COMMITTEE ON MUTAGENICITY OF CHEMICALS IN FOOD, CONSUMER PRODUCTS AND THE ENVIRONMENT (COM)

COMMITTEE ON CARCINOGENICITY OF CHEMICALS IN FOOD, CONSUMER PRODUCTS AND THE ENVIRONMENT (COC)

JOINT COM & COC STATEMENT ON MUTAGENICITY AND CARCINOGENICITY OF MALACHITE GREEN (MG) AND LEUCOMALACHITE GREEN (LMG)

COM/04/S4 & COC/04/S7 - December 2004

INTRODUCTION

1. Malachite green (MG) is a cationic triphenylmethane dyestuff used in a number of industries including fish farming. Its use in fish for human consumption was banned in the EU in June 2002 but residues continue to be found in fish.

2. In 1999 the COM provided advice to the COT on the mutagenicity of malachite green and its lipophilic metabolite leucomalachite green (LMG). The COT had asked for advice in particular on the results from 32P post-labelling studies investigating DNA adduct formation in the liver of rats and mice from 28 day repeated dose toxicity studies. These studies were part of the US National Toxicology programme, and were the range-finding studies prior to initiating carcinogenicity bioassays on MG and LMG.

MUTAGENICITY

COM Advice 1999

3. In 1999 the COM agreed the following conclusions regarding the mutagenicity of MG and LMG, which were incorporated in the 1999 COT statement on MG and LMG in farmed fish.1

Malachite Green

i. There is one report of a Salmonella assay done to an acceptable protocol; malachite green oxalate (>90% pure) was shown to induce mutations in TA98 in the presence of an exogenous metabolic activation system.2 There is also some evidence of clastogenicity in Chinese hamster lung cells.3 MG should thus be regarded as having mutagenic potential.

ii. Negative results were obtained in a poorly reported bone marrow micronucleus test in mice using a single oral dose of malachite green oxalate (>90% pure) at the MTD (75% of the LD50).2 Members agreed that the high dose level of 37.5 mg/kg was adequate, but it was difficult to assess the value of the negative results in the absence of appropriate information on bone marrow toxicity or data to show that MG and/or metabolites reached the bone marrow.

iii. Recent 32P-post-labelling studies using a 28 day dietary exposure and carried out with NTP range - finding studies (used in designing carcinogenicity bioassays) indicate that MG induces DNA adducts in the liver of rats and mice.4,5

iv. The Committee concluded that although a limited negative in-vivo micronucleus test was available, the results of the recently conducted 32P- post-labelling studies indicated that it would be prudent to assume that MG may be a potential in-vivo mutagen.

Leucomalachite green

i. The Committee were concerned that only limited information was available on LMG. The lack of information on LMG prevents an adequate mutagenicity assessment for this compound.

ii. The Committee recommended that in-vitro studies in bacteria for gene mutations, in mammalian cells for clastogenicity and a mammalian cell assay for gene mutation (preferably the mouse lymphoma assay), conducted according to current OECD guidelines, should be undertaken as an important step to evaluating the mutagenic potential of LMG.

iii. The Committee, however, concluded that the results of the recently conducted post labelling studies4,5 indicated that it would be prudent to assume that LMG may also be a potential in-vivo mutagen.

Updated advice from COM 2004

4. The results of the NTP carcinogenicity bioassays on MG and LMG were published in 2004.6 The NTP report in addition included the results of a number of new studies to investigate the mutagenicity of these 2 compounds, most of which had also been published separately. The COM reviewed these new data in 2004 with a view to updating their conclusions on the mutagenicity of these two compounds.

5. New data were now available from in-vitro studies to investigate the ability of MG and LMG to induce mutations in the Salmonella assay, the CHO/hprt assay, and an in-vitro comet assay.6,7 Negative results were obtained except for the comet assay with MG which was positive.

6. The 32P post-labelling studies considered by the COM in 1999 have now been published in full. Male F344 rats and female B6C3F1 mice were fed 9, 100 and 600 ppm MG or 0, 96 or 580 ppm LMG in the diet for 28 days.8 32P post-labelling analysis of liver DNA indicated a single adduct, or co-eluting adducts, with both compounds, with the level of adducts increasing significantly as a function of dose. In rats the level of adducts seen was similar with MG and LMG. However in the mice MG gave a clear dose-related increase in binding which was slightly lower than that seen in the rats, whereas LMG produced only very low levels of adducts, of doubtful significance. In later studies with female Big Blue rats and mice LMG again produced evidence of DNA adduct formation in the rat but not the mouse.9

7. The ability of MG or LMG to induce micronuclei in peripheral blood or in hprt mutations in the spleen was also investigated in the above studies; negative results were obtained.6,9 The Committee noted that these studies, which used repeated exposure over 4-32 weeks, did not optimise the chances of detecting a mutagenic response.

8. Studies to investigate induction of lacI mutations by LMG in the liver of female Big Blue rats gave equivocal results.10 Samples were obtained at 3 time periods (4, 16, 32 weeks) at 5 dose levels. An increase in lacI mutations was seen at only a single time point (16 weeks) and only at the top dose level. Since lacI mutations were not expected to decline with continued exposure to compounds the nature of the DNA sequence alterations was investigated in DNA from the isolated positive result. When corrected for clonality there was no significant difference between the LMG treated and the control frequency. The authors considered that this isolated positive was an artifact due to the disproportionate expansion of spontaneous lacI mutations.

9. Studies to investigate cII mutations induced by LMG in the liver of Big Blue B6C3F1 mice did, however, give a positive result.6 At a dietary level of 408 ppm LMG produced a statistically significant (p <0.05) increase in mutations. Further analysis showed that these contained a spectrum of mutations that was distinct from that in the control animals. In contrast MG did not produce any significant increase in such mutations in the mice. In similar studies in Big Blue rats, LMG did not produce any increase in cII mutation frequency in liver DNA.

10. Members considered the lack of concordance between the results of studies investigation DNA adducts and mutations in transgenic mice and the observation of an apparently specific mutation in the cII transgene in mice represented an unusual data set and hence there was uncertainty in deriving conclusions from these studies.

11. The COM agreed to revise their 1999 conclusions regarding the mutagenicity of MG and LMG as follows:

Malachite green

i. There is one report of a Salmonella assay done to an acceptable protocol; MG oxalate (>90% pure) was shown to induce mutations in TA98 in the presence of an exogenous metabolic activation system.2 There is also some evidence of clastogenicity in Chinese Hamster Lung cells.3 MG has also been shown to produce DNA damage in CHO cells using the Comet assay.6 MG should thus be regarded as having mutagenic potential.

iii. 32P post-labelling studies using a 28 day dietary exposure have indicated that MG induces DNA adduct(s) in the liver of both F344 rats and B6C3F1 mice.7 This has been confirmed in a separate study using 16 weeks dietary exposure.1 MG did not induce micronuclei in peripheral lymphocytes nor hprt mutations in splenic lymphocytes in the 28 day study; the experimental design of these studies did not, however, optimise the chances of obtaining a positive response and no definite conclusions can be drawn.

iv. In view of the demonstration of DNA adduct formation in samples from both rats and mice, MG should be regarded as an in-vivo mutagen.

Leucomalachite green

i. Data are now available on the in-vitro mutagenicity of LMG from limited Salmonella and CHO/HGPRT assays, and also from a Comet assay in CHO cells.7 Negative results were obtained. However in view of the limitations of these studies, and the fact that no data are available on clastogenicity, it is not possible to make an adequate assessment of the mutagenic potential of LMG from these data.

ii. 32P Post-labelling studies using a 28 day dietary exposure have indicated that LMG induces a low level of DNA adduct(s) in the liver of F344 rats; only a marginal response was however seen in B6C3F1 mice, and no conclusions can be drawn from these data.8 This has been confirmed in a separate unpublished study in mice using 16 weeks dietary exposure; there was no evidence for DNA adduct formation in the liver.6 LMG did not induce micronuclei in peripheral lymphocytes nor hprt mutations in splenic lymphocytes in the 28 day study,9 but the experimental design of these studies did not optimise the chances of obtaining a positive response and no definite conclusions can be drawn.

iii. Gene mutation studies have indicated that LMG can induce mutations in vivo in liver DNA. Studies in Big Blue F344 rats gave equivocal results. LMG produced an increase in lacI mutations only at the highest dose tested (543 ppm in diet) and at a single time point (16 weeks). No increase was seen after 32 weeks. Evidence was provided to suggest that this isolated positive may have been due to disproportionate expansion of spontaneous lacI mutations.9,10 However studies in female Big Blue B6C3F1 mice indicated that LMG produced an increase in cII mutant frequency in liver DNA of mice treated with 408 ppm LMG in the diet.6 Furthermore it was shown that the spectrum of mutation seen in the DNA was distinct from that of the control mice. These data provide evidence of in-vivo mutagenicity at the target site in the carcinogenicity bioassay.

iv. In view of the demonstration of the induction of mutations in liver DNA of female B6C3F1 mice, LMG should be regarded as an in-vivo mutagen.

CARCINOGENICITY

COC Advice 2004

12. The COC considered the results of the NTP carcinogenicity studies on MG and LMG in June 2004. They noted that prior to the publication of the NTP bioassay data on MG and LMG there had been no data available to make any meaningful assessment of the carcinogenicity of these compounds.

13. The carcinogenicity of MG was investigated in female F344 rats and in female B6C3F1 mice, compound being given in the diet for 104 weeks. It was noted that the rationale for testing only in females was that this had been shown in range finding studies to be the most sensitive gender. In the study in rats groups of 48 female rats were given diets containing 0, 100, 300 and 600 ppm MG (equivalent to average daily doses of approximately 0, 1, 21 and 43 mg/kg bw/day). Survival in all groups was comparable, but mean body weight gain was slightly lower at 300 and 600 ppm (approximately 10% reduction compared to controls). At autopsy it was noted that the relative liver weights were increased at the top dose. In the study in mice groups of 48 animals were fed diets containing 0, 100, 225 and 450 ppm MG (equivalent to average daily doses of approximately 0, 15, 33 and 67 mg/kg bw/day). Survival was again comparable in all groups but there was a slight reduction in body weight gain (5-10%) at the top dose. Effects on relative kidney weight were noted at autopsy.

14. The carcinogenicity of LMG was investigated in both male and female F344 rats and in female mice, the compound being given in the diet for 104 weeks. Groups of 48 male and female rats were fed diets containing 0, 91, 272 or 543 ppm LMG (equivalent to daily doses of 0, 5, 15 and 30 mg/kg bw/day in the males and 0, 6, 17 and 35 mg LMG/kg bw/day in the females). Survival was comparable in all groups (except for an increase in males given 272 ppm) but there was a reduction in body weight gain at the top dose (25% in females, 10-15% in males). At autopsy liver weight were noted to be increased in the males at the two higher doses, as were relative liver weights in the females.

15. In the study in female mice groups of 48 were given diets containing 0, 91, 204 or 408 ppm LMG (equivalent to average daily doses of 0, 13, 31 and 63 mg LMG/kg bw/day). Survival and body weight gain were comparable in all groups. At autopsy a decrease in relative kidney weight was noted.

16. Regarding the evidence for carcinogenicity in these bioassays the Committee agreed with the NTP conclusions, as modified by their peer review panel. They agreed the following conclusions regarding the carcinogenicity of MG and LMG.

Conclusions regarding carcinogenicity of malachite green

i. There was equivocal evidence of carcinogenicity in female F344 rats based on an increase in thyroid gland tumours (adenoma or carcinoma combined) hepatocellular adenomas and mammary gland carcinomas.

ii. There was no evidence of carcinogenic activity in female B6C3F1 mice.

iii. Overall there was no convincing evidence for any carcinogenic effect with malachite green in these studies.

Carcinogenicity of leucomalachite green

i. There was evidence of carcinogenic activity in female B6C3F1 mice based on an increase in hepatocellular adenoma or carcinoma combined.

ii. There was equivocal evidence of carcinogenic activity in male F344 rats based on an increase in interstitial cell adenoma of the testes and the occurrence of thyroid gland follicular cell adenoma or carcinoma (combined).

iii. There was equivocal evidence of carcinogenic activity in female F344 rats based on an increased incidence of hepatocellular adenoma or carcinoma (combined).

OVERALL CONCLUSIONS REGARDING MUTAGENICITY AND CARCINOGENICITY OF MG AND LMG

17. The COM concluded that both MG and LMG should be regarded as in vivo mutagens.

18. The COC concluded that the only convincing evidence for any carcinogenic effect of MG or LMG in the NTP bioassays was for LMG in female mice, based on an increase in hepatocellular adenoma or carcinoma combined.

19. The COC considered the possible mechanisms by which LMG induced tumours in the liver of the female mice. It was noted that the overall tumour profile was not that which would be expected of a genotoxic carcinogen, with activity being limited to effects in the liver of the female mouse; furthermore this was mainly due to an increase in adenomas. However it was also noted that there was no evidence from the NTP studies to support any non-genotoxic mechanism. In view of this, and taking into account the views of the COM, the Committee agreed that it was not possible to discount a genotoxic mechanism for the induction of the liver tumours in female mice and it would therefore be prudent to regard LMG as a genotoxic carcinogen.

December 2004

REFERENCES

1. COT Malachite Green and Leucomalachite Green in farmed fish. Annual Report of COT/COC/COM PP23-27(1999).

2. Clemmenson S, Jensen J C, Jensen N J et al. Toxicological studies on malachite green. Arch. Toxicol. 56, 43 - 5 (1984).

3. Ishidite M. Application of chromosome aberration tests in-vitro. Tests Courts Cancer. Quo Vades Symp. 57 - 79 (1981).

4. NTP (USA). Notification of approval protocol. 2 year bioassay in rats administered MG and LMG in the diet. Project. 212801. July 1998.

5. Culp S J, Blankenship L R, Kusewitt D F et al. Metabolic changes occurring in mice and rats fed LMG. Proc. 88th Ann. Meeting of Am. Soc. For Cancer Research, San Diego. P. 121 (1997).

6. NTP Technical Report TR-527 Toxicology and Carcinogenesis Studies of Malachite Green and Leucomalachite Green in F344 rats and B6C3F1 mice (feed studies) NTP US Dept Health and Human Services(2004).

7. Fessard V, Godard T, Huet S et al. Mutagenicity of MG and LMG in in-vitro tests. J. Appl. Toxicol 19, 421 - 30 (1999).

8. Culp S J, Blankenship L R, Kusewitt D F et al. Toxicity and metabolism of MG and LMG during short term feeding to Fischer 344 rats and B6C3F1 mice. Chem. Biol. Interactions. 122, 153 - 70 (1999).

9. Culp S J, Beland F A, Heflich R H et al. Mutagenicity and carcinogenicity in relation to DNA adduct formation in rats fed LMG. Mut Res 506/7, 55 - 63 (2002).

10. Majanatha M G, Shelton S D, Bishop M et al. Analysis of mutations and bone marrow micronuclei in Big Blue rats fed LMG. Mut. Res. 547, 5 - 18 (2004).