Dr J Clements
Professor C Cooper
Professor P Farmer
Dr N Gooderham
Ms M Langley
Dr I Mitchell
Professor D Phillips
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:
Dr R Brown (PSD)
Mr A Browning (VMD)
Dr A Smith (HSE)
In attendance :
Mr G Kowalczyk (DH)
CONTENTS
Item
Paragraph
1.
Apologies for absence / Announcements
1
2.
Minutes of meeting held on 12th October 2000
5
3.
Matters arising:
3.1 Publication of COM guidance on a strategy for testing
6
4.
Phosphine: Update on 1997 conclusions
9
5.
In-vivo comet assay; data on 208 chemicals including
dichlorvos and dimethoate
14
6.
1,3-Dichloropropanol and 2,3-dichloropropanol
20
7.
Review of risk procedures used by the Government's advisory
committees dealing with food safety (for discussion)
24
8.
Risk assessment of mutagens; adoption of
approach based on absence of thresholds.
28
9.
International workshop on the
categorisation of mutagens.
32
10.
Any other business
36
ITEM 1: APOLOGIES FOR ABSENCE
1. Apologies for absence were received from Professors John Ashby, David
Tweats and Dr Dick Shillaker (for whom Dr Richard Brown (PSD) deputised).
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 review
of risk procedures used by advisory committee dealing with food safety
(MUT/01/8) before the discussion paper on risk assessment (MUT/01/2).
4. Members were informed that the paper on human lung cancer and p 53:
The interplay between mutagenesis and selection (MUT/01/5) would be considered
at the April meeting in order to include the most recently published information
on this subject.
ITEM 2: MINUTES OF THE MEETING HELD ON 12th OCTOBER 2000 (MUT/MIN/2000/3)
5. Minutes were agreed subject to one minor typographical change.
ITEM 3: MATTERS ARISING
6. The Chairman informed members that the COM Guidance on a Strategy of
testing Chemicals for Mutagenicity had been published on the 12th January
2001 and that the document had also been placed on the COM website. He
commented that it had been well received.
7. The Chairman noted that an Ad Hoc meeting of the EU Standing Committee
on Plant Protection Products had recommended an ADI and AOEL could be
set for carbendazim and had accepted the COM advice that the aneugenicity
of carbendazim was threshold related.
8. The Chairman reported on his attendance at a working group of the EU
Scientific Committee on Food (SCF) on 3-MCPD. The COM/COC conclusions
that 3-MCPD was a non-genotoxic carcinogen in the rat had been accepted.
The SCF Working Group had debated the necessity for an uncertainty factor
of a 1000 for the kidney tumours in the rat as suggested by COC, and had
agreed to consult further on the evaluation of the carcinogenicity data
before reaching a final conclusion on an uncertainty factor.
ITEM 4: PHOSPHINE: UPDATE ON 1997 CONCLUSIONS (MUT/01/1 and Addendums
1 and 2)
9. Dr Clements declared a non-specific non-personal interest. The Chairman
noted that this related to some work undertaken over 10 years ago and
considered that Dr Clements could take full part in the discussion.
10. Phosphine is used as a pesticide to fumigate grain and as a rodenticide.
The COM considered the mutagenicity of phosphine in 1997. Although in-vitro
data indicated it had mutagenic potential, in-vivo data in rodents
(bone marrow, liver and germ cell) were negative. However a clastogenicity
study by Garry et al (Science, 246, 251-255, 1989) in fumigant
workers in Minnesota did give rise to concern, although it was recognised
that this study had limitations. The COM recommended that consideration
be given to a feasibility study in UK pesticide applicators using modern
methods for assessing genotoxic effects. This recommendation was passed
by PSD to industry. After detailed consideration of this request the industry
view was that such a study would be impractical due to the small numbers
of workers involved and the fact that nearly all were also potentially
exposed to another mutagen, methyl bromide. Industry have suggested a
study of those workers involved in the manufacture of phosphine, outside
the 'closed' area where full PPE is worn. However the numbers are relatively
small (about 20) and the exposures low. In addition a new inhalation carcinogenicity
bioassay in the rat which had been conducted to acceptable regulatory
standards and which was negative had now become available. The Committee
was asked to review its conclusions reached in 1997 in the light of this
new information. Members were also asked to note the most recent estimates
of exposure to phosphine during the manufacture of phosphides which had
been tabled for information.
11. Members discussed the available mutagenicity data and reaffirmed that
that phosphine was highly reactive and agreed that the mutagenicity data
provided some evidence for positive results in-vitro but the available
in-vivo assays were negative. The Committee agreed that the negative
rat inhalation carcinogenicity bioassay provided additional reassurance
with regard to possible site of contact mutagenicity. It was noted that
there was no evidence that phosphine had a direct interaction with DNA,
and that any mutagenic effect in-vitro might be related to the
production of reactive oxides which could react with cellular components.
It came to the notice of the Committee that phosphine has been reported
to induce oxidative DNA damage in-vitro and in-vivo.
12. Members recalled that exposures to phosphine in the studies reported
by Garry et al indicated concentration of phosphine up to 4.1 ppm.
The Committee noted a recent communication received by the secretariat
from the Lawrence Livermore Laboratory USA in respect of a study which
had been published as an abstract only in 1996, which reported that the
negative results of cytogenetic screening study in workers potentially
exposed to phosphine during fumigation work, was probably due to the efficiency
of exposure reduction provided by Personal Protective Equipment worn by
the operators. The Committee agreed that occupational exposure to phosphine
was significantly reduced in fumigators and other workers exposed to phosphine
by the use of Personal Protective Equipment. It was therefore unlikely
that any occupational groups could be currently identified within the
UK where an appropriate biomonitoring study could be undertaken. Members
were aware that the use of phosphine as a pesticide in the UK was expected
to increase in the future as a result of restrictions to be enforced with
respect to the use of other fumigants (methyl bromide). The Committee
was made aware of proposals to measure exposure in workers undertaking
fumigation operations and those re-entering grain stores. The Committee
agreed that it would like to see a review of any results that became available.
Members commented that exposure of workers to phosphine during the manufacture
of electronics might represent a separate occupational group which could
be considered in any further studies of exposure to phosphine.
13. The Committee concluded that additional new data, (namely negative
rat inhalation carcinogenicity study), together with the negative in-vivo
mutagenicity data in bone marrow and liver in animals provided sufficient
reassurance regarding the in-vivo mutagenicity of phosphine, taken
together with the information relating to very low potential exposure
arising from pesticides use. Thus the COM's original suggestion for consideration
of a biomonitoring study in UK pesticide applicators was no longer necessary.
The Committee noted that use of phosphine was likely to increase in the
future and asked to be informed of any results of exposure measurements
made available to the UK regulatory authorities.
ITEM 5: IN VIVO COMET ASSAY: DATA ON 208 CHEMICALS AND ADVICE ON DICHLORVOS
(AND DIMETHOATE) (MUT/01/4) addendum to MUT/01/4 and MUT/01/10 (tabled)
14. The Committee considered a recent report on a substantial data set
on the in-vivo comet assay (Sasaki Y F et al, 2000. Critical
Rev. Toxicol. Vol 30, p 629-799). Comet assay results were given for 8
organs in the mouse with 208 chemicals for which carcinogenicity bioassay
data were available. This assay is given specific consideration in the
revised COM guidance, where it is stated that earlier problems regarding
the distinction between cytotoxic and genotoxic chemicals have now largely
been resolved. Overall the assay does appear to give a high correlation
with in-vitro genotoxicity (i.e. Ames positives); but there are
some results that give rise for concern. Twenty percent of rodent non-carcinogens
were positive. There was poor correlation between compounds positive in
the bone marrow in the Comet assay and positive in the bone marrow mouse
micronucleus test (26 compounds positive in both, but 37 negative in comet
and positive in micronucleus test, and 9 positive in the Comet assay and
negative in the micronucleus test).
15. Members considered that the approach adopted by Sasaki et al
to 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. In this respect members highlighted the results obtained
with a number of polycyclic aromatic hydrocarbons and commented that positive
results had been reported in the new Comet assay data for some PAHs (eg
pyrene) which were considered to be non-carcinogenic in rodents.
16. Members concluded that the new data did not affect the advice given
in the COM Guidance document regarding the in-vivo Comet assay
which should be regarded as a secondary test for use in specific situations
not as a primary screen for mutagenic activity. It was considered useful
for following up positive in-vitro mutagenicity tests, and to have
the advantage of being able to look at every tissue in-vivo in
one assay.
Dichlorvos
17. With respect to Dichlorvos, members noted that this chemical contained
a structural alert for mutagenicity and was a direct acting in-vitro
mutagen. Members noted that negative results had been documented in a
number of in-vivo assays reported to be adequate but on the data
available it was not possible to assess tissue exposure. 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 new Comet assay.
The purpose of this review would be to evaluate Dichlorvos but it would
also enhance understanding of the Comet assay. The Committee was of the
view that the potential mutagenic activity of chemicals which produce
dichlorvos in-vivo (in particular trichlorofon) should be reviewed.
Dimethoate
18. With respect to dimethoate, COM members noted that the critical mutagenicity
studies (unpublished) for pesticides approvals were submitted by the Dimethoate
Task Force (DTF). Data refer to a specific batch of test material used
to support pesticides approved in the UK under the Control of Pesticides
Regulations (1986). These studies show that dimethoate was mutagenic in-vitro
(in Salmonella typhimurium TA100 and Escherichia coli WP/uvrA both in
presence and absence of S-9 and in a hepatocyte UDS assay). Negative results
were produced in the unpublished in-vivo assays submitted by DTF (rat
bone marrow cytogenetics, mouse bone marrow micronucleus, rat liver UDS,
and mouse dominant lethal). Members were aware that the ACP review noted
that there are a number of positive in-vivo assays in the published literature.
Less weight had been placed on these assays in view of limited knowledge
of test material purity and the adequacy of test methods used. The COM
agreed that it would be appropriate to suggest that the Dimethoate Task
Force undertake an in-vivo Comet assay with test material conforming to
the specification used in other assays submitted by the DTF.
19. The COM also agreed that industry should also be asked to provide
information on which impurities were responsible for producing positive
results in the published studies with dimethoate.
ITEM 6: 1,3-DICHLOROPROPAN-2-OL (1,3-DCP) AND 2,3- DICHLOROPROPANOL
(2,3 DCP) (MUT/01/6 AND MUT/01/7)
20. Members were asked to consider the papers on 1,3-DCP and 2,3-DCP together.
These closely related compounds are structurally similar to 3-MCPD considered
by COM at its previous meeting. The committee was informed that exposure
might occur from drinking water and that there are, at present, no mandatory
levels for chloropropanols other than 3-MCPD. For consistency DH wished
to obtain formal advice from COM on these two compounds, to inform the
Drinking Water Inspectorate (DWI) in this area. Exposures can also occur
from certain foodstuffs.
21. The Committee considered the limited mutagenicity data available on
both compounds. In both cases in-vitro data indicate that the two
compounds had mutagenic potential, but there were no data available in
whole animals to enable any conclusions to be reached on whether this
activity could be expressed in-vivo.
22. Members agreed that metabolism of 1,3-DCP was likely to produce the
reactive epoxide metabolite that could damage DNA. Members suggested that
a survey of the levels of this group of compounds in food would be useful.
23. The Committee concluded that it would be prudent to regard 1,3-DCP
and 2,3-DCP as potentially genotoxic in-vivo and agreed that both
compounds should be tested for genotoxicity in-vivo using the approach
set out in the COM guidelines. In the first instance this should be in
the in-vivo bone marrow micronucleus assay.
ITEM 7: REVIEW OF RISK PROCEDURES USED BY THE GOVERNMENT ADVISORY COMMITTEES
DEALING WITH FOOD SAFETY (MUT/01/8)
24. Members were asked to consider the report by Sir Robert May on the
review of risk procedures used by the Government's advisory committees
dealing with food safety. The Chairman had drafted an initial response
for COM which were outlined in Annex 4.
25. Members considered that due to the complex nature of some of the work
undertaken by the Committee, that flexibility was required rather than
a formalised approach to risk Assessment. It was agreed that the COM guidelines
recently published were suitable as a format for Committee discussions.
26. The Committee agreed that on occasions improved communication between
the COM, COC and COT would be useful. It was suggested that these committees
could receive each other's minutes.
27. It was also suggested that joint meetings and cross-membership could
be usefully pursued. Members agreed that the secretariat should identify
potential ideas for a joint scientific meeting of all three committees.
ITEM 8: RISK ASSESSMENT OF MUTAGENS: ADOPTION OF AN APPROACH BASED
ON ABSENCE OF THRESHOLDS (MUT/01/2)
28. The Committee recalled that during the discussion on the COM guidelines
members had identified risk assessment as a priority area of future work.
The Committee was reminded that the COM adopted a precautionary approach,
which assumed that there is no threshold of action for in-vivo
mutagens. Any exposure is considered to result in increased risk, although
possibly very small. The Committee has in the past recognised two exceptions
to this generalisation relating to aneugenicity identification by tubulin
inhibitors and the rapid detoxication of hydroquinone and phenol via the
oral route. Some key papers from the EEMS 1998 symposium on threshold-mediated
mechanisms in mutagenicity were made available to the Committee.
29. The Committee agreed that it was important to base its discussion
on chemicals rather than using the often quoted example of radiation.
Members confirmed that the formation of DNA adducts represented a potential
mutagenic hazard. In the absence of specific information on the conversion
of a DNA adduct to a mutation, it had to be assumed that the formation
of DNA adducts represented a potential mutagenic risk. Members discussed
the conversion of propylene oxide DNA adducts to mutagenic lesions but
agreed that in most cases where there was no specific mechanistic information
relating to the chemical under consideration, it had to be assumed that
the formation of a DNA adduct equated to a mutagenic hazard. Members agreed
that quantitative data in DNA adducts could not be used to assess risk
of mutagenicity, except in the rare situations where appropriate data
were available for cancer target organs and the conversion of DNA adducts
to mutagenic lesions had been investigated.
30. Members discussed the concept of a threshold for mutagenicity and
agreed that there were essentially two distinct types of mechanisms that
could be invoked in respect of thresholds, ie through binding to protein
targets (such as microtubules, DNA synthetases, topoisomerases) or metabolic
detoxification of promutagenic chemicals or DNA adducts. If there were
specific data on a chemical to invoke such mechanisms then the possibility
of threshold could be considered. Members agreed that data on threshold-related
mechanisms would in most instances come from in-vitro studies.
Any observed NOEL from such in-vitro mechanistic studies could
be used to inform in a risk assessment but it was unlikely that the data
could be used in a quantitative way. Members reaffirmed that it might
be possible to compare mutagenic potency of chemicals on the basis of
the results of in-vivo studies provided appropriate kinetic data
were available, but it would not be possible to undertake such ranking
exercises on the basis of in-vitro data.
31. Members agreed that a short statement outlining the COM conclusions
should be drafted.
ITEM 9: INTERNATIONAL WORKSHOP ON THE CATEGORISATION OF MUTAGENS (MUT/01/3)
32. The COM reviewed the recently agreed the Globally Harmonised Scheme
( GHS) and agreed criteria for mutagens. The system was similar to that
of the EU except that it only contains 2 classes (1 and 2) category 1
being divided into known mutagens (1a) and those that should be regarded
as if they induce heritable mutations in germ cells of humans. Category
2 mutagens are those which cause concern for man owing to the possibility
that they may induce heritable mutations in the germ cells of humans.
Thus category 1B on the GHS was equivalent to category 2 on the EU system,
and category 2 on the GHS was equivalent to category 3 on the EU system.
33. The "detailed" criteria for the GHS classification were
similar to those in the EU systems with 2 exceptions. For category 1b
mutagens, data from positive in-vivo heritable germ cell tests
were needed or data from positive in-vivo somatic cell assays plus
evidence that the substance had the potential to cause mutations in germ
cells eg from mutagenicity/genotoxicity tests in germ cells in-vivo,
or by demonstrating the ability of the substance to interact with the
genetic material of germ cells (this was in line with earlier COM recommendations).
For category 2 mutagens in the GHS, activity from somatic cell in in-vivo
assays for mutagenicity was needed, or from genotoxicity assays in-vivo
supported by positive in-vitro data.
34. It was also noted that chemicals which were positive in-vitro
in mammalian mutagenicity assays and which show chemical structure activity
relationships to known germ cell mutagens, should be considered for classification
as category 2 mutagens. Members agreed that this proposal went further
than earlier COM advice.
35. Most members agreed that the proposals recently adopted by the GHS
for classifying chemicals as mutagens were reasonable. It was felt that
the importance of in-vivo somatic cell mutagens needed to be recognised
when classifying chemicals as carcinogens. It was agreed that this should
be brought up at the October meeting. One member questioned the emphasis
on germ line mutations, as opposed to somatic cell mutations, when classifying
chemicals as mutagens.
ITEM 10: ANY OTHER BUSINESS
36. There was no other business discussed.
ITEM 11: DATE OF NEXT MEETING
37. April 26, 2001
ACTIONS
Item
Action
Responsibility
4
Phosphine Draft statement for ACP. Circulate to Members.
Secretariat
5
Comet Assay Prepare statement on general aspects of
assay and on dichlorvos and dimethoate.
Secretariat in liaison with HSE, PSD and
VMD.
6
Dichloropropanols Draft statement. Prepare paper for
COC
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