago. Fine needle aspiration cytology confirmed the presence of Hürthle cells, raising the possibility of a Hürthle cell neoplasm. The patient underwent staging and surgery. Histology showed Hürthle cell carcinoma and the patient underwent adjuvant therapy. The literature on Hürthle cell neoplasms is reviewed.
Patient and methods
The case of Hürthle cell carcinoma is reported in a 79-year-old man who presented with a rapidly increasing lump on the left side of his neck, having had a right hemithyroidectomy for colloid goitre 24-years-ago. Fine needle aspiration cytology confirmed the presence of Hürthle cells, raising the possibility of a Hürthle cell neoplasm. The patient underwent staging and surgery. Histology showed Hürthle cell carcinoma and the patient underwent adjuvant therapy. The literature on Hürthle cell neoplasms is reviewed.Conclusions Fine needle aspiration cytology may recognise Hürthle cell lesion but final diagnosis of carcinoma depends upon histological confirmation of vascular or capsular invasion. Staging and surgery in Hürthle cell carcinoma are similar to follicular carcinoma of thyroid with favourable outcome despite the controversy regarding the histological classification and adjuvant therapy. Elderly patients with Hürthle cell carcinoma need to be made aware of their poorer prognosis and should be offered more radical treatment.
Conclusions
Fine needle aspiration cytology may recognise Hürthle cell lesion but final diagnosis of carcinoma depends upon histological confirmation of vascular or capsular invasion. Staging and surgery in Hürthle cell carcinoma are similar to follicular carcinoma of thyroid with favourable outcome despite the controversy regarding the histological classification and adjuvant therapy. Elderly patients with Hürthle cell carcinoma need to be made aware of their poorer prognosis and should be offered more radical treatment.

Background The natural history of Hürthle cell carcinoma (HCC) is not well understood. It accounts for <5% of all differentiated thyroid malignancies. Hürthle cells are characterised by eosinophilic cytoplasm with trabecular/follicular growth pattern. [ 1 ]. Oncocytes are seen in follicular cell carcinoma but in HCC oncocytes represent more than 75% of cells, which exhibit a rather more trabecular growth pattern [ 2 ]. There is much debate regarding its clinical behaviour and little is known about the long-term survival of patients with HCC. Some studies have reported a relatively benign course while others have found the tumour to behave aggressively [ 3 - 6 ]. Most studies show that advanced age (>45), male sex, size of primary tumour (>4 cm), degree of invasion and recurrence are poor prognostic indicators [ 6 - 8 ]. Fine needle aspiration cytology is a good predictor of Hürthle cell neoplasm but is of little diagnostic value in evaluating HCC, since for a tumour to be deemed malignant one has to show vascular or capsular invasion [ 9 ]. Intraoperative frozen sections have a low predictive value. Udelsman  et al  found that in 96.4% cases with follicular neoplasm of thyroid, frozen section was neither informative nor cost-effective [ 10 ]. Well-encapsulated HCC run a favourable course while locally advanced HCC are associated with higher mortality and should be treated aggressively [ 4 , 11 ]. In a well-differentiated thyroid carcinoma death resulting from local disease is unusual and most die of distant metastases [ 12 ]. We report a case of a Hürthle cell carcinoma presenting in the left lobe of thyroid following a right hemithyroidectomy for a colloid goitre 24 years ago.
Background
The natural history of Hürthle cell carcinoma (HCC) is not well understood. It accounts for <5% of all differentiated thyroid malignancies. Hürthle cells are characterised by eosinophilic cytoplasm with trabecular/follicular growth pattern. [ 1 ]. Oncocytes are seen in follicular cell carcinoma but in HCC oncocytes represent more than 75% of cells, which exhibit a rather more trabecular growth pattern [ 2 ]. There is much debate regarding its clinical behaviour and little is known about the long-term survival of patients with HCC. Some studies have reported a relatively benign course while others have found the tumour to behave aggressively [ 3 - 6 ]. Most studies show that advanced age (>45), male sex, size of primary tumour (>4 cm), degree of invasion and recurrence are poor prognostic indicators [ 6 - 8 ]. Fine needle aspiration cytology is a good predictor of Hürthle cell neoplasm but is of little diagnostic value in evaluating HCC, since for a tumour to be deemed malignant one has to show vascular or capsular invasion [ 9 ]. Intraoperative frozen sections have a low predictive value. Udelsman  et al  found that in 96.4% cases with follicular neoplasm of thyroid, frozen section was neither informative nor cost-effective [ 10 ]. Well-encapsulated HCC run a favourable course while locally advanced HCC are associated with higher mortality and should be treated aggressively [ 4 , 11 ]. In a well-differentiated thyroid carcinoma death resulting from local disease is unusual and most die of distant metastases [ 12 ].et alWe report a case of a Hürthle cell carcinoma presenting in the left lobe of thyroid following a right hemithyroidectomy for a colloid goitre 24 years ago.Case presentation A 79-year-old male was referred in March 2003 with a lump on the left side of his neck. The patient had noted a sudden increase in the size of the lump over the preceding two months. He did not report any neck pressure symptoms, weight loss or anorexia. His past history included right partial thyroidectomy for a solitary nodule (colloid goitre) in 1978 and repair of abdominal aortic aneurysm in 1994. He had suffered myocardial infarction in 1995 and had an episode of acute coronary insufficiency in January 2003. His recent coronary angiograms showed an occluded left anterior descending artery and echocardiogram revealed good left ventricular function. He was a non-smoker and consumed alcohol in moderation. He had been taking warfarin, diltiazem MR, lisinopril, uniphyllin, glyceryl trinitrate tablets and buccal suscard. On examination he had left sided goitre extending superiorly into the posterior triangle and inferiorly into the retrosternal space, with variable consistency. The trachea was deviated to the right and there was cervical lymphadenopathy on the left side. Systemic examination was unremarkable and fine needle aspiration of thyroid gland showed presence of Hürthle cells. Computerised tomographic (CT) scan with contrast enhancement (figure  1  & 2 ) of the neck and thorax revealed large left sided thyroid goitre with significant mediastinal extension. It showed mixed attenuation with foci of calcification peripherally. There was a 3 cm complex mass on the left side of the neck, posterior to the carotid sheath structures and deep to the sternomastoid, indicative of lymph node metastases. Thyroid profile and routine blood investigations were unremarkable. Figure 1 Superior extension of left goitre with 3 cm diameter complex mass deep to sternomastoid, posterior to carotid sheath. Note the displacement of larynx to the right. Figure 2 Mediastinal extension of left goitre. Based on the above findings radical surgery was planned. On exploration of the neck we confirmed left goitre with intrathoracic extension and enlarged lymph nodes under the sternocleidomastoid close to the jugulodigastric muscle and surrounding the carotid sheath. There was no remnant thyroid tissue seen on the right side following the previous thyroid surgery. Left hemithyroidectomy with modified neck dissection (lymphadenectomy, preserving all vessels and nerves) was performed. Macroscopic examination of the thyroid lobe showed a well defined solid pale brown mass approximately 8 cm in maximum dimension, surrounded by a narrow rim of preserved thyroid tissue. The lymph node specimen comprised of several nodules of partly necrotic tissue. Microscopic examination showed the thyroid lobe containing a Hürthle cell neoplasm, which was mostly encapsulated, with foci of capsular and vascular invasion. The two lymph nodes revealed metastatic Hürthle cell carcinoma. [pT3, N1a, Mx], (Figure  3  & 4 ). Figure 3 Photomicrograph showing capsular invasion (Haematoxylin and Eosin ×200) Figure 4 photomicrograph showing Hürthle cell note the eosinophilic cytoplasm and prominent nucleoli (Haematoxylin and Eosin ×500). The patient had adjuvant therapy with oral radioiodine 131 (3060 MBq Sodium Iodine). He was put on a daily dose of 100 mcg of thyroxine. This was to be followed by a second dose of 5911 MBq of radioactive iodine six months from the time of the first dose.
Case presentation
A 79-year-old male was referred in March 2003 with a lump on the left side of his neck. The patient had noted a sudden increase in the size of the lump over the preceding two months. He did not report any neck pressure symptoms, weight loss or anorexia. His past history included right partial thyroidectomy for a solitary nodule (colloid goitre) in 1978 and repair of abdominal aortic aneurysm in 1994. He had suffered myocardial infarction in 1995 and had an episode of acute coronary insufficiency in January 2003. His recent coronary angiograms showed an occluded left anterior descending artery and echocardiogram revealed good left ventricular function. He was a non-smoker and consumed alcohol in moderation. He had been taking warfarin, diltiazem MR, lisinopril, uniphyllin, glyceryl trinitrate tablets and buccal suscard.On examination he had left sided goitre extending superiorly into the posterior triangle and inferiorly into the retrosternal space, with variable consistency. The trachea was deviated to the right and there was cervical lymphadenopathy on the left side. Systemic examination was unremarkable and fine needle aspiration of thyroid gland showed presence of Hürthle cells. Computerised tomographic (CT) scan with contrast enhancement (figure  1  & 2 ) of the neck and thorax revealed large left sided thyroid goitre with significant mediastinal extension. It showed mixed attenuation with foci of calcification peripherally. There was a 3 cm complex mass on the left side of the neck, posterior to the carotid sheath structures and deep to the sternomastoid, indicative of lymph node metastases. Thyroid profile and routine blood investigations were unremarkable.Based on the above findings radical surgery was planned. On exploration of the neck we confirmed left goitre with intrathoracic extension and enlarged lymph nodes under the sternocleidomastoid close to the jugulodigastric muscle and surrounding the carotid sheath. There was no remnant thyroid tissue seen on the right side following the previous thyroid surgery. Left hemithyroidectomy with modified neck dissection (lymphadenectomy, preserving all vessels and nerves) was performed. Macroscopic examination of the thyroid lobe showed a well defined solid pale brown mass approximately 8 cm in maximum dimension, surrounded by a narrow rim of preserved thyroid tissue. The lymph node specimen comprised of several nodules of partly necrotic tissue. Microscopic examination showed the thyroid lobe containing a Hürthle cell neoplasm, which was mostly encapsulated, with foci of capsular and vascular invasion. The two lymph nodes revealed metastatic Hürthle cell carcinoma. [pT3, N1a, Mx], (Figure  3  & 4 ).The patient had adjuvant therapy with oral radioiodine 131 (3060 MBq Sodium Iodine). He was put on a daily dose of 100 mcg of thyroxine. This was to be followed by a second dose of 5911 MBq of radioactive iodine six months from the time of the first dose.Discussion Hürthle cell carcinomas are heterogeneous neoplasms that display a wide range of biological behaviour and accounts for less than 5% of all differentiated thyroid malignancies. The term HCC should be restricted to tumours with more than 75% of oncocytic cells [ 2 ]. Oncocytes are seen in follicular thyroid cell carcinoma and in papillary thyroid cell carcinoma [ 13 , 14 ]. On one hand patients with HCC live for years with slow growing tumour and lymphatic metastases and on the other hand, patients die of highly aggressive tumour with haematogenous spread. Our patient had several indicators for poor prognosis such as his advanced age, male gender, large tumour size (8 cm), extra thyroid extension and nodal metastasis. Interestingly enough the patient had no pressure symptoms despite marked deviation of larynx, trachea and oesophagus, which may be due to previous right hemithyroidectomy. In elderly patients with sudden enlargement of neck mass and pre-existing thyroid conditions such as benign thyroid nodule, goitre (as in our case), Grave's disease or differentiated thyroid carcinoma, one has to bear in mind anaplastic thyroid carcinoma (ATC). In ATC local compression symptoms such as hoarseness, strider, dyspnoea and dysphagia occur as a rule [ 15 - 17 ]. In aggressive type of HCC haematogenous spread has been noted, but in ATC, at presentation patients are quite likely to have distant metastases involving lung, bone, brain and soft tissues [ 15 , 16 ]. Our patient had undergone fine needle aspiration cytology, which revealed Hürthle cells. Since the lesion was rapidly growing with mediastinal extension and nodal involvement, the patient underwent staging and left hemithyroidectomy with modified neck dissection. Histology confirmed HCC based on vascular and capsular invasion. Intraoperative frozen sections have low predictive value and are particularly not a sensitive test for diagnosing HCC therefore this was not carried out [ 10 ]. McIvor et al have clearly shown that FNAC can easily recognise the tumour as Hürthle cell lesion [ 9 ]. Cases with suspicious histology and over 50 years of age carry a high risk of cancer [ 18 ]. In the management of HCC the primary mode of treatment is surgical, ranging from hemithyroidectomy to total thyroidectomy. Larger tumours (>T2) require total thyroidectomy and lymphadenectomy if lymph nodes are involved [ 8 ]. Adjuvant radioiodine treatment or external beam radiotherapy is used for widely invasive carcinoma or locally advanced disease [ 8 ]. Several reports in literature have identified contra lateral foci of carcinoma in 40–70% of cases of HCC [ 11 , 19 ]. HCC is less responsive to radioactive iodine therapy [ 20 ] and taking into account the aggressive behaviour, it has been suggested that every Hürthle cell tumour greater than 2 cm should be treated by total thyroidectomy [ 21 ]. In 1990 they showed that recurrent disease was noted in 17% of patients treated with total thyroidectomy compared to 59% in cases where a more limited procedure was carried out [ 21 , 22 ]. Other authors support the role of total thyroidectomy as there is 15 to 35% incidence of multiple foci in HCC [ 23 ]. There are several reasons favouring the use of  131 I remnant ablation after near-total thyroidectomy [ 24 ]. First, presence of thyroid remnant can obscure  131 I uptake in cervical or lung metastases [ 25 , 26 ]. Second, distant (lung) metastases may be seen only on the post treatment whole body scan after remnant ablation [ 27 ]. Finally, remnant ablation may destroy residual normal follicular cells, which may become malignant [ 28 ] and any occult cancer that may recur years later. Radioiodine therapy has no overall effect on mortality but subgroup analysis has shown that those patients who receive radioactive iodine for adjuvant ablation of remnant thyroid tissue have lower mortality rate compared with patients who either did not receive treatment or in whom the indication was the presence of residual disease [ 29 ]. Radioiodine uptake in the elderly is much lower. Schlumberger and colleagues noted  131 I uptake at metastatic sites in only 53% of patients over 40 years of age, compared to 90% in patients below the age of 40 [ 30 ]. Univariate analysis indicated that older age and large tumour size predicted worse survival rates due to aggressive nature of the tumour (extra glandular invasion and multifocal disease). One recent series reviewed medical records of patients between the years 1944 and 1995. Of the 89 HCC cases studied, 29% had only undergone lobectomy as initial treatment and 50% had undergone partial resection. Of the three quarters of the patients in this series who received radioactive iodine, only 38% of patients with known metastases showed positive uptake [ 29 ]. Another study clearly suggested that treatment with  131 I to ablate the thyroid remnant and to treat residual disease were independent prognostic variables that favourably influenced recurrence, distant recurrence, and cancer death rates [ 24 ]. Our patient received radioactive iodine treatment in the postoperative period. He has been followed up with whole body scans (Fig  5  and  6 ), which indicate his response to adjuvant radioactive iodine therapy. He is on 125 mcg thyroxine in order to maintain a TSH level of less than 0.01 mIU/L and FT4 at the upper limit of normal (8–28 pmol/L). Figure 5 Whole body scan on November 3, 2003 following  131 I ablation therapy on 28 th  October 2003, with 3060 MBq Sodium Iodine ( 131 I). Increased uptake is seen in the region of the thyroid bed. No abnormal accumulation was noted elsewhere. Figure 6 Whole body scan on 19 th  April 2004 following  131 I ablation therapy on 13 th  April 2004 with 5911 MBq Sodium Iodine ( 131 I). Two small focal area of uptake are seen in the thyroid bed. Low uptake focal area in the left lateral aspect of the neck, could possibly represent activity in a cervical node. Stojdinovic  et al  have treated 56 patients with HCC between the years 1940 and 2000 [ 8 ]. Of these patients 23(41%) had minimally invasive disease with no evidence of extra thyroid invasion (T2 N0 M0) and 33(56%) had widely invasive HCC. Primary mode of treatment was surgery ranging from lobectomy and isthumusectomy to total thyroidectomy with cervical lymphadenectomy in presence of lymph node involvement. Some patients received adjuvant radioiodine or external beam radiotherapy for widely invasive carcinoma. Study end points were relapse free survival and disease specific survival. They reported 8 years survival rate of 100% and 58% for low and high-risk cancers respectively. In their entire study cohort age was not found to predict the outcome but the most significant factor was widely invasive carcinoma. Khafif  et al  in their series (42 patients with HCC between 1957–1997) used radioiodine in patients with distant metastases; none had thyroid remnant ablation with radioactive iodine [ 4 ]. They reported an overall survival rate of 90.5% and noted that age, size of tumour and extent of resection adversely affected the prognosis. Hürthle cell lesion can be easily picked up on FNAC but to make a diagnosis of HCC one has to demonstrate vascular or capsular invasion. Intraoperative frozen sections have low predictive value and cases with advanced age (over 50), rapid enlargement of lump and palpable nodes should be regarded with high index of suspicion for presence of HCC. HCC or other differentiated carcinomas of thyroid in the elderly patients are generally more aggressive with less favourable prognosis compared to younger patients. They should be offered total thyroidectomy and selective lymph node dissection (when lymph nodes are involved) followed by ablative radioiodine therapy, provided they can withstand the above treatment. Coexisting medical disorders should be recognized and managed effectively prior to surgery [ 31 ]. Further research is needed to clarify the role of adjuvant radioiodine therapy in the management of HCC.
Discussion
Hürthle cell carcinomas are heterogeneous neoplasms that display a wide range of biological behaviour and accounts for less than 5% of all differentiated thyroid malignancies. The term HCC should be restricted to tumours with more than 75% of oncocytic cells [ 2 ]. Oncocytes are seen in follicular thyroid cell carcinoma and in papillary thyroid cell carcinoma [ 13 , 14 ]. On one hand patients with HCC live for years with slow growing tumour and lymphatic metastases and on the other hand, patients die of highly aggressive tumour with haematogenous spread.Our patient had several indicators for poor prognosis such as his advanced age, male gender, large tumour size (8 cm), extra thyroid extension and nodal metastasis. Interestingly enough the patient had no pressure symptoms despite marked deviation of larynx, trachea and oesophagus, which may be due to previous right hemithyroidectomy. In elderly patients with sudden enlargement of neck mass and pre-existing thyroid conditions such as benign thyroid nodule, goitre (as in our case), Grave's disease or differentiated thyroid carcinoma, one has to bear in mind anaplastic thyroid carcinoma (ATC). In ATC local compression symptoms such as hoarseness, strider, dyspnoea and dysphagia occur as a rule [ 15 - 17 ]. In aggressive type of HCC haematogenous spread has been noted, but in ATC, at presentation patients are quite likely to have distant metastases involving lung, bone, brain and soft tissues [ 15 , 16 ].Our patient had undergone fine needle aspiration cytology, which revealed Hürthle cells. Since the lesion was rapidly growing with mediastinal extension and nodal involvement, the patient underwent staging and left hemithyroidectomy with modified neck dissection. Histology confirmed HCC based on vascular and capsular invasion. Intraoperative frozen sections have low predictive value and are particularly not a sensitive test for diagnosing HCC therefore this was not carried out [ 10 ]. McIvor et al have clearly shown that FNAC can easily recognise the tumour as Hürthle cell lesion [ 9 ]. Cases with suspicious histology and over 50 years of age carry a high risk of cancer [ 18 ].In the management of HCC the primary mode of treatment is surgical, ranging from hemithyroidectomy to total thyroidectomy. Larger tumours (>T2) require total thyroidectomy and lymphadenectomy if lymph nodes are involved [ 8 ]. Adjuvant radioiodine treatment or external beam radiotherapy is used for widely invasive carcinoma or locally advanced disease [ 8 ].Several reports in literature have identified contra lateral foci of carcinoma in 40–70% of cases of HCC [ 11 , 19 ]. HCC is less responsive to radioactive iodine therapy [ 20 ] and taking into account the aggressive behaviour, it has been suggested that every Hürthle cell tumour greater than 2 cm should be treated by total thyroidectomy [ 21 ]. In 1990 they showed that recurrent disease was noted in 17% of patients treated with total thyroidectomy compared to 59% in cases where a more limited procedure was carried out [ 21 , 22 ]. Other authors support the role of total thyroidectomy as there is 15 to 35% incidence of multiple foci in HCC [ 23 ].There are several reasons favouring the use of  131 I remnant ablation after near-total thyroidectomy [ 24 ]. First, presence of thyroid remnant can obscure  131 I uptake in cervical or lung metastases [ 25 , 26 ]. Second, distant (lung) metastases may be seen only on the post treatment whole body scan after remnant ablation [ 27 ]. Finally, remnant ablation may destroy residual normal follicular cells, which may become malignant [ 28 ] and any occult cancer that may recur years later.Radioiodine therapy has no overall effect on mortality but subgroup analysis has shown that those patients who receive radioactive iodine for adjuvant ablation of remnant thyroid tissue have lower mortality rate compared with patients who either did not receive treatment or in whom the indication was the presence of residual disease [ 29 ]. Radioiodine uptake in the elderly is much lower. Schlumberger and colleagues noted  131 I uptake at metastatic sites in only 53% of patients over 40 years of age, compared to 90% in patients below the age of 40 [ 30 ]. Univariate analysis indicated that older age and large tumour size predicted worse survival rates due to aggressive nature of the tumour (extra glandular invasion and multifocal disease). One recent series reviewed medical records of patients between the years 1944 and 1995. Of the 89 HCC cases studied, 29% had only undergone lobectomy as initial treatment and 50% had undergone partial resection. Of the three quarters of the patients in this series who received radioactive iodine, only 38% of patients with known metastases showed positive uptake [ 29 ]. Another study clearly suggested that treatment with  131 I to ablate the thyroid remnant and to treat residual disease were independent prognostic variables that favourably influenced recurrence, distant recurrence, and cancer death rates [ 24 ]. Our patient received radioactive iodine treatment in the postoperative period. He has been followed up with whole body scans (Fig  5  and  6 ), which indicate his response to adjuvant radioactive iodine therapy. He is on 125 mcg thyroxine in order to maintain a TSH level of less than 0.01 mIU/L and FT4 at the upper limit of normal (8–28 pmol/L).Stojdinovic  et al  have treated 56 patients with HCC between the years 1940 and 2000 [ 8 ]. Of these patients 23(41%) had minimally invasive disease with no evidence of extra thyroid invasion (T2 N0 M0) and 33(56%) had widely invasive HCC. Primary mode of treatment was surgery ranging from lobectomy and isthumusectomy to total thyroidectomy with cervical lymphadenectomy in presence of lymph node involvement. Some patients received adjuvant radioiodine or external beam radiotherapy for widely invasive carcinoma. Study end points were relapse free survival and disease specific survival. They reported 8 years survival rate of 100% and 58% for low and high-risk cancers respectively. In their entire study cohort age was not found to predict the outcome but the most significant factor was widely invasive carcinoma.et alKhafif  et al  in their series (42 patients with HCC between 1957–1997) used radioiodine in patients with distant metastases; none had thyroid remnant ablation with radioactive iodine [ 4 ]. They reported an overall survival rate of 90.5% and noted that age, size of tumour and extent of resection adversely affected the prognosis.et alHürthle cell lesion can be easily picked up on FNAC but to make a diagnosis of HCC one has to demonstrate vascular or capsular invasion. Intraoperative frozen sections have low predictive value and cases with advanced age (over 50), rapid enlargement of lump and palpable nodes should be regarded with high index of suspicion for presence of HCC. HCC or other differentiated carcinomas of thyroid in the elderly patients are generally more aggressive with less favourable prognosis compared to younger patients. They should be offered total thyroidectomy and selective lymph node dissection (when lymph nodes are involved) followed by ablative radioiodine therapy, provided they can withstand the above treatment. Coexisting medical disorders should be recognized and managed effectively prior to surgery [ 31 ]. Further research is needed to clarify the role of adjuvant radioiodine therapy in the management of HCC.Competing interests None declared.
Competing interests
None declared.Authors' contribution MRH  managed the patient, searched the literature and drafted the manuscript LI:  did the histological study, and contributed to pathological aspects in the present study DG:  Managed the patient, conceptualise the present report, edited the manuscript and coordinated after reviewing the manuscript
Authors' contribution
MRH  managed the patient, searched the literature and drafted the manuscriptMRHLI:  did the histological study, and contributed to pathological aspects in the present studyLI:DG:  Managed the patient, conceptualise the present report, edited the manuscript and coordinated after reviewing the manuscriptDG:


The mouse spot test, an  in vivo  mutation assay, has been used to assess a number of chemicals. It is at present the only  in vivo  mammalian test system capable of detecting somatic gene mutations according to OECD guidelines (OECD guideline 484). It is however rather insensitive, animal consuming and expensive type of test. More recently several assays using transgenic animals have been developed. From data in the literature, the present study compares the results of  in vivo  testing of over twenty chemicals using the mouse spot test and compares them with results from the two transgenic mouse models with the best data base available, the  lacI  model (commercially available as the Big Blue ®  mouse), and the  lacZ  model (commercially available as the Muta™ Mouse). There was agreement in the results from the majority of substances. No differences were found in the predictability of the transgenic animal assays and the mouse spot test for carcinogenicity. However, from the limited data available, it seems that the transgenic mouse assay has several advantages over the mouse spot test and may be a suitable test system replacing the mouse spot test for detection of gene but not chromosome mutations  in vivo .in vivoin vivoin vivolacIlacZin vivo

Background This is the second presentation from a project for the International Programme on Chemical Safety (IPCS) evaluating the possible use of transgenic animal mutagenicity assays in toxicity testing and mechanistic research. Part I, preceeding this article, discussed comparison of effects of chemicals using certain transgenic assays with results using the bone marrow micronucleus test. The assessment of the potential genotoxicity of chemicals  in vivo  is important for both the verification and confirmation of intrinsic mutagenicity and for establishing the mode of action of chemical carcinogens. Although the present trend is to reduce animal testing,  in vitro  data must be confirmed by testing in  in vivo  conditions which take into account whole animal processes like absorption, tissue distribution, metabolism and excretion of the chemical and its metabolites, and overall toxicity [ 1 ]. In the mid 1980s, the mouse spot test [ 2 ] was suggested as a complementary  in vivo  test to the bacterial mutagenicity assay for detection of mutagenic substances and as a confirmatory test for the identification of carcinogens [ 3 ]. The mouse spot test has been used to assess a number of chemicals (see e.g.  Additional file 1 , see separate file). It is at present the only  in vivo  mammalian test system capable of detecting somatic gene mutations according to OECD guidelines (OECD guideline 484 [ 4 ]). However to achieve an acceptable sensitivity, a large number of animals are necessary and it is therefore an expensive type of test and seldom used. More recently assays using transgenic animals have been developed for testing  in vivo  gene mutagenicity. The two transgenic mouse models with the best data base available are the  lacI  model (commercially available as the Big Blue ®  mouse), and the  lacZ  model (commercially available as the Muta™ Mouse). The present study compares the results of  in vivo  testing of a number of chemicals using the mouse spot test and compares it with results from these two transgenic mouse models. Descriptions of test systems Mouse spot test In the spot test, mouse embryos which are heterozygous for different recessive coat colour genes, are treated  in utero  at gestation day 9–11 with the test substance. The exposed embryo at gestation day 10 contains about 150–200 melanoblasts and each melanoblast has 4 coat colour genes under study [ 2 , 5 ]. The  in utero  exposure may result in an alteration or loss of a specific wild-type allele in a pigment precursor cell resulting in a colour spot in the coat of the adult animal. The frequency of spots is compared with the frequency in sham-exposed controls [ 2 , 4 ]. In the mouse spot test there are 4 possible mechanisms by which the recessive coat-colour alleles can be expressed: 1) gene mutation in the wild-type allele, 2) deficiency (large or small) of a chromosomal segment involving the wild-type allele, 3) nondisjunctional (or other) loss of the chromosome carrying the wild-type allele and 4) somatic recombination (marker gene then homozygous) [ 5 ]. Gene mutagenic but also clastogenic effects are detected by this test system. Transgenic mouse models The transgenic mutation test systems the  lacI  model (Big Blue ®  mouse), and the  lacZ  model (Muta™ Mouse) are described in detail in the preceding article: Mutagenicity testing with transgenic mice. Part I: Comparison with the mouse bone marrow micronucleus test
Background
This is the second presentation from a project for the International Programme on Chemical Safety (IPCS) evaluating the possible use of transgenic animal mutagenicity assays in toxicity testing and mechanistic research. Part I, preceeding this article, discussed comparison of effects of chemicals using certain transgenic assays with results using the bone marrow micronucleus test.The assessment of the potential genotoxicity of chemicals  in vivo  is important for both the verification and confirmation of intrinsic mutagenicity and for establishing the mode of action of chemical carcinogens. Although the present trend is to reduce animal testing,  in vitro  data must be confirmed by testing in  in vivo  conditions which take into account whole animal processes like absorption, tissue distribution, metabolism and excretion of the chemical and its metabolites, and overall toxicity [ 1 ]. In the mid 1980s, the mouse spot test [ 2 ] was suggested as a complementary  in vivo  test to the bacterial mutagenicity assay for detection of mutagenic substances and as a confirmatory test for the identification of carcinogens [ 3 ]. The mouse spot test has been used to assess a number of chemicals (see e.g.  Additional file 1 , see separate file). It is at present the only  in vivo  mammalian test system capable of detecting somatic gene mutations according to OECD guidelines (OECD guideline 484 [ 4 ]). However to achieve an acceptable sensitivity, a large number of animals are necessary and it is therefore an expensive type of test and seldom used. More recently assays using transgenic animals have been developed for testing  in vivo  gene mutagenicity. The two transgenic mouse models with the best data base available are the  lacI  model (commercially available as the Big Blue ®  mouse), and the  lacZ  model (commercially available as the Muta™ Mouse). The present study compares the results of  in vivo  testing of a number of chemicals using the mouse spot test and compares it with results from these two transgenic mouse models.in vivoin vitroin vivoin vivoin vivoin vivolacIlacZin vivoDescriptions of test systems Mouse spot test In the spot test, mouse embryos which are heterozygous for different recessive coat colour genes, are treated  in utero  at gestation day 9–11 with the test substance. The exposed embryo at gestation day 10 contains about 150–200 melanoblasts and each melanoblast has 4 coat colour genes under study [ 2 , 5 ]. The  in utero  exposure may result in an alteration or loss of a specific wild-type allele in a pigment precursor cell resulting in a colour spot in the coat of the adult animal. The frequency of spots is compared with the frequency in sham-exposed controls [ 2 , 4 ]. In the mouse spot test there are 4 possible mechanisms by which the recessive coat-colour alleles can be expressed: 1) gene mutation in the wild-type allele, 2) deficiency (large or small) of a chromosomal segment involving the wild-type allele, 3) nondisjunctional (or other) loss of the chromosome carrying the wild-type allele and 4) somatic recombination (marker gene then homozygous) [ 5 ]. Gene mutagenic but also clastogenic effects are detected by this test system. Transgenic mouse models The transgenic mutation test systems the  lacI  model (Big Blue ®  mouse), and the  lacZ  model (Muta™ Mouse) are described in detail in the preceding article: Mutagenicity testing with transgenic mice. Part I: Comparison with the mouse bone marrow micronucleus test
Descriptions of test systems
Mouse spot test In the spot test, mouse embryos which are heterozygous for different recessive coat colour genes, are treated  in utero  at gestation day 9–11 with the test substance. The exposed embryo at gestation day 10 contains about 150–200 melanoblasts and each melanoblast has 4 coat colour genes under study [ 2 , 5 ]. The  in utero  exposure may result in an alteration or loss of a specific wild-type allele in a pigment precursor cell resulting in a colour spot in the coat of the adult animal. The frequency of spots is compared with the frequency in sham-exposed controls [ 2 , 4 ]. In the mouse spot test there are 4 possible mechanisms by which the recessive coat-colour alleles can be expressed: 1) gene mutation in the wild-type allele, 2) deficiency (large or small) of a chromosomal segment involving the wild-type allele, 3) nondisjunctional (or other) loss of the chromosome carrying the wild-type allele and 4) somatic recombination (marker gene then homozygous) [ 5 ]. Gene mutagenic but also clastogenic effects are detected by this test system.
Mouse spot test
In the spot test, mouse embryos which are heterozygous for different recessive coat colour genes, are treated  in utero  at gestation day 9–11 with the test substance. The exposed embryo at gestation day 10 contains about 150–200 melanoblasts and each melanoblast has 4 coat colour genes under study [ 2 , 5 ]. The  in utero  exposure may result in an alteration or loss of a specific wild-type allele in a pigment precursor cell resulting in a colour spot in the coat of the adult animal. The frequency of spots is compared with the frequency in sham-exposed controls [ 2 , 4 ].in uteroin uteroIn the mouse spot test there are 4 possible mechanisms by which the recessive coat-colour alleles can be expressed: 1) gene mutation in the wild-type allele, 2) deficiency (large or small) of a chromosomal segment involving the wild-type allele, 3) nondisjunctional (or other) loss of the chromosome carrying the wild-type allele and 4) somatic recombination (marker gene then homozygous) [ 5 ]. Gene mutagenic but also clastogenic effects are detected by this test system.Transgenic mouse models The transgenic mutation test systems the  lacI  model (Big Blue ®  mouse), and the  lacZ  model (Muta™ Mouse) are described in detail in the preceding article: Mutagenicity testing with transgenic mice. Part I: Comparison with the mouse bone marrow micronucleus test
Transgenic mouse models
The transgenic mutation test systems the  lacI  model (Big Blue ®  mouse), and the  lacZ  model (Muta™ Mouse) are described in detail in the preceding article: Mutagenicity testing with transgenic mice. Part I: Comparison with the mouse bone marrow micronucleus testlacIlacZMethods Data presented in this documentation are the results of an extensive literature research. Concerning data on transgenic mouse assays only primary literature was used. Data on the mouse spot test were extracted from reliable reviews on this item or from primary literature. For all other data informations from secondary literature or data banks were used.
Methods
Data presented in this documentation are the results of an extensive literature research. Concerning data on transgenic mouse assays only primary literature was used. Data on the mouse spot test were extracted from reliable reviews on this item or from primary literature. For all other data informations from secondary literature or data banks were used.Results and Discussion Comparison of the mouse spot test with transgenic mouse model systems In the literature search chemicals have been identified that had been tested using the spot test  and  the Muta™ mouse assay (n = 20) or the Big Blue ®  mouse assay (n = 9) or both transgenic mutation assays (n = 8). The results (including references) are given in  Additional file 1 . The results on 15 out of 20 substances (2-acetylaminofluorene, acrylamide, benzo[ a ]pyrene, 1,3-butadiene, cyclophosphamide, ethylmethanesulfonate,  N -ethyl- N -nitrosourea,  N -methyl- N '-nitro- N -nitrosoguanidine,  N -methyl- N -nitrosourea, 4-nitroquino-line-1-oxide,  N -nitrosodiethylamine,  N -nitrosodimethylamine, procarbazine, 4-acetylaminofluorene and  N -propyl- N -nitrosourea) showed agreement between the Muta™ mouse and the mouse spot test. No agreement was seen with 5 out of 20 substances (4-acetylaminofluorene, 2-amino-3-methylimidazo(4,5-f)quinoline (IQ), hydrazine, mitomycin C, trichloroethylene). The positive results obtained with the Big Blue ®  mouse assay agreed with results in the mouse spot test for 7 out of 9 substances (2-acetylaminofluorene, benzo[ a ]pyrene, 1,3-butadiene, cyclophosphamide,  N -ethyl- N -nitrosourea,  N -methyl- N -nitrosourea,  N -nitrosodimethylamine); one (di-(2-ethylhexyl)phthalate) was negative in both test systems and only one (methyl methanesulfonate) showed no agreement between the two test systems. With two exceptions, 4-acetylaminofluorene and N-propyl-N-nitrosourea (discussed later), all of the tested substances showed also clearly positive results in  in vitro  gene mutation assays (exception of 1,3-butadiene, negative results) and in the majority of  in vivo  studies on this endpoint. Further they induced carcinogenic effects in long-term studies on mice. Although no data on carcinogenicity on mice is available on  N -propyl- N -nitrosourea, this substance might also be included in the category mentioned above, since carcinogenic effects were reported in rats [ 113 ] and  in vitro  gene mutation assays revealed clearly positive results. The following substances did  not  show agreement between results in the mouse spot test and transgenic mouse assays or negative results were reported in both test systems (see  Additional file 1 ). These are therefore discussed in more detail here; for references see Additional file  1 . Table 1 Characteristics of the Muta™ mouse assay and the Big Blue ®  mouse assay for predicting mouse carcinogenicity in comparison with the mouse spot test Term# Calculation* for the mouse spot test Calculation* for Muta™ and/or Big Blue ®  mouse combined ** Sensitivity 84% (16/18) 79% (15/18) Specificity 0 (0/0) 0 (0/0) Positive predictability 100% (16/16) 100% (15/15) Negative predictability 0 (0/2) 0 (0/3) Overall accuracy 84% (16/18) 79% (15/18) # Sensitivity = % of carcinogens with a positive result in the specified test system (STS) Specificity = % of noncarcinogens with a negative result in the STS Positive predictivity = % of positive results in the STS that are carcinogens Negative predictivity = % of negative results in the STS that are noncarcinogens Overall accuracy = % of chemicals tested where STS results agree with the carcinogenicity results *: carcinogens with genotoxic and nongenotoxic mechanisms were considered but not substances without data on carcinogenicity; only data on mice were used **: judged as positive in transgenic assays if positive in one of the two test systems For methylmethanesulfonate, the weak positive results were judged as positive. Trichloroethylene was not included in the calculation (inconclusive results in the mouse spot test). 4-Acetylaminofluorene This substance showed mutagenic activity in the Muta™ mouse assay [ 19 ] but negative results in the mouse spot test [ 12 , 13 ]. No data on carcinogenicity are available on 4-acetylaminofluorene. However, data on two  in vitro  test systems indicated gene mutagenic activity supporting results in the transgenic assay [ 15 - 18 ]. 2-Amino-3-methylimidazo(4,5-f)quinol (IQ) IQ is mutagenic in the Muta™ mouse assay [ 28 ] but negative results were obtained in the mouse spot test [ 29 ]. This negative result in the mouse spot test is in contrast to all other  in vivo  gene mutation assays on rodents and insects which revealed positive results [ 27 ]. Furthermore, gene mutagenic activity was detected in  in vitro  test systems and carcinogenic effects were observed in long-term studies on mice [ 27 ]. The results in the Muta™ mouse assay are in accordance with these data. Di-(2-ethylhexyl)phthalate Negative results in the mouse spot test [ 51 ] are in agreement with the negative Big Blue ®  assay [ 11 ]. Furthermore no gene mutagenic or questionable activity was reported in  in vitro  tests and in tests on Drosophila. Carcinogenic effects were obtained in studies on mice but nongenotoxic mechanisms are presumed. Hydrazine This substance induced mutagenic effects in the mouse spot test [ 72 ] but negative results were observed in the Muta™ mouse assay [ 71 ]. Other  in vivo  as well as  in vitro  test systems revealed gene mutagenic effects [ 70 ]. Increased tumor incidences were observed in carcinogenicity studies on mice. Overall, the mouse spot test but not the Muta™ mouse assay reflects data on genotoxicity and carcinogenicity. However, a single exposure was used in the Muta™ mouse assay [ 71 ]. Studies on other  in vivo  genotoxicity endpoints have shown generally negative results after single exposure but genotoxic activity after repeated application, for example the mouse bone marrow micronucleus assay was positive [ 20 ]. It is possible that positive results may be found using another experimental design in the Muta™ mouse assay e.g. repeated exposure. Methyl methanesulfonate Only weak mutagenic effects were observed in the Muta™ mouse [ 19 , 57 , 75 - 77 ] and negative results in the Big Blue ®  mouse [ 63 - 65 , 78 ]. In the mouse spot test this carcinogenic substance is mutagenic [ 3 ] as well as in other gene mutation assays  in vitro  and  in vivo  [ 73 , 74 ]. However, there is evidence that the chromosome mutagenic activity is detectable at much lower doses than the gene mutagenic activity. Tinwell et al. [ 19 ] have shown in Muta™ mice a weak gene mutagenic effect in the liver but no effect in the bone marrow. The same dose induced in these animals a significant increase in bone marrow micronuclei indicating clear clastogenic activity. However, the transgenic mutation assay is less suitable for detection of these effects [ 1 ]. Mitomycin C No mutagenic activity was observed in the Muta™ mouse assay after single application and ambiguous results after repeated exposure [ 93 ] but positive results were obtained with the mouse spot test [ 2 , 3 ] and other gene mutation assays  in vitro  and  in vivo  with this carcinogenic substance [ 90 - 92 ]. The reason for this discrepancy is similar to that presumed for methyl methanesulfonate above. Clastogenicity in bone marrow but no gene mutagenic activity in liver and bone marrow has been shown in the same animals in the Muta™ mouse assay combined with a micronucleus assay [ 93 ]. However, using another experimental design for detection of gene mutations in the Muta™ mouse assay (dose level up to the MTD, repeated exposure) positive results might be obtained. Trichloroethylene Also with this carcinogenic substance, no mutagenicity was detected in the Muta™ mouse assay [ 117 ], the mouse spot test was positive [ 3 ], but this result is possibly related to contaminations with epoxides [ 116 ]. Further  in vitro  and  in vivo  assays on gene mutation resulted in weak positive, questionable, or negative effects [ 116 ]. Results in chromosome mutation assays are equivocal. However, a further (simple) reason for this discrepancy between the Muta™ mouse assay and the mouse spot test might be that the MTD was not reached in the Muta™ mouse assay presented by Douglas et al. [ 117 ]. In general, from the studies on genotoxic carcinogens given above, the results do not seem to give a preference for either the spot test or transgenic mouse model system. However, considering the mechanisms of action of specific substances there is some evidence, that the mouse spot test detects gene mutations as well as chromosome mutations whereas the transgenic mouse assays are restricted to gene mutations. Evidence for this hypothesis has been shown with the examples methyl methanesulfonate, mitomycin C, and trichloroethylene. In the mouse spot test, there are four possible mechanisms by which the recessive coat-colour alleles can be expressed (see introduction) including gene and chromosome mutations. Although the chromosome mutations have to survive several mitoses to cause the expression of the recessive allele [ 118 ], there is evidence that also predominantly clastogenic substances might result in a positive mouse spot test. In contrast, the transgenic mutation assays detected point mutations and maximal small deletions and insertions [ 1 ]. Predictivity of the transgenic animal assays and the mouse spot test for carcinogenicity The sensitivity, specificity and predictivity of carcinogenicity for the transgenic mouse model (Muta™ mouse assay and the Big Blue ®  mouse assay combined) and the mouse spot test are documented in Table  1 . Data on 18 substances (see  Additional file 1 ) are available on carcinogenicity in mice  and  mutagenic effects in transgenic mice as well as mutagenic effects in the mouse spot test (trichloroethylene not included because of inconclusive results in the mouse spot test). Although the data pool is not sufficient for a comprehensive comparison, there is some indication, that no significant differences were detectable between the two test systems. Advantages and disadvantages of both test systems Sensitivity of the test system In comparison to models using endogenous genes like the target genes in the mouse spot test, the spontaneous mutant frequency in transgenic animals is relatively high. This might be due to the fact that bacterial DNA is the target gene (high methylation rate) and/or the transgene is silent and no transcription related repair occurs as in endogenous genes which are more efficiently repaired [ 1 ]. However, comparing the number of cells and genes at risk at the time of exposure, the mouse spot test is numerical inferior to the transgenic mouse mutation assays. In the mouse spot test, the exposed embryo at gestation day 10 contains about 150–200 melanoblasts and each melanoblast has 4 coat colour genes under study [ 2 , 5 ]. In the transgenic Big Blue ®  mouse, for example, 30–40 copies of the target gene (the constructed λLIZα shuttle vector) are integrated on chromosome 4 of  each  cell of the animal [ 1 ]. Other factors To achieve an acceptable sensitivity, a large number of animals are necessary in the mouse spot test. Many pregnant dams have to be in one treatment group to get a sufficient number of surviving F1-animals, since the test substance may induce maternal and developmental toxicity. Fahrig [ 2 ] suggested that 30–40 pregnant mice are needed per treatment group for evaluation of spots in the progeny. At least 150 F1-mice are recommended for the concurrent vehicle control [ 5 ] and at least two dose groups are used (OECD guideline 484 [ 4 ]). Therefore, the mouse spot test is an expensive type of  in vivo  test. In contrast, in transgenic mutation assays ca. 20 animals (3 dose groups and 1 concurrent vehicle control group in laboratories which already established this test system) are recommended per species and gender [ 119 - 121 ]. In the mouse spot test the discrimination between spots of mutagenic and non-mutagenic origin may be problematic [ 2 ]. A comparison of both test systems is presented in Table  2 . Table 2 Advantages and Disadvantages of mouse spot test compared to the transgenic Big Blue ®  and Muta™ mouse assays Mouse spot test  [2-5] Transgenic mouse mutation assay [1, 122] Age restriction Exposure restricted to embryos at gestation day 9–11 Usually less than 3 months Toxicokinetics and metabolism Restrictions in toxicokinetics: test substance reaches the fetal melanoblasts after administration to the dams and absorption of the test substance itself or the toxic metabolites via the placenta No further barrier like the placenta after absorption and distribution Target tissue Restricted to melanoblasts No tissue restriction; analysis of mutagenic potency in different organs Type of mutation Detects 1) gene mutation, 2) large or small deletions, 3) loss of the chromosome carrying the wild-type allele and 4) somatic recombination (marker gene then homozygous) Detects 1) gene mutation, 2) small deletions or insertions Dependency of effects on application route Only systemic effects can be detected; no application route specific effects For different routes systemic as well as local mutagenic effects can be detected Target gene/cell 4 genes per cell in ca. 200 melanocytes Ca. 40 (Big Blue) or ca. 80 (Muta™ mouse) copies of the transgene per nucleus of each cell of the organismk Number of animals Animal consuming test system Not more than 5 animals per gender per dose necessary Specificity of test system Discrimination between spots of mutagenic and non-mutagenic origin may be problematically Identifying and isolating mutated genes with a high specificity Characterisation of mutations by molecular methods Less suitable for identification of mutations in DNA analysis due to size of the genes detection of the "molecular signature" of a particular mutagen by DNA sequence analysis with standardized methods Possibility of parallel investigation of several genetic endpoints No combination with other genotoxic endpoints possible The transgenic mouse assay can be combined with other  in vivo  genotoxic endpoints in the same animal: e.g. micronuclei, chromosomal aberration, unsheduled DNA synthesis, sister chromatid exchange Endogenous versus foreign target gene The mouse spot test shows an in situ end point (expression of the target genes) Target genes are integrated parts of foreign DNA and consequently no "normal" mutational target Costs Expensive type of  in vivo  test Less expensive
Results and Discussion
Comparison of the mouse spot test with transgenic mouse model systems In the literature search chemicals have been identified that had been tested using the spot test  and  the Muta™ mouse assay (n = 20) or the Big Blue ®  mouse assay (n = 9) or both transgenic mutation assays (n = 8). The results (including references) are given in  Additional file 1 . The results on 15 out of 20 substances (2-acetylaminofluorene, acrylamide, benzo[ a ]pyrene, 1,3-butadiene, cyclophosphamide, ethylmethanesulfonate,  N -ethyl- N -nitrosourea,  N -methyl- N '-nitro- N -nitrosoguanidine,  N -methyl- N -nitrosourea, 4-nitroquino-line-1-oxide,  N -nitrosodiethylamine,  N -nitrosodimethylamine, procarbazine, 4-acetylaminofluorene and  N -propyl- N -nitrosourea) showed agreement between the Muta™ mouse and the mouse spot test. No agreement was seen with 5 out of 20 substances (4-acetylaminofluorene, 2-amino-3-methylimidazo(4,5-f)quinoline (IQ), hydrazine, mitomycin C, trichloroethylene). The positive results obtained with the Big Blue ®  mouse assay agreed with results in the mouse spot test for 7 out of 9 substances (2-acetylaminofluorene, benzo[ a ]pyrene, 1,3-butadiene, cyclophosphamide,  N -ethyl- N -nitrosourea,  N -methyl- N -nitrosourea,  N -nitrosodimethylamine); one (di-(2-ethylhexyl)phthalate) was negative in both test systems and only one (methyl methanesulfonate) showed no agreement between the two test systems. With two exceptions, 4-acetylaminofluorene and N-propyl-N-nitrosourea (discussed later), all of the tested substances showed also clearly positive results in  in vitro  gene mutation assays (exception of 1,3-butadiene, negative results) and in the majority of  in vivo  studies on this endpoint. Further they induced carcinogenic effects in long-term studies on mice. Although no data on carcinogenicity on mice is available on  N -propyl- N -nitrosourea, this substance might also be included in the category mentioned above, since carcinogenic effects were reported in rats [ 113 ] and  in vitro  gene mutation assays revealed clearly positive results. The following substances did  not  show agreement between results in the mouse spot test and transgenic mouse assays or negative results were reported in both test systems (see  Additional file 1 ). These are therefore discussed in more detail here; for references see Additional file  1 . Table 1 Characteristics of the Muta™ mouse assay and the Big Blue ®  mouse assay for predicting mouse carcinogenicity in comparison with the mouse spot test Term# Calculation* for the mouse spot test Calculation* for Muta™ and/or Big Blue ®  mouse combined ** Sensitivity 84% (16/18) 79% (15/18) Specificity 0 (0/0) 0 (0/0) Positive predictability 100% (16/16) 100% (15/15) Negative predictability 0 (0/2) 0 (0/3) Overall accuracy 84% (16/18) 79% (15/18) # Sensitivity = % of carcinogens with a positive result in the specified test system (STS) Specificity = % of noncarcinogens with a negative result in the STS Positive predictivity = % of positive results in the STS that are carcinogens Negative predictivity = % of negative results in the STS that are noncarcinogens Overall accuracy = % of chemicals tested where STS results agree with the carcinogenicity results *: carcinogens with genotoxic and nongenotoxic mechanisms were considered but not substances without data on carcinogenicity; only data on mice were used **: judged as positive in transgenic assays if positive in one of the two test systems For methylmethanesulfonate, the weak positive results were judged as positive. Trichloroethylene was not included in the calculation (inconclusive results in the mouse spot test). 4-Acetylaminofluorene This substance showed mutagenic activity in the Muta™ mouse assay [ 19 ] but negative results in the mouse spot test [ 12 , 13 ]. No data on carcinogenicity are available on 4-acetylaminofluorene. However, data on two  in vitro  test systems indicated gene mutagenic activity supporting results in the transgenic assay [ 15 - 18 ]. 2-Amino-3-methylimidazo(4,5-f)quinol (IQ) IQ is mutagenic in the Muta™ mouse assay [ 28 ] but negative results were obtained in the mouse spot test [ 29 ]. This negative result in the mouse spot test is in contrast to all other  in vivo  gene mutation assays on rodents and insects which revealed positive results [ 27 ]. Furthermore, gene mutagenic activity was detected in  in vitro  test systems and carcinogenic effects were observed in long-term studies on mice [ 27 ]. The results in the Muta™ mouse assay are in accordance with these data. Di-(2-ethylhexyl)phthalate Negative results in the mouse spot test [ 51 ] are in agreement with the negative Big Blue ®  assay [ 11 ]. Furthermore no gene mutagenic or questionable activity was reported in  in vitro  tests and in tests on Drosophila. Carcinogenic effects were obtained in studies on mice but nongenotoxic mechanisms are presumed. Hydrazine This substance induced mutagenic effects in the mouse spot test [ 72 ] but negative results were observed in the Muta™ mouse assay [ 71 ]. Other  in vivo  as well as  in vitro  test systems revealed gene mutagenic effects [ 70 ]. Increased tumor incidences were observed in carcinogenicity studies on mice. Overall, the mouse spot test but not the Muta™ mouse assay reflects data on genotoxicity and carcinogenicity. However, a single exposure was used in the Muta™ mouse assay [ 71 ]. Studies on other  in vivo  genotoxicity endpoints have shown generally negative results after single exposure but genotoxic activity after repeated application, for example the mouse bone marrow micronucleus assay was positive [ 20 ]. It is possible that positive results may be found using another experimental design in the Muta™ mouse assay e.g. repeated exposure. Methyl methanesulfonate Only weak mutagenic effects were observed in the Muta™ mouse [ 19 , 57 , 75 - 77 ] and negative results in the Big Blue ®  mouse [ 63 - 65 , 78 ]. In the mouse spot test this carcinogenic substance is mutagenic [ 3 ] as well as in other gene mutation assays  in vitro  and  in vivo  [ 73 , 74 ]. However, there is evidence that the chromosome mutagenic activity is detectable at much lower doses than the gene mutagenic activity. Tinwell et al. [ 19 ] have shown in Muta™ mice a weak gene mutagenic effect in the liver but no effect in the bone marrow. The same dose induced in these animals a significant increase in bone marrow micronuclei indicating clear clastogenic activity. However, the transgenic mutation assay is less suitable for detection of these effects [ 1 ]. Mitomycin C No mutagenic activity was observed in the Muta™ mouse assay after single application and ambiguous results after repeated exposure [ 93 ] but positive results were obtained with the mouse spot test [ 2 , 3 ] and other gene mutation assays  in vitro  and  in vivo  with this carcinogenic substance [ 90 - 92 ]. The reason for this discrepancy is similar to that presumed for methyl methanesulfonate above. Clastogenicity in bone marrow but no gene mutagenic activity in liver and bone marrow has been shown in the same animals in the Muta™ mouse assay combined with a micronucleus assay [ 93 ]. However, using another experimental design for detection of gene mutations in the Muta™ mouse assay (dose level up to the MTD, repeated exposure) positive results might be obtained. Trichloroethylene Also with this carcinogenic substance, no mutagenicity was detected in the Muta™ mouse assay [ 117 ], the mouse spot test was positive [ 3 ], but this result is possibly related to contaminations with epoxides [ 116 ]. Further  in vitro  and  in vivo  assays on gene mutation resulted in weak positive, questionable, or negative effects [ 116 ]. Results in chromosome mutation assays are equivocal. However, a further (simple) reason for this discrepancy between the Muta™ mouse assay and the mouse spot test might be that the MTD was not reached in the Muta™ mouse assay presented by Douglas et al. [ 117 ]. In general, from the studies on genotoxic carcinogens given above, the results do not seem to give a preference for either the spot test or transgenic mouse model system. However, considering the mechanisms of action of specific substances there is some evidence, that the mouse spot test detects gene mutations as well as chromosome mutations whereas the transgenic mouse assays are restricted to gene mutations. Evidence for this hypothesis has been shown with the examples methyl methanesulfonate, mitomycin C, and trichloroethylene. In the mouse spot test, there are four possible mechanisms by which the recessive coat-colour alleles can be expressed (see introduction) including gene and chromosome mutations. Although the chromosome mutations have to survive several mitoses to cause the expression of the recessive allele [ 118 ], there is evidence that also predominantly clastogenic substances might result in a positive mouse spot test. In contrast, the transgenic mutation assays detected point mutations and maximal small deletions and insertions [ 1 ].
Comparison of the mouse spot test with transgenic mouse model systems
In the literature search chemicals have been identified that had been tested using the spot test  and  the Muta™ mouse assay (n = 20) or the Big Blue ®  mouse assay (n = 9) or both transgenic mutation assays (n = 8). The results (including references) are given in  Additional file 1 .andThe results on 15 out of 20 substances (2-acetylaminofluorene, acrylamide, benzo[ a ]pyrene, 1,3-butadiene, cyclophosphamide, ethylmethanesulfonate,  N -ethyl- N -nitrosourea,  N -methyl- N '-nitro- N -nitrosoguanidine,  N -methyl- N -nitrosourea, 4-nitroquino-line-1-oxide,  N -nitrosodiethylamine,  N -nitrosodimethylamine, procarbazine, 4-acetylaminofluorene and  N -propyl- N -nitrosourea) showed agreement between the Muta™ mouse and the mouse spot test. No agreement was seen with 5 out of 20 substances (4-acetylaminofluorene, 2-amino-3-methylimidazo(4,5-f)quinoline (IQ), hydrazine, mitomycin C, trichloroethylene).aNNNNNNNNNNNThe positive results obtained with the Big Blue ®  mouse assay agreed with results in the mouse spot test for 7 out of 9 substances (2-acetylaminofluorene, benzo[ a ]pyrene, 1,3-butadiene, cyclophosphamide,  N -ethyl- N -nitrosourea,  N -methyl- N -nitrosourea,  N -nitrosodimethylamine); one (di-(2-ethylhexyl)phthalate) was negative in both test systems and only one (methyl methanesulfonate) showed no agreement between the two test systems.aNNNNNWith two exceptions, 4-acetylaminofluorene and N-propyl-N-nitrosourea (discussed later), all of the tested substances showed also clearly positive results in  in vitro  gene mutation assays (exception of 1,3-butadiene, negative results) and in the majority of  in vivo  studies on this endpoint. Further they induced carcinogenic effects in long-term studies on mice.in vitroin vivoAlthough no data on carcinogenicity on mice is available on  N -propyl- N -nitrosourea, this substance might also be included in the category mentioned above, since carcinogenic effects were reported in rats [ 113 ] and  in vitro  gene mutation assays revealed clearly positive results.NNin vitroThe following substances did  not  show agreement between results in the mouse spot test and transgenic mouse assays or negative results were reported in both test systems (see  Additional file 1 ). These are therefore discussed in more detail here; for references see Additional file  1 .Characteristics of the Muta™ mouse assay and the Big Blue ®  mouse assay for predicting mouse carcinogenicity in comparison with the mouse spot test# Sensitivity = % of carcinogens with a positive result in the specified test system (STS)Specificity = % of noncarcinogens with a negative result in the STSPositive predictivity = % of positive results in the STS that are carcinogensNegative predictivity = % of negative results in the STS that are noncarcinogensOverall accuracy = % of chemicals tested where STS results agree with the carcinogenicity results*: carcinogens with genotoxic and nongenotoxic mechanisms were considered but not substances without data on carcinogenicity; only data on mice were used**: judged as positive in transgenic assays if positive in one of the two test systemsFor methylmethanesulfonate, the weak positive results were judged as positive.Trichloroethylene was not included in the calculation (inconclusive results in the mouse spot test).4-Acetylaminofluorene This substance showed mutagenic activity in the Muta™ mouse assay [ 19 ] but negative results in the mouse spot test [ 12 , 13 ]. No data on carcinogenicity are available on 4-acetylaminofluorene. However, data on two  in vitro  test systems indicated gene mutagenic activity supporting results in the transgenic assay [ 15 - 18 ].
4-Acetylaminofluorene
This substance showed mutagenic activity in the Muta™ mouse assay [ 19 ] but negative results in the mouse spot test [ 12 , 13 ]. No data on carcinogenicity are available on 4-acetylaminofluorene. However, data on two  in vitro  test systems indicated gene mutagenic activity supporting results in the transgenic assay [ 15 - 18 ].in vitro2-Amino-3-methylimidazo(4,5-f)quinol (IQ) IQ is mutagenic in the Muta™ mouse assay [ 28 ] but negative results were obtained in the mouse spot test [ 29 ]. This negative result in the mouse spot test is in contrast to all other  in vivo  gene mutation assays on rodents and insects which revealed positive results [ 27 ]. Furthermore, gene mutagenic activity was detected in  in vitro  test systems and carcinogenic effects were observed in long-term studies on mice [ 27 ]. The results in the Muta™ mouse assay are in accordance with these data.
2-Amino-3-methylimidazo(4,5-f)quinol (IQ)
IQ is mutagenic in the Muta™ mouse assay [ 28 ] but negative results were obtained in the mouse spot test [ 29 ]. This negative result in the mouse spot test is in contrast to all other  in vivo  gene mutation assays on rodents and insects which revealed positive results [ 27 ]. Furthermore, gene mutagenic activity was detected in  in vitro  test systems and carcinogenic effects were observed in long-term studies on mice [ 27 ]. The results in the Muta™ mouse assay are in accordance with these data.in vivoin vitroDi-(2-ethylhexyl)phthalate Negative results in the mouse spot test [ 51 ] are in agreement with the negative Big Blue ®  assay [ 11 ]. Furthermore no gene mutagenic or questionable activity was reported in  in vitro  tests and in tests on Drosophila. Carcinogenic effects were obtained in studies on mice but nongenotoxic mechanisms are presumed.
Di-(2-ethylhexyl)phthalate
Negative results in the mouse spot test [ 51 ] are in agreement with the negative Big Blue ®  assay [ 11 ]. Furthermore no gene mutagenic or questionable activity was reported in  in vitro  tests and in tests on Drosophila. Carcinogenic effects were obtained in studies on mice but nongenotoxic mechanisms are presumed.in vitroHydrazine This substance induced mutagenic effects in the mouse spot test [ 72 ] but negative results were observed in the Muta™ mouse assay [ 71 ]. Other  in vivo  as well as  in vitro  test systems revealed gene mutagenic effects [ 70 ]. Increased tumor incidences were observed in carcinogenicity studies on mice. Overall, the mouse spot test but not the Muta™ mouse assay reflects data on genotoxicity and carcinogenicity. However, a single exposure was used in the Muta™ mouse assay [ 71 ]. Studies on other  in vivo  genotoxicity endpoints have shown generally negative results after single exposure but genotoxic activity after repeated application, for example the mouse bone marrow micronucleus assay was positive [ 20 ]. It is possible that positive results may be found using another experimental design in the Muta™ mouse assay e.g. repeated exposure.
Hydrazine
This substance induced mutagenic effects in the mouse spot test [ 72 ] but negative results were observed in the Muta™ mouse assay [ 71 ]. Other  in vivo  as well as  in vitro  test systems revealed gene mutagenic effects [ 70 ]. Increased tumor incidences were observed in carcinogenicity studies on mice. Overall, the mouse spot test but not the Muta™ mouse assay reflects data on genotoxicity and carcinogenicity. However, a single exposure was used in the Muta™ mouse assay [ 71 ]. Studies on other  in vivo  genotoxicity endpoints have shown generally negative results after single exposure but genotoxic activity after repeated application, for example the mouse bone marrow micronucleus assay was positive [ 20 ]. It is possible that positive results may be found using another experimental design in the Muta™ mouse assay e.g. repeated exposure.in vivoin vitroin vivoMethyl methanesulfonate Only weak mutagenic effects were observed in the Muta™ mouse [ 19 , 57 , 75 - 77 ] and negative results in the Big Blue ®  mouse [ 63 - 65 , 78 ]. In the mouse spot test this carcinogenic substance is mutagenic [ 3 ] as well as in other gene mutation assays  in vitro  and  in vivo  [ 73 , 74 ]. However, there is evidence that the chromosome mutagenic activity is detectable at much lower doses than the gene mutagenic activity. Tinwell et al. [ 19 ] have shown in Muta™ mice a weak gene mutagenic effect in the liver but no effect in the bone marrow. The same dose induced in these animals a significant increase in bone marrow micronuclei indicating clear clastogenic activity. However, the transgenic mutation assay is less suitable for detection of these effects [ 1 ].
Methyl methanesulfonate
Only weak mutagenic effects were observed in the Muta™ mouse [ 19 , 57 , 75 - 77 ] and negative results in the Big Blue ®  mouse [ 63 - 65 , 78 ]. In the mouse spot test this carcinogenic substance is mutagenic [ 3 ] as well as in other gene mutation assays  in vitro  and  in vivo  [ 73 , 74 ]. However, there is evidence that the chromosome mutagenic activity is detectable at much lower doses than the gene mutagenic activity. Tinwell et al. [ 19 ] have shown in Muta™ mice a weak gene mutagenic effect in the liver but no effect in the bone marrow. The same dose induced in these animals a significant increase in bone marrow micronuclei indicating clear clastogenic activity. However, the transgenic mutation assay is less suitable for detection of these effects [ 1 ].in vitroin vivoMitomycin C No mutagenic activity was observed in the Muta™ mouse assay after single application and ambiguous results after repeated exposure [ 93 ] but positive results were obtained with the mouse spot test [ 2 , 3 ] and other gene mutation assays  in vitro  and  in vivo  with this carcinogenic substance [ 90 - 92 ]. The reason for this discrepancy is similar to that presumed for methyl methanesulfonate above. Clastogenicity in bone marrow but no gene mutagenic activity in liver and bone marrow has been shown in the same animals in the Muta™ mouse assay combined with a micronucleus assay [ 93 ]. However, using another experimental design for detection of gene mutations in the Muta™ mouse assay (dose level up to the MTD, repeated exposure) positive results might be obtained.
Mitomycin C
No mutagenic activity was observed in the Muta™ mouse assay after single application and ambiguous results after repeated exposure [ 93 ] but positive results were obtained with the mouse spot test [ 2 , 3 ] and other gene mutation assays  in vitro  and  in vivo  with this carcinogenic substance [ 90 - 92 ]. The reason for this discrepancy is similar to that presumed for methyl methanesulfonate above. Clastogenicity in bone marrow but no gene mutagenic activity in liver and bone marrow has been shown in the same animals in the Muta™ mouse assay combined with a micronucleus assay [ 93 ]. However, using another experimental design for detection of gene mutations in the Muta™ mouse assay (dose level up to the MTD, repeated exposure) positive results might be obtained.in vitroin vivoTrichloroethylene Also with this carcinogenic substance, no mutagenicity was detected in the Muta™ mouse assay [ 117 ], the mouse spot test was positive [ 3 ], but this result is possibly related to contaminations with epoxides [ 116 ]. Further  in vitro  and  in vivo  assays on gene mutation resulted in weak positive, questionable, or negative effects [ 116 ]. Results in chromosome mutation assays are equivocal. However, a further (simple) reason for this discrepancy between the Muta™ mouse assay and the mouse spot test might be that the MTD was not reached in the Muta™ mouse assay presented by Douglas et al. [ 117 ]. In general, from the studies on genotoxic carcinogens given above, the results do not seem to give a preference for either the spot test or transgenic mouse model system. However, considering the mechanisms of action of specific substances there is some evidence, that the mouse spot test detects gene mutations as well as chromosome mutations whereas the transgenic mouse assays are restricted to gene mutations. Evidence for this hypothesis has been shown with the examples methyl methanesulfonate, mitomycin C, and trichloroethylene. In the mouse spot test, there are four possible mechanisms by which the recessive coat-colour alleles can be expressed (see introduction) including gene and chromosome mutations. Although the chromosome mutations have to survive several mitoses to cause the expression of the recessive allele [ 118 ], there is evidence that also predominantly clastogenic substances might result in a positive mouse spot test. In contrast, the transgenic mutation assays detected point mutations and maximal small deletions and insertions [ 1 ].
Trichloroethylene
Also with this carcinogenic substance, no mutagenicity was detected in the Muta™ mouse assay [ 117 ], the mouse spot test was positive [ 3 ], but this result is possibly related to contaminations with epoxides [ 116 ]. Further  in vitro  and  in vivo  assays on gene mutation resulted in weak positive, questionable, or negative effects [ 116 ]. Results in chromosome mutation assays are equivocal. However, a further (simple) reason for this discrepancy between the Muta™ mouse assay and the mouse spot test might be that the MTD was not reached in the Muta™ mouse assay presented by Douglas et al. [ 117 ].in vitroin vivoIn general, from the studies on genotoxic carcinogens given above, the results do not seem to give a preference for either the spot test or transgenic mouse model system.However, considering the mechanisms of action of specific substances there is some evidence, that the mouse spot test detects gene mutations as well as chromosome mutations whereas the transgenic mouse assays are restricted to gene mutations. Evidence for this hypothesis has been shown with the examples methyl methanesulfonate, mitomycin C, and trichloroethylene. In the mouse spot test, there are four possible mechanisms by which the recessive coat-colour alleles can be expressed (see introduction) including gene and chromosome mutations. Although the chromosome mutations have to survive several mitoses to cause the expression of the recessive allele [ 118 ], there is evidence that also predominantly clastogenic substances might result in a positive mouse spot test. In contrast, the transgenic mutation assays detected point mutations and maximal small deletions and insertions [ 1 ].Predictivity of the transgenic animal assays and the mouse spot test for carcinogenicity The sensitivity, specificity and predictivity of carcinogenicity for the transgenic mouse model (Muta™ mouse assay and the Big Blue ®  mouse assay combined) and the mouse spot test are documented in Table  1 . Data on 18 substances (see  Additional file 1 ) are available on carcinogenicity in mice  and  mutagenic effects in transgenic mice as well as mutagenic effects in the mouse spot test (trichloroethylene not included because of inconclusive results in the mouse spot test). Although the data pool is not sufficient for a comprehensive comparison, there is some indication, that no significant differences were detectable between the two test systems.
Predictivity of the transgenic animal assays and the mouse spot test for carcinogenicity
The sensitivity, specificity and predictivity of carcinogenicity for the transgenic mouse model (Muta™ mouse assay and the Big Blue ®  mouse assay combined) and the mouse spot test are documented in Table  1 . Data on 18 substances (see  Additional file 1 ) are available on carcinogenicity in mice  and  mutagenic effects in transgenic mice as well as mutagenic effects in the mouse spot test (trichloroethylene not included because of inconclusive results in the mouse spot test).Although the data pool is not sufficient for a comprehensive comparison, there is some indication, that no significant differences were detectable between the two test systems.Advantages and disadvantages of both test systems Sensitivity of the test system In comparison to models using endogenous genes like the target genes in the mouse spot test, the spontaneous mutant frequency in transgenic animals is relatively high. This might be due to the fact that bacterial DNA is the target gene (high methylation rate) and/or the transgene is silent and no transcription related repair occurs as in endogenous genes which are more efficiently repaired [ 1 ]. However, comparing the number of cells and genes at risk at the time of exposure, the mouse spot test is numerical inferior to the transgenic mouse mutation assays. In the mouse spot test, the exposed embryo at gestation day 10 contains about 150–200 melanoblasts and each melanoblast has 4 coat colour genes under study [ 2 , 5 ]. In the transgenic Big Blue ®  mouse, for example, 30–40 copies of the target gene (the constructed λLIZα shuttle vector) are integrated on chromosome 4 of  each  cell of the animal [ 1 ]. Other factors To achieve an acceptable sensitivity, a large number of animals are necessary in the mouse spot test. Many pregnant dams have to be in one treatment group to get a sufficient number of surviving F1-animals, since the test substance may induce maternal and developmental toxicity. Fahrig [ 2 ] suggested that 30–40 pregnant mice are needed per treatment group for evaluation of spots in the progeny. At least 150 F1-mice are recommended for the concurrent vehicle control [ 5 ] and at least two dose groups are used (OECD guideline 484 [ 4 ]). Therefore, the mouse spot test is an expensive type of  in vivo  test. In contrast, in transgenic mutation assays ca. 20 animals (3 dose groups and 1 concurrent vehicle control group in laboratories which already established this test system) are recommended per species and gender [ 119 - 121 ]. In the mouse spot test the discrimination between spots of mutagenic and non-mutagenic origin may be problematic [ 2 ]. A comparison of both test systems is presented in Table  2 . Table 2 Advantages and Disadvantages of mouse spot test compared to the transgenic Big Blue ®  and Muta™ mouse assays Mouse spot test  [2-5] Transgenic mouse mutation assay [1, 122] Age restriction Exposure restricted to embryos at gestation day 9–11 Usually less than 3 months Toxicokinetics and metabolism Restrictions in toxicokinetics: test substance reaches the fetal melanoblasts after administration to the dams and absorption of the test substance itself or the toxic metabolites via the placenta No further barrier like the placenta after absorption and distribution Target tissue Restricted to melanoblasts No tissue restriction; analysis of mutagenic potency in different organs Type of mutation Detects 1) gene mutation, 2) large or small deletions, 3) loss of the chromosome carrying the wild-type allele and 4) somatic recombination (marker gene then homozygous) Detects 1) gene mutation, 2) small deletions or insertions Dependency of effects on application route Only systemic effects can be detected; no application route specific effects For different routes systemic as well as local mutagenic effects can be detected Target gene/cell 4 genes per cell in ca. 200 melanocytes Ca. 40 (Big Blue) or ca. 80 (Muta™ mouse) copies of the transgene per nucleus of each cell of the organismk Number of animals Animal consuming test system Not more than 5 animals per gender per dose necessary Specificity of test system Discrimination between spots of mutagenic and non-mutagenic origin may be problematically Identifying and isolating mutated genes with a high specificity Characterisation of mutations by molecular methods Less suitable for identification of mutations in DNA analysis due to size of the genes detection of the "molecular signature" of a particular mutagen by DNA sequence analysis with standardized methods Possibility of parallel investigation of several genetic endpoints No combination with other genotoxic endpoints possible The transgenic mouse assay can be combined with other  in vivo  genotoxic endpoints in the same animal: e.g. micronuclei, chromosomal aberration, unsheduled DNA synthesis, sister chromatid exchange Endogenous versus foreign target gene The mouse spot test shows an in situ end point (expression of the target genes) Target genes are integrated parts of foreign DNA and consequently no "normal" mutational target Costs Expensive type of  in vivo  test Less expensive
Advantages and disadvantages of both test systems
Sensitivity of the test system In comparison to models using endogenous genes like the target genes in the mouse spot test, the spontaneous mutant frequency in transgenic animals is relatively high. This might be due to the fact that bacterial DNA is the target gene (high methylation rate) and/or the transgene is silent and no transcription related repair occurs as in endogenous genes which are more efficiently repaired [ 1 ]. However, comparing the number of cells and genes at risk at the time of exposure, the mouse spot test is numerical inferior to the transgenic mouse mutation assays. In the mouse spot test, the exposed embryo at gestation day 10 contains about 150–200 melanoblasts and each melanoblast has 4 coat colour genes under study [ 2 , 5 ]. In the transgenic Big Blue ®  mouse, for example, 30–40 copies of the target gene (the constructed λLIZα shuttle vector) are integrated on chromosome 4 of  each  cell of the animal [ 1 ].
Sensitivity of the test system
In comparison to models using endogenous genes like the target genes in the mouse spot test, the spontaneous mutant frequency in transgenic animals is relatively high. This might be due to the fact that bacterial DNA is the target gene (high methylation rate) and/or the transgene is silent and no transcription related repair occurs as in endogenous genes which are more efficiently repaired [ 1 ]. However, comparing the number of cells and genes at risk at the time of exposure, the mouse spot test is numerical inferior to the transgenic mouse mutation assays. In the mouse spot test, the exposed embryo at gestation day 10 contains about 150–200 melanoblasts and each melanoblast has 4 coat colour genes under study [ 2 , 5 ]. In the transgenic Big Blue ®  mouse, for example, 30–40 copies of the target gene (the constructed λLIZα shuttle vector) are integrated on chromosome 4 of  each  cell of the animal [ 1 ].eachOther factors To achieve an acceptable sensitivity, a large number of animals are necessary in the mouse spot test. Many pregnant dams have to be in one treatment group to get a sufficient number of surviving F1-animals, since the test substance may induce maternal and developmental toxicity. Fahrig [ 2 ] suggested that 30–40 pregnant mice are needed per treatment group for evaluation of spots in the progeny. At least 150 F1-mice are recommended for the concurrent vehicle control [ 5 ] and at least two dose groups are used (OECD guideline 484 [ 4 ]). Therefore, the mouse spot test is an expensive type of  in vivo  test. In contrast, in transgenic mutation assays ca. 20 animals (3 dose groups and 1 concurrent vehicle control group in laboratories which already established this test system) are recommended per species and gender [ 119 - 121 ]. In the mouse spot test the discrimination between spots of mutagenic and non-mutagenic origin may be problematic [ 2 ]. A comparison of both test systems is presented in Table  2 . Table 2 Advantages and Disadvantages of mouse spot test compared to the transgenic Big Blue ®  and Muta™ mouse assays Mouse spot test  [2-5] Transgenic mouse mutation assay [1, 122] Age restriction Exposure restricted to embryos at gestation day 9–11 Usually less than 3 months Toxicokinetics and metabolism Restrictions in toxicokinetics: test substance reaches the fetal melanoblasts after administration to the dams and absorption of the test substance itself or the toxic metabolites via the placenta No further barrier like the placenta after absorption and distribution Target tissue Restricted to melanoblasts No tissue restriction; analysis of mutagenic potency in different organs Type of mutation Detects 1) gene mutation, 2) large or small deletions, 3) loss of the chromosome carrying the wild-type allele and 4) somatic recombination (marker gene then homozygous) Detects 1) gene mutation, 2) small deletions or insertions Dependency of effects on application route Only systemic effects can be detected; no application route specific effects For different routes systemic as well as local mutagenic effects can be detected Target gene/cell 4 genes per cell in ca. 200 melanocytes Ca. 40 (Big Blue) or ca. 80 (Muta™ mouse) copies of the transgene per nucleus of each cell of the organismk Number of animals Animal consuming test system Not more than 5 animals per gender per dose necessary Specificity of test system Discrimination between spots of mutagenic and non-mutagenic origin may be problematically Identifying and isolating mutated genes with a high specificity Characterisation of mutations by molecular methods Less suitable for identification of mutations in DNA analysis due to size of the genes detection of the "molecular signature" of a particular mutagen by DNA sequence analysis with standardized methods Possibility of parallel investigation of several genetic endpoints No combination with other genotoxic endpoints possible The transgenic mouse assay can be combined with other  in vivo  genotoxic endpoints in the same animal: e.g. micronuclei, chromosomal aberration, unsheduled DNA synthesis, sister chromatid exchange Endogenous versus foreign target gene The mouse spot test shows an in situ end point (expression of the target genes) Target genes are integrated parts of foreign DNA and consequently no "normal" mutational target Costs Expensive type of  in vivo  test Less expensive
Other factors
To achieve an acceptable sensitivity, a large number of animals are necessary in the mouse spot test. Many pregnant dams have to be in one treatment group to get a sufficient number of surviving F1-animals, since the test substance may induce maternal and developmental toxicity. Fahrig [ 2 ] suggested that 30–40 pregnant mice are needed per treatment group for evaluation of spots in the progeny. At least 150 F1-mice are recommended for the concurrent vehicle control [ 5 ] and at least two dose groups are used (OECD guideline 484 [ 4 ]). Therefore, the mouse spot test is an expensive type of  in vivo  test.in vivoIn contrast, in transgenic mutation assays ca. 20 animals (3 dose groups and 1 concurrent vehicle control group in laboratories which already established this test system) are recommended per species and gender [ 119 - 121 ].In the mouse spot test the discrimination between spots of mutagenic and non-mutagenic origin may be problematic [ 2 ].A comparison of both test systems is presented in Table  2 .Advantages and Disadvantages of mouse spot test compared to the transgenic Big Blue ®  and Muta™ mouse assaysMouse spot test  [2-5]Transgenic mouse mutation assay [1, 122]in vivoin vivoConclusions Although the mouse spot test is a standard genotoxicity test system according to the OECD guidelines, this system has seldom been used for detection of somatic mutations  in vivo  in the last decades. This is partly due to considerations of cost effectiveness and number of animals needed for testing but also for toxicological considerations. The usefulness of the mouse spot test in toxicology is limited by restrictions in toxicokinetics, sensitivity, target cell/organ, and molecular genetics. From the limited data available, it seems that the transgenic mouse assay has several advantages over the mouse spot test and may be a suitable test system replacing the mouse spot test for detection of gene but not chromosome mutations  in vivo .
Conclusions
Although the mouse spot test is a standard genotoxicity test system according to the OECD guidelines, this system has seldom been used for detection of somatic mutations  in vivo  in the last decades. This is partly due to considerations of cost effectiveness and number of animals needed for testing but also for toxicological considerations. The usefulness of the mouse spot test in toxicology is limited by restrictions in toxicokinetics, sensitivity, target cell/organ, and molecular genetics. From the limited data available, it seems that the transgenic mouse assay has several advantages over the mouse spot test and may be a suitable test system replacing the mouse spot test for detection of gene but not chromosome mutations  in vivo .in vivoin vivoAuthor's contributions UW was the main author. The other authors were involved in the discussions, writing small parts of text and in final preparation of the manuscript.
Author's contributions
UW was the main author. The other authors were involved in the discussions, writing small parts of text and in final preparation of the manuscript.Supplementary Material Additional File 1 Results in the transgenic mouse assay versus mouse spot test Click here for file
Supplementary Material
Additional File 1
Results in the transgenic mouse assay versus mouse spot testClick here for file