Professor J Ashby
Dr J Clements
Professor C Cooper
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 Shillaker (PSD)
Dr A Smith (HSE)
In attendance :
Dr K Murphy (HSE)
CONTENTS
Item
Paragraph
1.
Apologies for absence
1
2.
Minutes of the meeting of 8 February 2001
4
3.
Matters arising:
3.1 Statement on the mutagenicity of 1,3-dichloropropanol
and 2,3-dichloropropanol
5
4.
Draft statement on risk assessment of in vivo
mutagens (and gentoxic carcinogens) MUT/01/11
6
5.
Code of practice for Scientific Advisory Committee:
2nd round of consultation MUT/01/16
9
6.
Review of the mutagenicity of dichlorvos MUT/01/12,14,15
(Sent 4/4/01 and Addendum MUT/01/12 sent 9/4/01)
11
7.
Tobacco induced lung carcinogenesis: the importance
of p53 mutations MUT/01/13
12
8.
Any other business
18
9.
Date of next meeting
19
ITEM 1: APOLOGIES FOR ABSENCE
1. Apologies for absence were received from Professor P Farmer and Dr
N Gooderham.
Announcements
2. Members were reminded of the need to declare any interests before discussion
of items.
3. The Chairman informed members that he wished to consider the Code of
Practice for Scientific Advisory Committees before the review of dichlorvos.
ITEM 2: MINUTES OF THE MEETING HELD ON 8TH FEBRUARY 2001
(MUT/MIN/2001/1)
4. The minutes were agreed subject to minor typographical amendments.
ITEM 3: MATTERS ARISING
5. Members were informed that a statement on 1,3-dichloropropanol and
2,3-dichloropropanol would soon be published on the website.
ITEM 4: DRAFT STATEMENT ON RISK ASSESSMENT OF IN VIVO MUTAGENS
(AND GENOTOXIC CARCINOGENS) (MUT/01/11)
6. The Committee was reminded that it had considered the principles of
risk assessment of chemical in-vivo mutagens at its last meeting.
The current precautionary approach, based on the assumption of the absence
of any threshold, was endorsed as the default. Two mechanistic exceptions
to this had previously been recognised namely, aneugenicity induction
by tubulin inhibitors (specifically methyl benzimadazole carbamates, benomyl,
carbendazim and thiophanate-methyl) and the rapid detoxication of hydroquinone
and phenol via the oral route. In view of the importance of this subject
and the fact that the COM have not comprehensively updated their guidelines,
only their strategy of testing, it is believed to be important to publish
a statement covering this area. The Committee was asked to agree the draft
statement (MUT/01/11).
7. Members expressed concern at whether it was clear that this document
addressed risk assessment as well as thresholds, or the absence of threshold.
It was agreed that an additional sentence should be added to the end of
the document to explain that if there were adequate evidence to support
a threshold mechanism, then it would be appropriate for regulatory authorities
to adopt a risk assessment approach based on identification of a critical
NOAEL and the use of uncertainty factors. It was agreed to delete the
table listing potential mechanisms since this did not concentrate on the
key aspects, and some of the listings were not appropriate. It was noted
that there would be a symposium on this topic later in the year.
8. The statement would be revised, circulated for comment and finalised
by Chairman's action.
ITEM 5: CODE OF PRACTICE FOR SCIENTIFIC ADVISORY COMMITTEE: 2ND
ROUND OF CONSULTATION (MUT/01/16)
9. Members recalled that they had previously commented on the first draft
of a Code of Practice for Scientific Advisory Committees. The second version
contained a number of suggested amendments and included discussion of
some topics, which arose from the findings of the Inquiry into BSE by
Lord Phillips. Members were asked for their comments on the new version,
which would be fed back to the Office of Science and Technology.
10. Members noted that the minutes of COM discussion were often highly
technical, and that there was need to make the information as comprehensible
as possible to the public, and that further consideration should be given
to this challenging issue. Regarding the proposal for a joint open meeting
of the COM/COC/COT, members noted that the topics of genomics and proteomics
would be useful as a scientific meeting, but would be of limited interest
to the general public. The Committee agreed that the format used by the
COT for stakeholder meetings and open scientific meetings would be appropriate
for the COC and COM. Members asked for more flexibility in assessing the
need for extra members of COM and to co-opt additional expertise when
required. The Committee accepted the practice that a consensus view was
reported in statements, but considered that it should be clearly stated
where one or several members disagreed with a conclusion. Members felt
that on appointment more information could be provided on the length of
service possible and of training opportunities. Members felt that the
Committee should be able to suggest topics where research was required,
although it was acknowledged that any proposal would have to be included
in the overall research priorities review process of the sponsoring Government
Department.
ITEM 6: REVIEW OF MUTAGENICITY OF DICHLORVOS (MUT/01/11,14,15, and
Addendum MUT/01/12)
11. Professor J Ashby declared a direct interest and was not present for
the discussion. Dr Benford (FSA) and Dr Clements declared lapsed non-personal,
non-specific interests. The Chairman noted 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.
12. Members recalled that at the last meeting of the COM in February 2001
consideration was given to a recently published report of results of numerous
chemicals in a Comet assay. Specific consideration was given to dichlorvos,
which was reported as positive, in contrast to the bulk of in-vivo
data on this material. The Committee considered that a full review of
the mutagenicity data was needed in order to assess the significance of
the results obtained in the Comet assay. Dichlorvos is currently being
reviewed by the ACP as part of the review programme for all organophosphates
and also by the Veterinary Medicines Directorate. A comprehensive review
of all the available mutagenicity data was provided in MUT/01/12 (and
addendum), Annex 1 of which was sections (toxicokinetics, mutagenicity,
and carcinogenicity) from the HSE review provided for the ACP. Although
the bulk of the in-vivo data were negative there were a number
of positive results, which were important to the COM evaluation. A number
of additional papers and comments had been submitted by industry (Annex
5 to MUT/01/12 and MUT/01/14,15 and 17).
13. The Chairman asked members to consider the in-vitro data first.
Members agreed that dichlorvos is a relatively weak methylating agent
(compared to methyl methanesulphonate; MMS). The Committee concurred with
the following assessment of the in-vitro mutagenicity studies.
Dichlorvos is mutagenic both in presence and absence of exogenous metabolism
in bacteria, yeast cells and in mammalian cell gene mutation assays, chromosome
aberrations assay, the in-vitro micronucleus test and sister chromatid
exchange assays. Positive results have been reported in in-vitro
UDS assays using human lymphocytes and human epithelial-like cells. Dichlorvos
has been shown to methylate nucleophiles and to induce strand breaks in
isolated DNA.
14. The Chairman asked Members to consider the available in-vivo
data. The Committee noted that there were a large number of in-vivo
studies available. Dichlorvos was negative in in-vivo mutagenicity
assays including mouse bone-marrow micronucleus (using ip route) bone-marrow
chromosome aberration studies in mice and hamsters (using oral and in
two studies (mice/hamster) inhalation exposure). Negative results were
also reported in SCE in mice and UDS assays (liver (rats)/forestomach
(mice)).
15. Members considered the results of a number of studies in detail where
positive results had been reported by the investigators. The Committee
agreed that dichlorvos induced micronuclei in keratinocytes in mice and
nuclear anomalies (including micronuclei) in hair follicles of mice following
the topical application to skin. Members agreed that the positive results
reported in abstract by Majeeth et al in a mouse bone-marrow micronucleus
assay could not be interpreted as insufficient information on methods
and results were available. The Committee considered that equivocal evidence
of chromosomal aberrations in bone-marrow smears had been reported in
a study where hamsters were given a single oral dose of up to half-the
LD50 of the formulation.
16. The Committee agreed that evidence for chromosomal aberrations had
been documented in the bone-marrow of rats following repeated oral dosing
with dichlorvos for 6 weeks (5 days/week). Members considered that the
methods used were satisfactory and noted that, although the adequacy of
reporting was limited, the results suggested a positive effect on numerical
chromosome aberrations. It was noted that a clear dose-response would
not necessarily be expected in this study as the dose range selected was
relatively narrow.
17. Regarding the recently published COMET assay, Members considered that
the approach adopted by Sasaki and colleagues to the mutagenicity testing
of 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
and colleagues had not been established and noted that it was not possible
to have concurrent evaluation of cytotoxicity using this method. In respect
of the study on dichlorvos, members agreed that the dose level chosen
(ca 80% of the LD50) was too high. Members agreed that, given
the limitations of the method used to assess COMETs and the excessive
dose level of dichlorvos that it would not be possible to differentiate
between cell necrosis, apoptosis and genotoxicity. Furthermore the positive
data in all tissues examined was unexpected. Members considered that it
was not possible to conclude that dichlorvos had mutagenic effects in
a wide range of tissues on the basis of these data. Thus, although the
authors suggested that dichlorvos had an in-vivo genotoxic effect,
the data were uninterpretable.
18. Members considered the in-vivo mutagenicity study in lacZ transgenic
mice (MutaTM Mouse) undertaken by Plesta and colleagues. The
authors had reported a statistically significant (3-fold) increase in
mutant frequency in the liver and a slight non-statistically significant
increase in mutant frequency in the bone-marrow following repeated dosing
with dichlorvos (5 x 11 mg/kg) given intraperitoneally. Members agreed
that the methods used for the assessment of mutation in these animals
was satisfactory and that the data were consistent with a mutagenic effect
of dichlorvos in-vivo. The Committee noted that the authors had
failed to identify any increase in tissue DNA adducts in transgenic mice
given a single intraperitoneal dose of either 4.4 mg/kg bw or 11 mg/kg
bw but agreed that the methods used by the authors were of low sensitivity
and it was unlikely that any increase in DNA adducts could have been detected
in the single dose studies using dichlorvos, since mutations had not been
observed following a single dose. Thus, Members commented that the levels
of DNA adducts at O6 and N7
in transgenic mice (MutaTM Mouse) following repeated dosing
with dimethylsulphate (10 x 6 mg/kg bw ip) were approximately 4-fold higher
than the limit of detection. Members considered that evaluation of DNA
adducts in dichlorvos treated animals after the repeat dosing regime might
have provided valuable information but these analyses had not been undertaken.
19. The Committee reached an initial conclusion that a pattern of mutagenic
effects had been documented in the in-vivo studies which suggested
that dichlorvos induced mutagenic effects in the skin following topical
application (ie at the site of contact) and in systemic tissues (liver
and bone-marrow) under conditions where repeated doses were administered.
20. The Chairman asked members to consider the further information provided
by industry. A number of papers had been submitted just prior to the COM
meeting. Members agreed that no substantive new mutagenicity data had
been submitted. The additional data from the Mouse lymphoma test undertaken
as part of the US NTP assessment of dichlorvos was consistent with other
assays and indicated a positive result in this assay. Regarding the specific
comments on the most recent in-vivo mutagenicity assays, members
agreed with the reservations proposed by industry regarding the interpretation
of COMET assay but did not agree with the views expressed regarding the
mutagenicity study in transgenic animals. In particular Members confirmed
that the conduct of the mutagenicity in transgenic mice had been adequately
conducted for the purpose of in-vivo hazard identification and
dichlorvos was clearly positive in the liver.
21. The Committee commented on the "Blue-Ribbon" evaluation
of dichlorvos completed in July 1998 and noted that differences in the
rate of methylation compared to the rate of phosphorylation could not
be used to discount a potential in-vivo mutagenic hazard of dichlorvos.
Additionally the role of phosphorylation in the induction of genotoxic
effects could not be discounted.
22. The Chairman asked members for comments on their interpretation of
the in-vivo mutagenicity data on dichlorvos taking all of the information
into account. The Committee agreed that dichlorvos should be regarded
as an in-vivo mutagen and that there was a potential risk of mutagenicity
at site of contact tissues and systemically following repeated exposure.
Members noted from the HSE evaluation document that retention of 14C-vinyl-labelled
dichlorvos in skin was recorded in a study where radiolabelled dichlorvos
was applied to the skin on the back of male rats. Members considered that
dichlorvos was mutagenic in-vivo under conditions where tissues
were exposed to sufficient quantities of dichlorvos before it was metabolised
to inactive compounds. However the Committee agreed that no threshold
could be assumed for the mutagenic activity of dichlorvos.
23. Members discussed the significance of the mutagenicity data with respect
to the available information on carcinogenicity. Members were aware that
there was some limited evidence for a carcinogenic effect in animals from
standard bioassays in rats and mice using gavage dosing; this related
to an increase in squamous cell papillomas and carcinomas of the forestomach
in female mice. Members noted that negative results had been obtained
in a single dose UDS assay in the forestomach of mice using gavage dosing.
However an increase in replicative DNA synthesis had been reported in
this study. Members considered that it was not possible to exclude a genotoxic
effect from these data given that repeat dosing would most likely be required
to identify any mutagenic effect of dichlorvos.
24. The Committee commented that trichlorfon which is used as a Veterinary
Medicine in some countries (not the UK) degraded to Dichlorvos in-vivo
and considered on the basis of the data reviewed in this statement that
there was good reason to conclude that trichlorfon was likely to have
in-vivo mutagenic activity.
25. The Committee reached an initial conclusion that dichlorvos should
be regarded as an in-vivo mutagen. Dichlorvos induced mutagenic
effects in the skin following topical application (ie at the site of contact)
and in systemic tissues (liver and bone-marrow) under conditions where
repeated doses were administered. The Committee agreed that no threshold
for the activity of dichlorvos could be assumed.
ITEM 7: TOBACCO INDUCED CARCINOGENESIS; THE IMPORTANCE OF P53 MUTATIONS
(MUT/01/3)
27. The Chairman declared a non-personal, non-specific interest. He invited
Professor Cooper to chair the discussion of this item.
28. Professor Cooper gave a short presentation to the Committee. He considered
that the P53 database now held by the WHO International Agency for Research
on Cancer (IARC) was a valuable tool for the investigation of mechanisms
of cancer, but limitations in the adequacy and accuracy of the information
held in the database needed to be acknowledged in research papers. He
noted that for most cancers it was generally acknowledged, that there
were a number of mutations leading to the development of tumours and thus
any conclusions with regard to the role of the P53 in the aetiology of
lung cancer needed to acknowledge this principle. Professor Cooper focused
his presentation on two of the papers which had been circulated to members,
namely Paschke T Mutagenesis15, 457-458, 2000 and Rodin
SN and Rodin AS, Proceedings of the National Academy of Sciences97, 12244-12249, 2000.
29. In respect of the paper from Paschke T, members agreed that it was
difficult to agree with the conclusions reached, namely that there were
no differences between smokers and non-smokers in respect of P53 patterns.
Although the authors had investigated the inconsistencies between the
different versions of the P53 database, they had not based the analysis
of the prevalence of G ----> T transversions on the original papers
and hence misclassification of smoking status was a likely confounding
factor. Members agreed that the subsequent analysis by Hainaut P and Pfeifer
G. Carcinogenesis, 22, 367-374, 2001, which was based on
assessment of smoking status from individual papers was a more appropriate
investigation. The COM agreed that the data supported the view that G
---> T transversions in the P53 gene were more prevalent in smokers.
30. Members heard a presentation from Professor Cooper on the approach
used by Rodin and Rodin to assess P53 mutation patterns of lung adenocarcinomas
from smokers and non-smokers. Members considered that the critical analysis
in the paper concerned the within-strand analysis of P53 mutation patterns.
The authors suggested that when complementary base-substitutions were
considered together in pairs (thus G ----> T and C --->A) there
were no apparent differences between the P53 mutation patterns in lung
tumours from smokers and non-smokers. Members considered that it was difficult
to evaluate the method used to assess within-strand P53 mutations and
observed that there was a potential inconsistency between the data reported
by Rodin and Rodin and the reactivity of benzo(a)pyrene epoxide metabolites
with DNA bases. It was agreed that a paper outlining member's comments
should be published to contribute to the debate on this important topic.
31. Members agreed that there were important reservations regarding the
rationale used by Rodin and Rodin. The Committee agreed that it was important
to record that there were over 3,000 chemicals in tobacco smoke and that
these included known genotoxic carcinogens such as the tobacco specific
nitrosamines which were also likely to be important in the aetiology of
tobacco-smoke induced lung carcinogenesis. Members agreed with Rodin and
Rodin that many of the chemicals in tobacco-smoke accentuated the formation
of tumours and acted as promoters. Overall it was agreed that a clear
conclusion in the role of mutations in P53 tobacco smoke induced lung
cancer should be published.
32. The Committee concluded that tobacco smoke contained many genotoxic
carcinogens, some of which are implicated in the aetiology of lung cancer
induced by smoking. The evidence from the study of mutational patterns
and hotspots in the P53 gene from lung tumours of smokers and non-smokers
suggests that carcinogens in tobacco smoke induce mutations in tumours.
This supports the view that tobacco smoke is demonstrably a genotoxic
carcinogen and any exposure is likely to be associated with some increased
risk of cancer (ie effects do not have a threshold).
ITEM 8: ANY OTHER BUSINESS
33. There was no other business discussed.
ITEM 9: DATE OF NEXT MEETING
34. October 11, 2001
ACTIONS
Item
Action
Responsibility
4. Risk Assessment
Finalise statement through Chair
Secretariat/ Chair
5. Code of Practice
Secretariat/Chair to write to OST with comments
Secretariat/Chair
6. Review of the mutagenicity of dichlorvos
Draft statement
Secretariat
7. Tobacco induced lung carcinogenesis; the importance
of p53 mutations
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