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Background 1. Malathion is an organophosphorous insecticide. It has been marketed in the UK for use in agriculture and horticulture since 1956. There were three products with approvals for use in agriculture and horticulture, home garden and use in pigeon lofts at the time when this review was initiated in January 2002. A number of products containing malathion are also licensed as human medicines for use in the control of head lice. 2. The Advisory Committee on Pesticides is reviewing the available toxicological information on malathion as part of its ongoing review of organophosphorous compounds. The ACP asked for advice from COM and COC on mutagenicity and carcinogenicity at its 289th meeting on 17 January 2002. There are inconsistent results in mutagenicity studies (both in-vitro and in-vivo) and there is evidence for the mutagenic activity of some impurities which may be present in some batches of technical malathion. There is also some limited evidence for tumourigenicity in rats (in particular the occurrence of benign nasal tumours in animals given high oral doses of technical grade malathion in the diet). 3. The Chairs of COM and COC agreed that a joint statement was required in view of the need for a full review of all mutagenicity and carcinogenicity data. 4. The COM undertook an initial consideration of the in confidence mutagenicity data provided by the pesticide data holder1-5 and available published information provided by the data holder6-55 at its 25 April 2002 meeting. A number of additional published papers on malathion 56-61and impurities present in technical grade malathion were also considered at this meeting.62-65 The COC reviewed the available carcinogenicity data on malathion which included in confidence reports (provided by the pesticide data holder regarding two studies in rats one in mice)66-68 and published reports of long-term bioassays in rats69,70 and mice69 at its 27 June 2002 meeting. Three long-term bioassays using malaoxon (the principle metabolite of malathion and also present in technical grade malathion as an impurity) were available which included two in rats71,72 and one in mice71 In addition the Committee also considered in confidence reports of Peer Reviews of the histology slides from the 1993-96 malathion bioassay in F344 rats73,74, the 1992-94 bioassay in B6C3F1 mice75 and some additional supplemental information for the 1992-1994 bioassay in B6C3F1 mice76 and the 1993-1996 bioassay of malaoxon in F344 rats77 provided by the Pesticide Data holder. A number of additional follow up reports from the contract laboratory concerning the 1993-96 bioassay of malathion in F344 rats were also reviewed.78-81 In addition the pesticide data holder submitted a response to questions from COC secretariat which provided an overall summary of the histology of the nasal tissue in animals with tumours and additional evaluation of the historical control data on nasal tumours in F344 rats and possible mechanisms for nasal tumours induced in F344 rats fed high doses of technical grade malathion.82 A published Peer Review of a number of published carcinogenicity bioassays was also available.83 The COC also considered expert reports from the EPA84 and a Scientific Advisory Panel85 established by EPA to review malathion. At its meeting of 10 October 2002 the COM considered some additional information provided in confidence by the pesticide data holder (a report of one additional in-vivo study86 and information on the potential for variation in impurities between different sources of malathion87) together with number of published studies not previously reviewed.88-92 The in confidence reports provided by the pesticides data holder contained appropriate Good Laboratory Practice and Quality Assurance statements. Introduction to review 5. The Committees had access to sections of the draft risk assessment prepared by the Pesticides Safety Directorate (PSD) for the ACP. This include evaluations of the absorption, distribution, metabolism and excretion and toxicology data on malathion. Information on the manufacturing process used by the pesticide data holder and the impurities present in commercial technical material was presented. 6. The Committees noted that malathion is an organophosphorothiolate compound and required activation to malaoxon which inhibits cholinesterases in mammals. It is rapidly absorbed orally and is rapidly metabolised and excreted (mainly in the urine) as metabolites. Dermal absorption studies in human volunteers suggested a moderate absorption (6-15%) depending on formulation and malathion concentration. The available information from a wide range of toxicity studies (including acute, sub acute, sub chronic and chronic studies and reproduction studies in a range of species) demonstrated that inhibition of cholinesterase is the most sensitive toxicological effect in all available studies. The toxicity of malathion can be influenced by the presence of impurities in the technical material. Thus, for example, data presented in the PSD draft risk assessment showed that isomalathion and OOS-trimethyl phosphorothioate can inhibit the detoxification (via carboxyesterases and glutathione) of malaoxon resulting in a potentiation of cholinesterase effects of this compound. It was notable that the acute toxicity of technical grade malathion differed between two manufacturing sources. In answer to questions raised by the COM, the pesticide data holder reported that the levels of impurities would vary according to quality of raw materials, reaction parameters and purification steps. In addition it was also reported that the level of one impurity (isomalathion) could increase during storage of technical grade malathion. Thus the possibility that impurities may have mutagenic potential needed to be considered. The Committee agreed that the specification of the test materials used in mutagenicity studies undertaken by the pesticide data holder had been adequately demonstrated. The evidence provided by the Pesticide Data Holder suggested that the impurities present in technical grade malathion from other manufacturers would be qualitatively similar but it was not possible to draw any conclusions on the levels of the individual impurities in the test materials used in published mutagenicity studies. COM review of mutagenicity In-vitro data on malathion 7. It was agreed that the primary objective of the COM review was to consider the mutagenic potential of commercially supplied (e.g. technical grade) malathion. Technical grade malathion used in many of the reported mutagenicity tests had a purity of between 93-96%, whilst in a small number of published studiesthe authors reported using highly purified malathion (³ 99%).8,38,39,51 The COM agreed that malathion was a DNA methylating agent.56 N7-Methyl guanine was the main identified adduct but others could not be excluded. There were also data to show that iso-malathion (an impurity formed during manufacture and storage) and OOO-trimethylphosphorothioate (a process impurity found in some batches of technical grade malathion) could alkylate nitro-benzyl-pyridine in-vitro.62 The Committees noted the proposed metabolism pathway in the draft risk assessment document prepared by the Pesticides Safety Directorate (PSD) and agreed that there were a number of malathion metabolites identified in metabolism studies, which could theoretically methylate DNA. 8. The Committee agreed that there was no evidence for mutagenicity of technical grade malathion in bacteria. The results in a number of tests in Salmonella typhimurium and Escherichia coli were negative.1,14,21,24,30,31 The significance of enhanced SOS DNA repair activity in Escherichia coli was unclear.50 The COM noted that only brief details of the studies in yeast were available,21,31 but considered that further evaluation of the data would be of limited value in view of the clear evidence for in vitro mutagenicity in mammalian cells (see next para). 9. Regarding in-vitro assays in mammalian cells, the COM agreed that a cytogenetics assay with technical grade malathion conducted by the pesticide approval holder, was positive in the presence and absence of endogenous metabolic activation at the high dose (which was moderately cytotoxic).4 The Committee reviewed the available published literature and agreed that details of purity of malathion used was absent in several studies. However, the COM agreed that positive results had been obtained in published studies in human lymphocytes, where malathion was of technical grade or of higher purity (³ 99%).23,27,51 Activity was reported both in the presence and absence of exogenous metabolic activation. Additionally, technical grade23,27 and purified malathion (³ 99% pure)38,39 had been shown to induce Sister Chromatid Exchanges in mammalian cells (CHO cells) and human lymphocytes and fibroblasts. The COM also noted that evidence for a gene mutation in the hprt locus in human lymphocytes had been published.41 The authors of this study noted that positive results had also been reported for different batches of technical grade malathion, which had contained slightly different levels of impurities. In a subsequent publication, these authors had provided limited information to suggest that malathion might induce specific mutations in the hprt gene in human lymphocytes. There was evidence for an increase in mutations at G:C base pairs and at GG dinucleotides in hprt gene in malathion treated cells compared to unexposed cells.89 The Committee agreed that the mouse lymphoma assay, recently conducted by the pesticide data holder using technical grade material, had also given positive results in the presence and absence of exogenous metabolic activation after a short 4-hour exposure period.3 However, a second trial using a 24-hour exposure period was negative (COM Members commented that the control mutation frequency in this study was relatively high and this might mask a weak positive response). 10. The COM agreed that the UDS assay in isolated hepatocytes with technical grade material, which had been submitted by the pesticide data holder, was negative.2 The Committee noted that although the top dose level (0.16µl/ml) had produced evidence of cytotoxicity, the dose might have been too low to detect any DNA damage induced by impurities rather than by malathion itself. The Committee noted that a recent COMET assay with malathion (98% pure) and using human peripheral blood lymphocytes had yielded negative results.12 However, no exogenous metabolic activation had been used and the exposure period (1hour) used in this study was shorter than expected for an adequate regulatory study (ca 3 or 24-h). 11. Overall the COM agreed that the available evidence was consistent with the conclusion that technical grade malathion (including its metabolites and impurities) induced the mutagenicity observed in in-vitro studies. In-vitro data on malaoxon 12. The Committee reviewed the available in-vitro mutagenicity data on malaoxon, the principle metabolite of malathion. Malaoxon had mutagenic activity in two mouse lymphoma assays in the absence of exogenous metabolic activation, and induced SCEs in CHO cells.64,65 The test material used in these assays was approximately 94-96% pure. Malaoxon (98% pure) had also induced DNA damage in human peripheral blood lymphocytes in one assay.63 13. The committee considered that the mutagenic activity seen in-vitro with technical grade malathion could, in part, be due to the metabolism of malathion to malaoxon. In-vivo data on malathion Studies submitted by the pesticide data holder 14. The Committee agreed that the oral rat bone marrow clastogenicity study, conducted by the pesticide data holder using high doses of technical grade material, had been adequately conducted and was negative.5 15. The Committee had reservations regarding the adequacy of the oral in-vivo liver UDS assay in rats submitted by the pesticide data holder86. Members were concerned with regard to the apparent positive response in animals 24 and 25 in the assay. The Committee did not accept the rationale provided by the contract laboratory that these positive responses were caused by technical errors (staining errors, the timing of dosing relative to other rats and/or use of control rats sampled 2-4 hours after dosing as controls for treated rats sampled 12-16 hours after dosing). Members also considered it feasible that a positive control substance had been dosed to these animals by mistake or there had been a mislabelling of the animals. Whilst such explanations may be plausible, there was a lack of direct evidence linking the positive effects in the two rats to any experimental error. However, it was noted that no evidence of mutagenicity was found in a repeated 12-16 hour sampling time experiment. Overall the COM agreed that no weight could be attributed to the study and that it should be repeated. Negative results in such a repeat study would remove any concerns that technical grade malathion was mutagenic to rats when dosed orally. Published Studies 16. The Committee was aware that there were a large number of published in-vivo studies which reported positive results for malathion. No definite conclusions could be drawn from the majority of these studies because the test material used was not of technical grade or the purity could not be determined and/or the methods used were inadequate by current standards.6,8,18-20,28,33,42,57,90 The Committee was aware that there were some studies which reported negative findings in-vivo with technical grade malathion17,46,52,53 but agreed that it was important to consider in detail the available studies where positive finding had been reported and where there was sufficient information reported to comment on the results.18,57,90 Oral administration 17. Giri et al (2002)57 documented limited evidence for an increase in chromosomal aberrations in bone-marrow of groups of 3 Swiss mice (sex not given) given either a single oral dose of 5 mg/kg bw or five daily doses of 2 mg/kg bw. A fixation time of 24 hours after the last dose was used. The test material used was a technical grade material manufactured in India.93 The Committee agreed that in view of the lack of lack of evidence for carcinogenicity in the long-term feeding carcinogenicity bioassay in mice, that there was no need for a further oral study in mice at this juncture. Dermal administration 18. The Committee considered the study by Dulout et al (1982)90 which had used both the dermal and intraperitoneal routes of dosing in bone-marrow micronucleus assays in mice. Members agreed it was difficult to explain why such a strong positive response had been documented following dermal application compared to intraperitoneal dosing. The test material (a technical grade material manufactured in Argentina) had been dissolved in corn oil which would have led to residual vehicle on the skin. Members noted that dermal absorption in human volunteers ranged from 5% to 15% depending on formulation and dilution with water. It was not possible to quantify the extent of dermal absorption in this study. There would have also been some oral ingestion arising by grooming of the animals, but his could not be quantified. Members noted that there was no evidence for a dose response following dermal administration in this study, but felt that the results could not be refuted and thus there was a need for a repeat study in mice. Intraperitoneal administration 19. The Committee agreed there was evidence from several studies for a mutagenic effect of technical grade malathion in the bone-marrow of mice given the test material by intraperitoneal administration.18,57,90 The Committee agreed that these data suggested technical grade malathion had mutagenic activity in-vivo in mice and reinforced the need to evaluate dermal exposure to technical grade malathion adequately. 20. Overall the Committee concluded that malathion was mutagenic in-vivo in mice dosed by the intraperitoneal route, and possibly also by the oral and dermal routes. Conversely, there was no evidence of in- vivo mutagenicity in rats dosed orally. These data may indicate a species-specific in-vivo mutagenicity in mice. Studies in humans exposed to malathion and other pesticides 21. The Committee discussed the available studies in humans exposed to malathion.14,29,35,44,47,49,54 It was noted that an increase in the incidence of micronuclei had been reported by the Californian Health Department, in a preliminary study of 13 applicators who used malathion in 1992.14,54 This result was not confirmed by a further study undertaken by the same group of researchers in 1993.14,54 Overall the Committee felt that no conclusions could be drawn from this study. The Committee considered that no conclusions could be derived from the other available published studies relating to monitoring of human exposure for mutagenic effects. The Committee concluded that appropriately designed studies of pesticide applicators using malathion, might be informative with regard to mutagenic activity in humans. Response of data holder to COM Evaluation (19/11/02 and 2/01/03) 22. The data holder provided some additional data on the 19 November 2002 on the in-vivo liver UDS assay in rats86, namely additional details of the conduct of the study and results in animals 24 and 25 and photomicrographs of slides from animal 24 and from a negative and positive control animal. It was noted that additional repeat investigations had been included in the study which had been triggered when technical difficulties concerning animals 24 and 25 had been noted. The data holder provided an additional in-vivo intraperitoneal micronucleus assay in the mouse.94 The data holder provided comments on the dermal in-vivo micronucleus assay in mice published by Dulout et al90. Additional photomicrographs and comments on the conduct of the rat liver UDS assay were provided by the pesticide data holder on 2 January 2003. COM comments 23. Regarding the new data on the rat liver UDS assay, COM members considered the additional data submitted on the 19/11/02 and on 2/01/03 and agreed that the assay was flawed. The arguments regarding the background staining of the slides did not match with the reported positive NNG values for animals 24 and 25. Overall members could not endorse this assay. The lack of evidence for a carcinogenic effect in livers of rats dosed with malathion is noted. However considering the clear mutagenicity of malathion in-vitro and in some other in-vivo assays, an adequate, technically defensible assay is considered appropriate. 24. Members noted the new intraperitoneal bone marrow micronucleus test which gave negative results and provided some reassurance, although the specification of the test material was uncertain and only 1000 PCEs had been scored.94 25. COM members acknowledged there are considerable problems with the conduct of study reported by Dulout et al 1982.86 However members felt that the pesticide data holder had not provided a rationale to suggest it was a false positive response and agreed that a repeat dermal assay was appropriate. 26. The COM considered other comments from the Data holder regarding the interpretation of human studies and the assessment of in-vitro studies with malathion. The Committee agreed that no alterations with regard to these areas of the statement were required. COM conclusions 27. The Committee agreed the following conclusions:
COC review of carcinogenicity 28. The Committee reviewed the conduct, adequacy and results from the available carcinogenicity bioassays in rats and mice where malathion or malaoxon had been administered in the diet.66-72 In reviewing these studies, the COC agreed that particular reference should be made to the evaluation of nasal tissue. The Committee commented that nasal tissue was generally not examined in carcinogenicity bioassays until around 1990. The Committee made the following comments on each of the long-term bioassays. Malathion bioassays in rats 29. NCI 1978, Osborne Mendel rat.69 The study authors had reported that there was an increase in thyroid follicular cell carcinomas/adenomas in females in the high dose group. A peer review of pathology had concluded that there was no significant increase in thyroid tumours.83 The COC noted there was no examination of nasal tissue, non-neoplastic pathology was reported in females but not males, there were no full necropsies of decedents and the control groups were too small. Overall it was agreed that this study was inadequate by contemporary standards and no conclusions could be drawn. 30. FDRL 1980, Sprague Dawley Rat.66 The study authors had not reported any evidence for a treatment related carcinogenic effect. The committee noted that there was no examination of nasal tissue. With this limitation noted, the committee considered the study had in general been adequately undertaken. 31. NCI 1979, Rat F344.71 There was an increase incidence in phaeochromocytomas in low dose but this was not found in high dose males, and the absence of adrenal medullary hyperplasia further questioned the significance of this finding. There was limited evidence for increased leukaemia in low dose males. It was agreed that this study provided no consistent evidence for a carcinogenic effect. The COC noted that examination of nasal tissue had not been undertaken in this study, but agreed that overall the study had in general been conducted adequately. 32. HLS 1993-1996, Rat F344.72 The COC agreed that this bioassay was conducted to modern standards and was the critical carcinogenicity study in the rat. Dietary levels used were 100 ppm (reduced to 50 ppm at month 3), 500 ppm, 6000 ppm and 12000 ppm. (Averaged dose levels (mg/kg bw/day) in males and females were respectively; 4/5, 29/35, 359/415, 739/868 mg/kg bw/day). The COC considered that the top two dose levels in males exceeded the Maximum Tolerated Dose (MTD) level. This was based on reduced mean body weight (-16.8% at month 18 in the 12000 ppm group and -11.1% at 6000 ppm) and statistically increased mortality compared to control animals. In females the MTD was exceeded at 12000 ppm (based on reduced mean body weight at 24 months; -16.8% and significant mortality compared to controls) but was not exceeded at 6000 ppm (mean reduction in body weight <5.1% compared to controls, 62% survival to termination). The study investigators had documented two nasal tumours in males (an olfactory epithelial adenoma in one animal at 6000 ppm and an olfactory epithelial carcinoma in one animal at 12000 ppm). The COC noted that an independent peer review of the nasal pathology had been undertaken.73 The COC concluded that the report of the peer review was satisfactory and agreed that the classsification of tumours used in this review should be used in the evaluation of this cancer bioassay. Following the peer review of the pathology, a consensus agreement had been reached that there were three nasal respiratory adenomas (one male and one female) at 12000 ppm and one female at 6000 ppm) and an olfactory epithelial adenoma in one male at 6000 ppm. These tumours were associated with severe non-neoplastic pathology in the nasal tissue at 6000 and 12000 ppm (including epithelial degeneration, hyperplasia and inflammation). The peer review also documented four tumours of the oral palate. The data holder had provided a concise summary of the neoplastic pathology in animals with nasal tumours.82 The COC observed that based on the relatively small laboratory historical control data, the incidence of nasal tumours was highly statistically significant. However it was evident from the EPA review and information supplied by the data holder that the background incidence of nasal tumours was unclear and depended upon the number of nasal sections used in bioassays.82,84 In modern bioassays this would comprise 5 sections whilst only two sections were used in the bioassays in the laboratory historical control database. The historical control range from 20 bioassays undertaken as part of the NTP program was 0-2% in both males and females. The Committee considered that overall there was evidence of a tumourigenic response to malathion in the nasal tissue of the rat but the observed tumours were induced in tissue which was subject to severe ongoing nasal inflammation. The COC agreed there was an increase in liver tumours at the two highest dose levels and that this was likely to be due to an effect of treatment on the background pathology of ageing animals. Malathion bioassays in mice 33. NCI 1978, Mouse B6C3F1.69 The COC observed that there was no examination of nasal tissue, there were no full necropsies of decedents and the control groups were too small. A statistically significant increase in combined hepatocellular carcinoma and neoplastic nodule were reported in males animals by trend analysis. No increase was reported when analysed by pair wise comparison or by life-time analysis. The Committee commented that the terminology used to describe histological lesions of the liver was now considered outdated and that there was a high incidence of hepatocellular carcinoma in historical control groups. The committee agreed that by contemporary standards this bioassay was inadequate. The Committee observed the evidence of coughing and wheezing in most high dose animals from week 72 which provided some evidence for an effect on the respiratory tract. 34. HLS 1992-1994, Mouse B6C3F1.68 The COC agreed that this bioassay had been conducted to modern standards. Dietary levels of 100 ppm, 800 ppm, 8,000 ppm, and 16000 ppm were used. (Averaged dose levels (mg/kg bw/day) in males and females were respectively;17.4/20.8, 143/167, 1476/1707, 2978/3448 mg/kg bw/day) The COC agreed that the Maximum Tolerated Dose level was exceeded at 16 000 ppm in both males (-20 % reduction in mean body weight compared to controls at week 78) and in females (-16% reduction in mean body weight compared to controls at week 78). The MTD was exceeded in males at 8000 ppm (-14% reduction in mean body weight compared to controls at week 78). The COC considered that the 8000 ppm dose level in females equated to the MTD (reduction in mean body weight for females was -9.7%). The study investigators reported the finding of hepatocellular adenoma, hepatocellular hypertrophy and increase liver weight in most mice (male and female) at 8000 ppm and 16000 ppm. A Peer review of histology in male mice essentially confirmed the liver findings reported by the study investigators.75 The Peer review group also identified significant and excessive non neoplastic pathology of the nasal tissue similar to that reported in F344 rats. The Committee agreed that the liver tumours were treatment related. Malaoxon bioassays in rats 35. NCI 1979, Rat F344.71 A statistically significant increase in thyroid C-cell adenomas and carcinomas were reported in females at the top dose. A Peer review had been undertaken.83 The Committee agreed with the conclusion reached by the peer review group that the evidence for a carcinogenic effect in the thyroid was equivocal. The Committee noted that examination of nasal tissue had not been undertaken in this study. The Committee noted the high dietary level was 1000 ppm, but agreed that, based on the preliminary 13 week study (no effects on body weight or histology at 2000 ppm), a higher dose level might have been used. 36. HLS 1993-1996, Rat F344.72 The COC agreed that this bioassay had been conducted to modern standards. Dietary levels of 20 ppm, 1000 ppm, and 2,000 ppm were used. (Averaged dose levels (mg/kg bw/day) in males and females were respectively; 1/1, 57/68, 114/141 mg/kg bw/day) The Committee considered that the Maximum Tolerated Dose level had not been exceeded in this study (-1.4% to -7.1% reduction in body weight compared to controls in males and -4.0% to -8.8% in females at termination). However a dose related increase in mortality was reported in males. A significant dose-related trend in mononuclear cell leukaemia was reported in males but no statistically significant increase was evident in pair wise comparisons with controls. The incidence of this tumour was within the historical control for the laboratory. A significant increase in testicular interstitial cell tumours was reported at 2000 ppm when the data were corrected for time to tumour identification. However the observed increase was within the historical control incidence. There was thus no evidence for any treatment related increase in neoplasia. The study investigators found significant non-neoplastic pathology of the nasal tissue and respiratory tract. This included significant purulent inflammation of the lungs. There was evidence of food particle deposition in many animals in this study. Malaoxon bioassay in mice 37. NCI 1979, Mouse B6C3F1.71 The COC noted that examination of nasal tissue had not been undertaken in this study. The Committee noted the high dietary level was 1000 ppm, but agreed that, based on the preliminary 13 week study (no effects on body weight at 2000 ppm), a higher dose level might have been used. No treatment related neoplasia were reported. Overall malathion carcinogenicty evaluation 38. The COC agreed that the assessment of the significance of the observed nasal tumours in F344 rats in the modern study undertaken by HLS USA was the critical neoplasm for discussion. The Committee agreed that the finding of liver tumours in F344 rats and B6C3F1 mice in the modern studies should also be evaluated in detail. The findings reported in earlier studies undertaken in the 1970s' and 80s' contributed little weight of evidence to the assessment of malathion. Nasal tumours in F344 rats 39. The COC noted that tumours of the nasal tissue in F344 rats were rare and that there were difficulties in assessment particularly with regard to the adequacy of historical control data from the laboratory. The COC noted the additional sectioning and microscopic evaluation of additional slides from the HLS USA study suggested that the US NTP historical control data for F344 rats might be more appropriate. Overall it was agreed that a tumourigenic response had been documented in this study. The COC observed that the observed tumours were all benign and included an olfactory epithelial adenoma arising from the Bowmans' gland in a male fed 6000 ppm and a respiratory epithelial adenoma in a male fed 12000 ppm. In females one animal at 6000 ppm and one at 12000 ppm had respiratory epithelial adenomas. All tumours were well defined and there was no evidence of pleomorphism or atypia.82 The Committee considered that the significance of these tumours had to be assessed against a background of severe inflammation, which exceeded that found in inhalation carcinogenicity studies with chemicals that were directly irritant to the nasal tissue. It was unclear from the HLS malathion study in F344 rats to what extent inhalation of food particles contributed to the induction of this inflammatory response in this study. The Committee agreed that direct irritant effects on the nasal passages was possible when animals were fed powdered diets. It was noted that the food pots used in the malathion study had some degree of covering which might have limited inhalation of food particles. It was noted that there was clear evidence to show the effect of food particles in the induction of localised inflammation of the nasal passages and the lungs in the malaoxon bioassay in F344 rats undertaken at the same laboratory as the critical malathion F344 rat bioassay. The Committee considered that the proposal from the data holder that de-esterification of malathion in nasal tissue to form acids was also potentially plausible. It was noted that nasal tissue would have appropriate metabolic capacity to metabolise malathion to its corresponding diacid metabolites. The Committee discussed the possibility of a genotoxic mechanism in the induction of nasal tumours. However the weight of evidence including the formation of tumours only at excessive doses, the evidence for severe prolonged localised inflammation in the target tissue, and the lack of multi-organ response suggested that a non-genotoxic mechanism was probable in this instance. Liver tumours in F344 rats and B6C3F1 mice 40. The Peer review pathology report of the Huntingdon Life Sciences bioassay in F344 rats conducted between 1993 and 1996 reported a consensus incidence of 5/70 (7.1%) hepatocellular adenomas in females at a dietary level of 12000 ppm (ca 868 mg/kg bw/day).74 The historical control incidence for the laboratory was 0-5.4% ( n= 254). The Peer review report noted that there were a number of non-neoplastic microscopic findings in the five animals with tumours, which included hypertrophy, congestion and vacuoloation. The COC agreed that the time of death and liver pathology seen in these animals would appear to be generally consistent with ageing pathology in this strain of rat. There was a significant increase in mortality in the female high dose group from month 18 to 24 (above that seen in controls) resulting in limited numbers of animals available at terminal sacrifice and thus the possibility existed that there might be some animals which could have developed liver tumours being missed. However there was no evidence of a dose response in females and only one hepatocellular adenoma was found in treated males (in a high dose male). All the available pathology evidence suggested that these tumours are most likely to be an effect of malathion on the background pathology of ageing F344 rats and are unlikely to be of significance for human health. 41. The COC commented that the increased incidence of hepatocellular adenomas in male and female B6C3F1 mice was associated with clearly overt increases in liver weight and hepatocellular hypertrophy and are unlikely to be significant to humans. It was suggested that, given the evidence for hepatocellular hypertrophy induced at doses exceeding the MTD, that it was highly likely that these tumours were induced through a non-genotoxic mechanism of technical grade malathion. Overall evaluation of malaoxon 42. The Committee concluded that there was no evidence for a carcinogenic effect of malaoxon, the main metabolite of malathion. COC conclusion 43. The COC drew the following overall conclusion:
Overall conclusion: Mutagenicity and Carcinogenicity of Malathion 44. The Committees agreed the following overall conclusion:
March 2003 REFERENCES 1. Traul KA (1988). Evaluation of CL 6601 in the bacterial/micorsome mutagenicity test . American Cyanamid Company. Inconfidence report dated 20/12/88. Laboratory report 114. MRID 40939302. 2. Pant K J (1990). Test for chemical induction of unscheduled DNA synthesis in rat primary hepatocyte cultures by autoradiography with AC 6,601. SITEK Research Laboratories, Rockville, MD20852. Inconfidence report dated 14/2/90. Study No 0125-5100. 3. Edwards CN (2001). Malathion Technical, in-vitro mammalian cell gene mutation test performed with mouse lymphoma cells (LY5178Y). Inconfidence report dated 12/10/2001. Lab report No 40413. 4. Edwards CN (2001). Malathion technical, in-vitro mammalian chromosome aberration test performed with human lymphocytes. Inconfidence report dated 3/9/2001. Lab report No 40412. 5. Gudi R (1990). Acute test for chemical induction of chromosome aberration in rat bone marrow cell in-vivo with AC 6,601. 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