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Background

1. Proquinazid (6-iodo-2-propoxy-3-propyl-3H-quinazolin-4-one) is a new fungicide intended for use in agriculture and viticulture providing control of powdery mildew in cereals and grapes. It is a novel class of fungicide acting by inhibiting the development of the appressorial germ tube. An application for first inclusion of proquinazid in Annex 1 of 91/414/EEC and for UK provisional approval (PPPR), in the product 'Proquinazid 200g/L EC', formulated as an emulsifiable concentrate containing 200g/L proquinazid was discussed at the 311th meeting of the Advisory Committee on Pesticides on 13 January 2005. The ACP deferred making a decision pending advice from the COC and COM with regard to the occurrence of cholangiocarcinoma in the rat carcinogenicity bioassay. The COC and COM have not been asked to review any other tumour reported in rodent carcinogenicity bioassays with proquinazid.

2. Proquinazid has slight solubility in water but is soluble in a range of organic solvents (Log K_ow is 5.5 at 25⁰C). It is slightly volatile. A number of batches were produced and used in the toxicology tests between 1997 and 2001 ranging from 96.4% w/w up to 97.9% w/w. Proquinazid is extensively absorbed from the gastrointestinal tract of rats and widely distributed in both single and repeated dose oral studies. It is noted that levels of radiolabel in bone marrow were reported to be equivalent to plasma and blood. Absorbed proquinazid was extensively metabolised and excreted via the bile and urine. Metabolism comprised predominantly of phenyl ring hydroxylation and hydroxylation at the propyl and propoxy side chains, as well as hydrolysis of some side chains. Major excretory metabolites included conjugates of these metabolites. One minor de-iodinated metabolite was identified.

Introduction to COM/COC review

3. The COM considered proquinazid at its meeting of the 26 May 2005. The COM had access to the individual study reports,^1-7 the evaluation prepared by PSD for the ACP⁸ and some additional information from the data holder provided in a power point presentation document. The data holder made a presentation to COM.^9-10

4. The COC considered proquinazid at its meeting of the 14 July 2005. The COC had access to the summary evaluation prepared by PSD for the ACP,⁸ the draft COM conclusions on proquinazid, detailed information on survival and pathology from the rat and mouse carcinogenicity bioassays^11,12 information from a one year study in dogs,¹³ the report of the Scientific Advisory Panel review of liver pathology from the rat and mouse carcinogenicity bioassays commissioned by the data holder,¹⁴ a mode of action assessment document for intestinal-type cholangiocarcinomas in the two year feeding study with proquinazid submitted by the data holder,¹⁵ and information from an internal memorandum on the hepatic pathology in the 2 year feeding study with proquinazid in rats.¹⁶ The data holder made a presentation to COC, which included photomicrographs of a number of histological sections of liver tissue from control and proquinazid treated rats.¹⁷ (An information paper on the pathogenesis of cholangiocarcinoma in humans and rats was drafted by the secretariat, http://www.advisorybodies.doh.gov.uk/pdfs/cc0510.pdf)

COM review of Mutagenicity (26 May 2005)

Presentation by data holder

5. The COM heard a presentation from DuPont on the mutagenicity of proquinazid. The purpose of the presentation was to provide DuPont's rationale on the adequacy of the genetic toxicology data base for proquinazid and to specifically discuss the results of the mammalian cell in-vitro chromosome aberration assay in human lymphocytes and the in-vitro mammalian cell gene hprt mutation assay in CHO cells. DuPont sought the committee's agreement that proquinazid is not genotoxic and further testing is unnecessary.

6. DuPont was aware of minor limitations in the in-vitro chromosomal assay and in-vitro gene mutation assay in mammalian cells. A battery of seven genotoxicity studies had been conducted with technical grade material during the period 1996-1998 (in-life dates of studies). The studies complied with the EU directive 91/414/EEC, the COM guidance of 1989, and OECD test guidelines (current at the time of testing). Two batches of technical material were evaluated (which had been produced by different manufacturing methods, the earlier batch DPX-KQ926-45 was 97% pure and the latter batch DPX-KQ926-75 was 97.9% pure). Tests undertaken with the 45 batch included the Ames test, the in-vitro chromosomal aberration assay, the in-vitro gene mutation assay in CHO cells (hprt) , the in-vitro UDS assay using primary rat hepatocytes, and an in-vivo bone marrow micronucleus (MN) assay. Tests undertaken with the 75 batch included an Ames test and an in-vivo bone marrow MN assay.

7. The presentation then focused on the two in-vitro mutagenicity studies where limitations had been identified. The design of and results obtained in the in-vitro chromosomal mutation assay in human lymphocytes were reviewed. The negative results and adequate performance of the positive control trials were noted. Some additional historical positive control data were presented (additional slide not in information tabled for members). COM members asked for further information on these additional positive control data The information was intended to show the lower limit of positive control responses in the test facility both before and after the in-life phase of the in-vitro chromosomal aberration assay. The data were consistent with those already provided to members and were generated from a number of tests with cyclophosphamide and MMC. DuPont noted that the study complied with an early proposed revision of the OECD guideline (ca 1996) and included a delayed harvest and that marked cytotoxicity was seen at the 24 h and 48 h harvests. In addition the consistent negative findings in this assay were supported by negative findings in two in-vivo bone marrow MN assays in mice in which oral dose levels up to 2g/kg bw had been tested with the occurrence of clinical signs of toxicity and/or mortality in mice and evidence of bone marrow exposure in the rat metabolism study.

8. The design of and results obtained in the in-vitro mammalian cell gene mutation assay (CHO/hprt) were reviewed. Two independent assays had been conducted for each treatment and a third trial had been performed with activation due to a lack of toxicity in the second trial. The study was a qualitative assessment of mutagenic potential. A positive response was obtained if the mutation frequency was > 40 x 10⁶ cells at two or more consecutive concentrations. Plating of 10⁶ cells was sufficient to detect a positive response. There was no evidence for a positive response in the assay. Mutation frequencies obtained with the test material were within the negative historical control range and there was a lack of reproducible dose-dependent increases. Positive controls produced marked increases in mutation frequency in all trials and it was noted that there was comparatively little variance in the positive control data between trials using the same treatment condition. DuPont had commented that all negative control mutation frequencies were within the range of historical controls for the test laboratory, the positive controls exhibited consistent responses despite the fluctuation in negative control frequencies and the negative findings were supported by a lack of genotoxicity in the battery of other in-vitro and in-vivo genotoxicity studies.

9. DuPont concluded that proquinazid does not pose a mutagenic concern. There were negative findings in a battery of seven in-vitro and in-vivo studies. There was no result to suggest a positive or weak positive response. Neither proquinazid, nor metabolites formed in rats, contain structural alerts for DNA reactivity. The studies were conducted to the prevailing guidelines at the time. The limitations noted by PSD with regard to the in-vitro chromosomal aberration assay and the mammalian cell gene mutation assay (CHO/hprt) did not impact on the overall genotoxic assessment of proquinazid. Thus further testing was unnecessary to conclude that proquinazid is not genotoxic.

COM consideration of presentation and mutagenicity data

10. The COM held a detailed discussion with the representatives of DuPont regarding the mutagenicity data and in particular the adequacy and conduct of the in-vitro chromosomal aberration assay in human lymphocytes and the in-vitro mammalian hprt gene mutation assay in CHO cells. The Committee agreed that as the COM and COC had been asked to evaluate the potential mechanism of cholangiocarcinomas seen in the rat long-term carcinogenicity bioassay with proquinazid, there was a need to have a high level of confidence in the mutagenicity data on proquinazid. Whilst members agreed that there was no convincing evidence for positive results in any of the tests, it was not possible to conclude that the in-vitro cytogenetic assay in human lymphocytes and the in-vitro hprt mammalian gene mutation assay in CHO cells were negative in view of the limitations. Members accepted that some reassurance was gained from the in-vivo bone marrow micronuclei assays in mice.

11. The COM discussed and reached the following conclusions with regard to the mutagenicity data on proquinazid.

Bacterial reverse mutation assay with batch DPX-KQ926-75 (97.9% pure). Date 18/11/98¹

12. Members agreed that this study had been adequately conducted and no further information was required.

Bacterial reverse mutation assay with batch DPX-KQ926-45 (97% pure). Date 28/02/97²

13. Members agreed that this study had been adequately conducted and no further information was required.

In-vitro mammalian cytogenicity test with batch DPX-KQ926-45 (97% pure). Date 26/02/97³

14. Members agreed that some aspects of the in-vitro chromosome aberration assay in human lymphocytes had been conducted in excess of requirements but considered that a prolonged exposure assay was required so that cells were exposed over the whole cell cycle. Members did not consider that the test had adequately covered this aspect or that the potential for aneugenicity had been examined. Members observed that the dose selection had resulted in high levels of cytotoxicity and that the positive control response reported (towards the lower limit of a very wide range, ca 6-80% cells with aberrations) limited the value of this assay. The Committee agreed that it would be advisable to request a continuous 20 h exposure in a mammalian cell chromosomal aberration assay (in human lymphocytes) in the absence of exogenous metabolic activation. This would ensure that all stages of the cell cycle were exposed. It would be appropriate to include estimation of polyploidy as an indicator of potential for aneugenicity. The study should be conducted to internationally accepted standards. The Committee was aware of the need to adequately explain the mode of action of the cholangiocarcinomas reported in the long-term bioassay of proquinazid in rats and agreed that this information would help to complete the in-vitro mutagenicity evaluation of proquinazid which was an important part of the carcinogenicity risk assessment.

In-vitro gene mutation in mammalian cells with batch DPX-KQ926-45. (97% pure). Date 22/05/97⁴

15. Members commented that the data from the in-vitro mammalian cell hprt gene mutation assay in CHO cells were consistent with the conclusions COM had previously published on practical difficulties in undertaking this assay. ( http://www.advisorybodies.doh.gov.uk/com/mut033.htm) There was considerable inter-trial variance in mutation frequency in the proquinazid assay and the finding of very low mutation frequencies in some trials (<0.6 x 10⁶ cells) limited the conclusions that could be drawn. Thus it was noted that the mutation frequency in the third trial in the acetone control and test material experiments was low at all dose levels. Members concluded that the data were not interpretable. The Committee agreed that a mouse lymphoma assay conducted to internationally accepted standards should be requested. The Committee was aware of the need to adequately explain the mode of action of the cholangiocarcinomas reported in the long-term bioassay of proquinazid in rats and agreed that this information was essential to complete the in-vitro mutagenicity evaluation of proquinazid which was an important part of the carcinogenicity risk assessment.

In-vitro unscheduled DNA synthesis with batch DPX-KQ926-45 (97% pure). Date 21/05/97. Minor editorial revision to report 22/02/99⁵

16. Members agreed that this study had been adequately conducted and no further information was required.

In-vivo bone marrow micronucleus in mice with batch DPX-KQ926-75 (97.9% pure) Date 14/01/99⁶

In-vivo bone marrow micronucleus in mice with batch DPX-KQ926-45 (97% pure) Date 22/02/99⁷

17 Members agreed that the data provided were indicative of negative findings in these assays. Members agreed the evidence for small increases in percentage micronucleated polychromatic erythrocytes seen at some doses most probably represented fluctuation in the background incidence of micronuclei. Members accepted the evidence of toxicity in these studies and information from the toxicological and toxicokinetic studies with proquinazid suggested that the bone-marrow would have been exposed to proquinazid and metabolites in these two studies.

COM conclusions from May 2005 meeting

18. Members considered that the company had provided some relevant information and comments but had not been convinced that the in-vitro mutagenicity test package was adequate. Members agreed that a key element of any proposal regarding mechanism of proquinazid induced cholangiocarcinoma in the rat would require an evaluation of mutagenicity and hence it was important to complete the mutagenicity test package.

19 The Committee considered that a mouse lymphoma assay should be conducted and that it was advisable to also conduct a continuous 20 h exposure in a mammalian cell chromosomal aberration assay (in human lymphocytes) in the absence of exogenous metabolic activation. Both studies should be conducted to internationally accepted standards. These studies were required to provide full information on the mutagenicity evaluation of proquinazid.

COM post meeting consideration of additional mouse lymphoma assay

20. The data holder submitted on 12 July 2005 the results of a new mouse lymphoma assay undertaken with proquinazid.¹⁸ The assay included an exposure of 3 hour (in presence and absence of exogenous metabolic activation) and a continuous 24 h exposure in the absence of metabolic activation. The results suggested that proquinazid was not mutagenic in this assay. A full report was not available during the postal consultation with COM members. The Committee considered the available data from the new mouse lymphoma assay by postal consultation and agreed there was no evidence for a mutagenic effect. It was also agreed that the full report should be considered by COM when available. The final report was submitted to COM members by postal consultation on the 22 August 2005. Members agreed that the submitted mouse lymphoma assay was acceptable and gave negative results.¹⁹

COC evaluation of cholangiocarcinoma in the rat (14 July 2005)

21. The COM heard a presentation from DuPont on the interpretation of cholangiocarcinoma reported in female rats fed a diet containing proquinazid as part of a long term carcinogenicity bioassay.^15,16 In brief, groups of 80 male and 80 female Cr1:CD®(SD) BR rats were fed diets containing 0,10, 30, 300, 600 (females), 1000 (males), 1200 (females) or 2000 ppm (males) for 104 weeks. Groups of five animals/sex were subject to interim haematology, clinical chemistry necropsy and histopathological evaluation at 1 week and 1 year of feeding. Survival at termination was increased at dose levels of 600 ppm and above. A statistically significant increase in intestinal-type cholangiocarcinoma was reported in female rats (8/60 at 600 ppm and 12/61 at 1200 ppm, cf none in concurrent control).¹¹

22. The representatives from DuPont were accompanied by Dr P Greaves (University of Leicester) who had been a member of a Scientific Advisory Panel (sponsored by DuPont). The Scientific Advisory Panel had reviewed the liver slides from the rat and mouse carcinogenicity bioassays. COC members questioned the representatives from DuPont and Dr Greaves during the presentation.

23. The Scientific Advisory Panel (SAP), had reviewed aspects of the liver pathology and a report had been finalised in November 2003. Dr Greaves had reviewed the slides again recently before the submission for the COC consideration was compiled.¹⁶ The SAP considered that the lesions seen in the female rats were not indicative of a carcinogenic risk for humans. The Panel's rationale had been that proquinazid was not genotoxic, that cholangiocarcinomas of the "intestinal" type occurred only in female rats fed 600 ppm or more at the end of the two-year study, the tumour type was linked to chronic hepatocellular injury with "cholangiofibrosis" and there was no risk of the lesion occurring in the absence of severe hepatic toxicity. A number of representative sections were shown to COC members. Features of the histopathology of the lesions seen at 1200 ppm included: cellular degeneration, inflammatory infiltrate, extensive necrosis, giant nuclei and fibrotic changes termed "cholangiofibrosis" by the original reporting pathologists. The cells within the lesions showed goblet cell differentiation and were therefore termed "intestinal-type". The cholangiofibrosis was not necessarily related to the portal tract and the lesions termed "cholangiocarcinoma" were quite unlike the usual pattern seen in human cholangiocarcinoma . COC members supported the view that the lesions were not neoplastic but were a florid inflammatory response to severe acute and chronic hepatocellular damage with marked reactive epithelial changes including cytoplasmic alterations, nuclear pleomorphism, and intestinal metaplasia. COC pathologists noted that there was little evidence of increased mitotic activity in the slides shown and aberrant mitoses were not a feature.

24. Dr Greaves confirmed that the SAP had reviewed virtually all the lesions diagnosed as cholangiocarcinomas and he had reviewed a proportion of them again recently. In answer to specific questioning, he agreed that, if chronic exposure to proquinazid was sufficiently high to induce similar severe liver toxicity in humans, there was a risk that similar lesions to that identified in rats could occur. He noted that liver toxicity was seen in female rats receiving 300 ppm proquinazid but was less severe than at 600 ppm. There had been individual variation in severity at 600 ppm. In answer to another question, DuPont representatives noted that the pigment seen in the liver sections had been shown to be PAS positive and was therefore likely to be lipofuschin as expected in the setting of cell degeneration and increased turnover. There was minimal deposition of stainable iron.

25. The Committee raised a question as to whether the role of oval cells in liver regeneration, as proposed in the MOA,¹⁵ is hypothetical or is more generally accepted. It was agreed that oval cell proliferation was seen in rodents when a regenerative stimulus was supplied but hepatocyte proliferation inhibited, for example by 2AAF.

COC consideration and conclusions

26. The committee concluded that it was uncertain that the lesions termed "cholangiocarcinomas" were truly neoplastic but accepted the conventionality of the term used by the study pathologist. However, it was noted that the pathology seen with proquinazid lacked the expected characteristics of that caused by a genotoxic carcinogen.

27. Members agreed that the MOA proposed by DuPont for the putative cholangiocarcinomas was plausible. They noted that the lesions were only seen at dose levels which caused severe liver toxicity and which exceeded the MTD. They further agreed that the lesions were relevant to humans in that they were a potential hazard. However, whether they were a risk in humans depended on the level of exposure.

28. Members agreed that the No Observed Adverse Effect Level (NOAEL) for putative cholangiocarcinoma was 16 mg/kg bw/day (300 ppm in female rats). They were informed that PSD had proposed an acceptable daily intake for proquinazid of 0.01 mg/kg bw, based on the overall No Observed Adverse Effect Level of 1.2 mg/kg bw/day derived from all the toxicology data on proquinazid and a safety factor of 100. Members confirmed that this would give an adequate margin of safety (1600) for cholangiocarcinoma.

Overall discussion of COM/COC on proquinazid

29. The COM has reviewed the available mutagenicity data on proquinazid (26/05/05). All of the studies were considered acceptable with the exception of the in-vitro mammalian mutagenicity assay in CHO cells (hprt locus) and it was also noted that an extended exposure of human lymphocytes in the absence of exogenous metabolic activation had not been undertaken as part of the assay for chromosomal aberrations. The COM was aware of a recently submitted in-vitro mouse lymphoma assay and agreed by postal consultation that the data were suggestive of a negative response (results submitted 12/07/05).¹⁸ The final report was submitted to COM members by postal consultation on the 22 August 2005. Members agreed that the submitted mouse lymphoma assay was acceptable and gave negative results. The COM agreed that it would not be necessary to complete the in-vitro mutagenicity test package by undertaking an extended exposure treatment of human lymphocytes for chromosomal aberrations in view of the totality of negative in-vitro and in-vivo mutagenicity data available on proquinazid if the MOA assessment for the cholangiocarcinoma which had been submitted to COC was deemed acceptable.

30. The COC considered the available carcinogenicity data and additional information on histopathology of the liver from the carcinogenicity bioassay in rats on 14/07/05. Members were unconvinced that the lesions reported as cholangiocarcinomas were truly neoplastic, but accepted that the study pathologists' diagnosis could be used for risk assessment. The COC agreed that a threshold approach to risk assessment could be used and agreed that there was a satisfactory margin of safety between the NOAEL for putative cholangiocarcinoma and the ADI proposed by PSD.

Overall conclusions of COM/COC on proquinazid

31. The COM concluded that the mutagenicity data submitted provided evidence that proquinazid is not an in-vitro or in-vivo mutagen. There were limitations in the adequacy of the submitted in-vitro gene mutation assay in mammalian cells (CHO, hprt) and the submitted in-vitro assay in human lymphocytes for chromosomal aberrations. The data holder submitted negative results from a mouse lymphoma assay which included both short-term (3 h) and extended exposure (24 h) treatments after the COM meeting (26/05/05). The full report of this study would need to be considered by COM when available. The final report was submitted to COM members by postal consultation on the 22 August 2005. Members agreed that the submitted mouse lymphoma assay was acceptable and gave negative results. The COM agreed that the advice for a further in-vitro study to examine chromosomal aberrations in human lymphocytes study would not be a requirement in view of the totality of negative mutagenicity data on proquinazid and if the MOA assessment for the cholangiocarcinoma which had been submitted to COC was deemed acceptable. (see para 32).

32. The COC concluded that it was uncertain that the lesions termed "cholangiocarcinomas" were truly neoplastic but accepted the conventionality, used by the study pathologist in the report of the carcinogenicity bioassay of proquinazid in rats, of classifying them as neoplastic. A satisfactory non-genotoxic Mode of Action explanation for the cholangiocarcinomas in rats had been submitted. The COC agreed that a threshold approach could be used for the risk assessment of cholangiocarcinoma reported in the rat.

September 2005
COM/05/S4
COC/05/S1

References

1. Cox L R (1998). In confidence report DPX-KQ926 technical: Bacterial reverse mutation test in Salmonella typhimurium and Escherichia coli. DuPont Haskell Laboratory, Newark DE USA, DuPont Report No: DuPont-1600.

2. Mathison B H (1997). In confidence report DPX-KQ926 technical: Mutagenicity testing in the Salmonella typhimurium and Escherichia coli plate incorporation assay. DuPont Haskell Laboratory, Newark DE USA, DuPont Report No: HL-1997-00040.

3. Gudi R (1999). In confidence report DPX-KQ926 technical: Mouse bone marrow micronucleus assay. Microbiological Associates, Inc, Rockville, Maryland USA, DuPont Report No: HLO 1100-96, Revision No: 1.

4. San R H C and Clarke J J (1997). In confidence report DPX-KQ926 technical: In vitro mammalian cell gene mutation test with an independent repeat assay. Microbiological Associates, Inc, Rockville, Maryland USA, DuPont Report No: HLO 1097-96.

5. San R H C (1999). In confidence report DPX-KQ926 technical: Unscheduled DNA synthesis in mammalian cells in vitro with an independent repeat assay. Microbiological Associates, Inc, Rockville, Maryland USA, DuPont Report No: HLO 1098-96, Revision No: 1.

6. Wun-Kim K (1999). In confidence report DPX-KQ926 technical: Mouse bone marrow micronucleus assay. DuPont Haskell Laboratory, Newark DE USA, DuPont Report No: DuPont-1791.

7. Gudi R and Schadly E (1999). In confidence report DPX-KQ926 technical: In vitro evaluation for chromosome aberrations in human lymphocytes. Microbiological Associates, Inc, Rockville, Maryland USA, DuPont Report No: HLO 1099-96, Revision No: 1.

8. PSD (2004). In confidence report Draft evaluation of Proquinazid, Volume 3 and 4, prepared for ACP meeting of 13 January 2005. ACP 8 (31/01/2005).

9. Bentley K S (2005). In confidence report DuPont response to the UK ACP review of proquinazid: Assessment of mutagenic potential. DuPont Haskell Laboratory, Newark DE USA, DuPont Report No: DuPont-16902.

10. Anon (2005). In confidence. DuPont power point presentation for genotoxicity review by COM, 24/04/05.

11. Malley L A (2002). In confidence report DPX-KQ926 technical: Combined chronic toxicity/oncogenicity two-year feeding study in rats, DuPont Haskell Laboratory, Newark DE USA, DuPont Report No:HL-1999-00644.

12. Donner E M (2002). In confidence report DPX-KQ926 technical: Oncogenicity eighteen-month feeding study in mice. DuPont Haskell Laboratory, Newark DE USA, DuPont Report No: HL-1998-00645.

13. Mertens J J W M (2002). In confidence report DPX-KQ926 technical: Chronic toxicity study one-year capsule study in dogs. WIL Research Laboratories, Inc (USA) and DuPont Haskell Laboratory, Newark, DE, USA, DuPont Report Number: HLO-1998-01341.

14. Mann P C, Gopinath C, Greaves P, Hardisty J F (2003). In confidence report. Scientific Advisory Panel. Review of the liver from mammalian toxicology studies with proquinazid DPX-KQ926-technical. Final report, Experimental Pathology Laboratories inc, November 18, 2003, DuPont project 13161.

15. Frame S R (2005). In confidence report Mode of action assessment for intestinal-type cholangiocarcinomas in the 2-year feeding study with proquinazid in rats. DuPont Haskell Laboratory, Newark DE USA, DuPont Report No: DuPont-17453.

16. Greaves P (2005). In confidence report Proquinazid: hepatic pathology in the 2-year rat feeding study. University of Leicester, Department of Cancer Studies and Molecular Medicine, Internal Memorandum.

17. Anon (2005). In confidence. DuPont Power point presentation on cholangiocarcinoma in rats fed proquinazid for COC review (08/07/05).

18. Anon (2005). In confidence. DuPont Power point presentation of results from mouse lymphoma assay with proquinazid. (12/07/05).

19. Ballantyne M (2005). DuPont-17121: Proquinazid (DPX-KQ926) Technical: Mutation at the thymidine kinase (tk) locus of mouse lymphoma L5178Y cells (MLA) using the microtitre fluctuation technique.

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