Dr J Clements
Professor C Cooper
Professor P Farmer
Dr N Gooderham
Ms M Langley
Dr I Mitchell
Professor D Phillips
Professor D Tweats
Secretariat:
Dr R J Fielder (Scientific DH)
Dr D Benford (Scientific FSA)
Mr J Battershill (Scientific DH)
Mr S Robjohns (Minutes)
Mr K N Mistry (Administrative)
Assessors:
Mr A Browning (VMD)
Mr D Jones (MCA)
Dr D Andrew (PSD)
Dr M Costigan (HSE)
In attendance :
Professor A Boobis (Deputy Chair ACP)
Dr K Murphy (HSE)
Mr B Groves (FSA)
In attendance on item 4:
Mr Ian Chant
Ms Ann Manley
Mrs Ann Taylor
Dr Barry Elliott
Mr Evert Slootweg
Dr Karel de Raat
CONTENTS
Item
Paragraph
1.
Apologies for absence/Announcements
1-3
2.
Minutes of the meeting of 26 April 2001
4
3.
Matters arising:
3.1 Tobacco induced lung carcinogenesis: the importance
of p53 mutations.
5
4.
COM review of dichlorvos
6-22
5.
Any other business
23
6.
Date of next meeting
24
ITEM 1: APOLOGIES FOR ABSENCE
1. There were no apologies for absence.
Announcements
2. Members were reminded of the need to declare any interests before
discussion of items.
3. The Committee was informed that this extraordinary meeting of COM had
been convened to finalise the Committee's evaluation of dichlorvos and
to consider the further data forwarded by industry and to listen to their
evaluation of the mutagenicity data on dichlorvos.
ITEM 2: MINUTES OF THE MEETING HELD ON 26th APRIL 2001 (MUT/MIN/2001/2)
4. The minutes were agreed subject to minor typographical amendments.
ITEM 3: MATTERS ARISING
5. Members were informed that a critical review of the paper by Rodin
SN and Rodin AS (Proceedings of the National Academy of Sciences97, 12244-12249, 2000) on the importance of p53 mutations in tobacco
induced carcinogenesis (MUT/01/3) was being drafted and would be circulated
to members before the next COM meeting (11th October 2001).
ITEM 4: COM REVIEW OF DICHLORVOS (MUT/01/19-25 and Addendum to MUT/01/21)
6. Dr Benford (FSA) and Dr Clements declared lapsed non-personal, non-specific
interests. The Chairman recalled that with respect to Dr Benford and Dr
Clements, the interests declared referred to studies on dichlorvos undertaken
approximately 10 years ago and that neither had any current interest in
the item under discussion.
Background to the Mutagenicity of Dichlorvos
7. The COM recalled the paper by Sasaki YF et al (Critical Reviews
in Toxicology, 30 (6), 629-799, 2000) on the performance of the in-vivo
COMET assay at its 8th February 2001 meeting. The Committee had agreed
that the results of this study supported the view outlined in the COM
revised guidelines namely, that the in-vivo Comet assay was of
particular use when following up the results of in-vitro positive
mutagenicity tests but was not appropriate as a general screen. Members
considered that the approach adopted by Sasaki of testing several hundreds
of chemicals had a number of drawbacks, for example, limited reporting
of signs of toxicity seen in animals. Members considered that the appropriateness
of the isolated nuclei method used by Sasaki et al had not been
established and noted that it was not possible to have concurrent evaluation
of cytotoxicity using this method. Hence there would be difficulties in
evaluating the significance of positive results with this method.
8. With respect to dichlorvos, members recalled that this chemical contained
a structural alert for mutagenicity and was a direct acting in-vitro
mutagen. The Committee had agreed that a full review of the mutagenicity
data was needed in order to assess the significance of the results obtained
in the new Comet assay. The purpose of such a review would be to evaluate
dichlorvos but it would also enhance understanding of the Comet assay.
Background to request for extraordinary meeting
9. Members recalled that a 1st draft statement was circulated for comments
after the 26 April 2001 meeting. The intention was to finalise the statement
for the ACP meeting of the 24 May 2001. As part of the procedures for
openness set out in the Annual Reports of the COT/COC/COM, the 1st draft
statement on dichlorvos was also forwarded to the data holders who made
a number of comments which are reproduced in MUT/01/24. The COM chair
in accordance with the procedures for openness considered comments from
data holders. Although there was no obligation to alter or change the
statement in any respect, in this instance it was felt that industry should
have an opportunity to present their arguments particularly with respect
to the COM's approach to the critical in-vivo studies and the prospect
of further relevant data particularly in respect of interpretation of
the study in transgenic animals and the postulation of a role for oxidative
damage in the production of the mutations observed.
10. The COM secretariat held a meeting with industry at the HSE offices
in Bootle on 20 June to discuss the preparation of documents for the COM.
The industry team was told that they would have the opportunity to see
a revised statement following the COM meeting on 23 July 2001 and to comment
over a very short period (2-3 days).
Consideration of written evidence
11. Members considered the papers forwarded by industry for this meeting.
The Chairman asked the Committee to identify the key areas for questioning.
These were identified as:
i) relative rates of hydrolysis compared to methylation of DNA by dichlorvos.
ii) the concept of a threshold for dichlorvos induced DNA methylation.
iii) the new data on oxidative damage in hepatocytes induced by dichlorvos
and relevance of these data to the response seen in the in-vivo
mutation study in transgenic animals
iv) the use of the intraperitoneal route for the assessment of direct
acting mutagenic effects in the liver.
v) The weight of evidence approach to the assessment of mutagenicity.
Industry presentation
12. The industry team comprised representatives of the data holders and
experts in mutagenicity assessment acting as consultants. The team distributed
a new set of overheads which differed in detail from those submitted to
the secretariat. The presentation lasted approximately 30 minutes. Members
raised a number of questions with the industry team. The team withdrew
from the room whilst Members discussed the comments raised by industry.
COM discussion of industry presentation
13. Industry accepted that dichlorvos was a direct acting in-vitro
mutagen but argued that since the rate of dichlorvos hydrolysis was many
orders of magnitude faster than DNA alkylation there was an effective
threshold for alkylation in-vivo. This was consistent with many
in-vivo negative studies covering a range of endpoints. Consideration
was then given to the four studies reported by the COM in the 1st draft
statement as giving positive results and which underpinned the conclusion
reached by COM.
14. The mouse hair follicle assay nuclear anomaly assay was not an appropriate
assay to identify genotoxicity and could not be used to detect mutagens.
This suggestion was agreed by the Committee but it was felt that the results
did indicate that the skin was a target organ for dichlorvos.
15. Industry's concerns with the mouse skin micronucleus assay were that
this was not a validated assay, and results were available from only one
laboratory. Other laboratories had difficulty with this test and no data
were available on sensitivity, specificity or reproducibility. The Committee
accepted that this was not a standard assay but noted that for supplementary
in-vivo assays, especially to investigate local site effects, novel
methods often provided useful data. Furthermore there was no convincing
explanation to discount this positive result.
16. Industry believed that the methodology used in the repeated dose rat
bone-marrow chromosome aberration study was inadequate to draw any conclusions
from the reported increase in numerical aberrations due to inadequate
methodology. It was also noted, by industry, that this study gave unique
results when compared to the other available repeat dose studies. The
Committee agreed that the method was limited but the results were indicative
of effects on numerical aberrations. They agreed it would be more appropriate
to refer to numerical aberrations rather than aneuploidy.
17. Finally, industry expressed concerns regarding the inappropriate study
design of the MutaTM Mouse study involving high doses with
the liver as the target. Industry suggested that the route of administration
(namely intraperitoneal) was fundamentally flawed for direct acting compounds.
There were also concerns about the excessively high dose level and the
lack of details to address the specification of the test material used.
The Committee felt that the methods used in this study had limitations
but the data were indicative of a mutagenic effect of dichlorvos at the
site-of-contact.
18. A major concern of industry was the lack of appropriate consideration
of weight of evidence with regard to the in-vivo mutagenicity studies.
However the Committee agreed that the positive data showed a pattern for
in-vivo site of contact mutagenicity and the systemic studies were
irrelevant for the evaluation of site-of-contact effects.
19. Members noted that no additional information was provided by industry
with regard to the potential for oxidative effects of dichlorvos in the
liver other than the paper appended to MUT/01/25.
20. Further consideration was given to the negative results in the single
dose UDS assay in the forestomach of mice using gavage dosing. The Committee
concluded that it was not possible to exclude genotoxic effects from these
data given the relative insensitivity of the method used as indicated
by the response with the positive control chemical, and that repeat dosing
would most likely be required to identify any mutagenic effects of dichlorvos
in this assay. In addition the Committee noted the views of the Chairman
and a member of the COC on the carcinogenicity bioassays in the mouse,
and in particular on the oesophageal and stomach tumours.
21. The Committee agreed that no specific reference to trichlorfon should
be
made in the revised statement. However, members considered that the mutagenicity
data of the related compounds, naled and trichlorfon should be reviewed.
The Veterinary Medicines Directorate welcomed this suggestion which would
be relevant to the current VPC consideration of naled as part of the non-sheep
dip organophosphate review and international considerations of trichlorfon.
22. The Committee concluded that dichlorvos should be regarded as an in-vivo
mutagen at the site-of-contact. High doses of dichlorvos induced mutagenic
effects in the skin following topical application and in the liver following
intraperitoneal dosing. The Committee agreed that dichlorvos induced tumours
of the forestomach in mice after gavage dosing; also that the oesophageal
tumours seen after dietary administration were probably related to dichlorvos
treatment. The Committee felt that it would be prudent to assume a genotoxic
mechanism on the basis of the available data. The Committee agreed that
in the absence of appropriate mechanistic data a precautionary approach
should be adopted and no threshold could be assumed for the mutagenic
and carcinogenic effects of dichlorvos.
Item 5: Any Other Business
23. Members asked if the secretariat could approach MCA for their views
on the possible nitrosation of nicotine derived from nicotine patches
applied to the skin.
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