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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2005 january 10th to 2005 march 14th
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study performed according to the OECD Guideline 471.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Lot No. DEV75332
Date Received: 07 January 2005
Physical Description: Off-white powder
Storage Conditions: Room temperature

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
The S9 homogenate was prepared from male Sprague-Dawley rats that had been injected (i.p.) with AroclorTM 1254 (200 mg per mL in corn oil) at 500 mg/kg.
Test concentrations with justification for top dose:
33.3 µg per plate,
100 µg per plate,
333 µg per plate,
1000 µg per plate,
3330 µg per plate,
5000 µg per plate.
Controlsopen allclose all
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Migrated to IUCLID6: 2.5 µg/plate TA98 with presence of S9 mix.
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Migrated to IUCLID6: 1.0 µg/plate TA98 in absence of S9 mix.
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene : 2.5 µg/plate TA100, TA1535, TA1537 and WP2uvrA(pKM101) in presence of S9 mix.
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Migrated to IUCLID6: 2.0 µg/plate TA100 and TA1535 in absence of S9 mix.
Positive controls:
yes
Positive control substance:
other: ICR-191 : 2.0 µg/plate TA1537 in absence of S9 mix.
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Migrated to IUCLID6: 2.0 µg/plate WP2uvrA(pKM101) in absence of S9 mix.
Details on test system and experimental conditions:
Tester Strains. Tester strains used were Salmonella typhimurium histidine auxotrophs TA98,
TAlOO, TA1535, and TA1537 as described by Ames et ai. (1975) and Escherichia coli
tryptophan auxotroph WP2uvrA as described by Green and Muriel (I 976) with the addition of
the pKMl01 plasmid.
In addition to a mutation in either the histidine or tryptophan operons, the tester strains contain
additional mutations that enhance their sensitivity to some mutagenic compounds. Mutation of
either the uvrA gene (Escherichia coli) or the uvrB gene (Salmonella typhimurium) results in a
deficient DNA excision repair system, which greatly enhances the sensitivity of these strains to
some mutagens. Since the uvrB deletion extends through the bio gene, Salmonella typhimurium
tester strains containing this deletion also require the vitamin biotin for growth.
Salmonella typhimurium tester strains also contain the rfa wall mutation, which results in loss of
one of the enzymes responsible for synthesis of part of the lipopolysaccharide barrier that forms
the surface of the bacterial cell wall. Resulting cell wall deficiency increases permeability to
certain classes of chemicals such as those containing large ring systems (e.g., benzo[a]pyrene)
otherwise excluded by a normal intact cell wall.
Tester strains TA98, TAl00, and WP2mrA(pKM1O1) also contain the pKM101 plasmid, which
further increases the sensitivity of these strains to some mutagens. The suggested mechanism by
which this plasmid increases sensitivity to mutagens is by modification of an existing bacterial
DNA repair polymerase complex involved with the mismatch-repair process.
Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to
histidine independence (prototrophy) by frameshift mutagens. Tester strains TAl 00, TA 1535,
and WP2uvrA(pKM1O1) are reverted from auxotrophy to prototrophy by base substitution
mutagens.
Source of Tester Strains. Salmonella typhimurium tester strains in use at Covance were
received directly from Dr, Bruce Ames, Department of Biochemistry, University of California,
Berkeley. Escherichia coli tester strain, WP2uvrA(pKM101), was received from The National
Collection of Industrial Bacteria, Torrey Research Station, Scotland (United Kingdom).
Frozen Permanent Stocks. Frozen permanent stocks were prepared by growing fresh overnight
cultures, adding DMSO (0.09 mL/mL of culture) and freezing away appropriately vialed aliquots.
Frozen permanent stocks of the tester strains were stored at -60°C to -80°C.
Master Plates. Master plates of tester strains were prepared by streaking each tester strain from
a frozen permanent stock onto minimal agar appropriately supplemented with either histidine and
biotin or tryptophan, and for strains containing the pKM101 plasmid, ampicillin. Tester strain
master plates were stored at >O°C to 10°C.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Positive controls validity:
valid
Additional information on results:
Test Article Handling
The test article was stored at room temperature. In solubility testing, the test article was observed
to form beigish-white, heterogeneous suspensions in deionized water at approximately 100 and
50 mg per mL. In solubility testing with DMSO, the test article was observed to form a
transparent, light yellow to yellow, non-viscous solution at approximately 100 mg per mL. For
this reason, DMSO (Acros Organics Lot Nos. A018490001, A019540701 and A019779001 ) was
selected as the vehicle. At 100 mg per mL, which was the most concentrated stock dilution
prepared for the mutagenicity assay, the test article was observed to form a transparent, dark
yellow to yellowish light orange, non-viscous solution. The test article remained in solution at
all succeeding lower dilutions prepared for the mutagenicity assay.
Dose Rangefinding Study
Doses tested in the mutagenicity assay were selected based on results of the dose rangefinding
study conducted on the test article using tester strains TA100 and WP2uvrA(pKM101) in the
presence and absence of S9 (one plate per dose). Ten doses of test article ranging from 6.67 to
5000 µg per plate were tested and results are presented in Table 1. These data were generated in
Trial A1 . No cytotoxicity was observed with either tester strain in the presence or absence of S9
mix as evidenced by no dose-related decreases in the number of revertants per plate and normal
bacterial background lawns. Test article precipitate was observed on the plates at 21 000 µg per
plate in the presence of S9 mix and at 23330 µg per plate in the absence of S9 mix.
Mutagenicity Assay
The mutagenicity assay results are presented in Tables 2 through 6. These data were generated in
Trials B 1, C 1 and D 1. Data are presented as individual plate counts (Tables 2,4, and 6) and as
mean revertants per plate k standard deviation (Tables 3,5, and 6) for each treatment and control
group.
Doses tested in the mutagenicity assay were 33.3, 100,333, 1000,3330, and 5000 µg per plate
with all tester strains in the presence and absence of S9 mix.
In the initial mutagenicity assay, Trial B1 (Tables 2 and 3), all data were acceptable and no
positive increase in the mean number of revertants per plate were observed with any of the tester
strains in either the presence or absence of S9 mix. Apparently dose-related decreases in the
number of revertants per plate were observed only with WP2uvrA(pKM101) at 23330 µg per
plate in the presence of S9 mix and at 21000 pg per plate in the absence of S9 mix. Test article
precipitate was observed on the plates at 21 000 µg per plate in the presence of S9 mix and at
23330 µg per plate in the absence of S9 mix (21 000 µg per plate with WP2uvrA(pKM101) in the
absence of S9 mix).
In the confirmatory mutagenicity assay, Trial C1 (Tables 4 and 5), the mean positive control
value for tester strain WP2uvrA(pKM101) in the presence of S9 mix did not exhibit at least a
3-fold increase over the mean vehicle control value. For this reason, the data generated with
tester strain WP2uvrApKM101) in the presence of S9 mix in Trial C1 were not used to evaluate
the test article. The test article was re-tested with tester strain WP2uvrA(pKM101) in the
presence of S9 mix in Trial D 1. All other data generated in Trial C 1 were acceptable and no
positive increases in the mean number of revertants per plate were observed with any of the tester
strains in either the presence or absence of S9 mix. Apparently dose-related decreases in the
number of revertants per plate were observed only with WP2uvrA(pKM101) at 23330 µg per
plate in the presence and absence of S9 mix. Test article precipitate was observed on the plates
at 21 000 µg per plate in the presence of S9 mix, obscuring the bacterial background lawns at
23330 µg per plate. In the absence of S9 mix, test article precipitate was observed on the plates
at 23330 µg per plate with TA98 and TA100, and at 21000 µg per plate with the other tester
strains.
In the repeat mutagenicity assay, Trial D1 (Table 6), all data were acceptable and no positive
increases in the mean number of revertants per plate were observed with tester strain
WP2uvrA(pKM101) in the presence of S9 mix. Apparently dose-related decreases in the number
of revertants per plate were observed with WP2uvrA(pKMIOl) at 21000 µg per plate in the
presence of S9 mix. Test article precipitate was observed on the plates at 21000 µg per plate in
the presence of S9 mix, obscuring the bacterial background lawns at 23330 µg per plate.
All criteria for a valid study were met (except as noted in the Protocol Deviation section).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The results of the Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay
with a Confirmatory Assay indicate that under the conditions of this study, Eastman Kodak
Company's test article did not cause a positive increase in the mean number of revertants per
plate with any of the tester strains in either the presence or absence of AroclorTM induced rat liver
(S9).