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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10/10/2012-26/03/2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to
Guideline:
other: the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF
Qualifier:
according to
Guideline:
other: the USA, EPA (TSCA) OPPTS harmonised guidelines.
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report):F-2200HM
- Physical state:Off-white powder
- Analytical purity: >99%
- Lot/batch No.:290110772
- Expiration date of the lot/batch: 12/2014
- Storage condition of test material:
- Other: ambient storage conditions in darkness

Method

Target gene:
hisD, hisG, hisC, trp
Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 μg/plate
Experiment 1: 50, 150, 500, 1500, 5000μg/plate
Experiment 2: 15, 50, 150, 500, 1500, 5000 μg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: fully soluble in DMSO, 100mg/ml in aceton
Controlsopen allclose all
Negative controls:
no
Solvent controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without metabolic activation. Strains: WP2uvrA, TA100, TA1535
Negative controls:
no
Solvent controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without metabolic activation. Strain TA1537
Negative controls:
no
Solvent controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without metabolic activation. Strain TA98
Negative controls:
no
Solvent controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene for strain TA100, TA1535, Ta1537, WP2uvrA
Remarks:
with metabolic activation
Negative controls:
no
Solvent controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with metabolic activation. Strain TA98
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
no
Positive control substance:
other: DMSO
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period:/
- Exposure duration:48h
- Expression time (cells in growth medium):48h


NUMBER OF REPLICATIONS:3

Rationale for test conditions:
According to test guidelines
Evaluation criteria:
1. A dose related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations
3. Biological relevance against in-house historical control ranges
4. Statistical analysis of data as determined by UKEMS
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accomponied by an out-of-historical range response).

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: The test item was non-toxic to the strains of bacteria used (TA100 and WP2uvrA).
The test item formulation and S9-mix used in this experiment were both shown to be sterile

COMPARISON WITH HISTORICAL CONTROL DATA: yes

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative

The test item, the substance was considered to be non-mutagenic under the conditions of this test.
Executive summary:

Introduction:The test method was designed to be compatible with the guidelines for

bacterial mutagenicity testing published by the major Japanese Regulatory Authorities
including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No.
471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC)
number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised
guidelines.
Methods. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and
Escherichia coli strain WP2uvrA were treated with the test item, F-2200HM, using both
the Ames plate incorporation and pre-incubation methods at up to six dose levels, in
triplicate, both with and without the addition of a rat liver homogenate metabolising
system (10% liver S9 in standard co-factors). The dose range for the range-finding test
was determined in a preliminary toxicity assay and was 15 to 5000 ~g/plate. The
experiment was repeated on a separate day (pre-incubation method) using an amended
dose range (50 to 5000 ~g/plate), fresh cultures of the bacterial strains and fresh test
item formulations. An additional dose level was included in the range-finding test to
allow for potential test item toxicity following reductions in revertant colony frequency in
the preliminary toxicity test.
Results. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant
colonies within the normal range. All of the positive control chemicals used in the test
induced marked increases in the frequency of revertant colonies, both with or without
metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix
were validated.
The test item caused no visible reduction in the growth of the bacterial background lawn
at any dose level and was, therefore, tested up to the maximum recommended dose
level of 5000 ~g/plate. A test item film (creamy in appearance) was noted from
1500 ~g/plate with an associated precipitate observed at 5000 ~g/plate. For the plates
dosed with S9-mix the film was noted to have spread and separated to a greater degree
than on plates dosed in the absence of S9-mix. Neither of these observations prevented
the scoring of revertant colonies.
PROJECT NUMBER: 41205168 PAGE 6
No toxicologically significant increases in the frequency of revertant colonies were
recorded for any of the bacterial strains, with any dose of the test item, either with or
without metabolic activation or exposure method. A small, statistically significant
increase in T A 1535 revertant colony frequency was observed in the presence of S9-mix
at 50 IJg/plate in the range-finding test. This increase was considered to be of no
biological relevance because there was no evidence of a dose-response relationship or
reproducibility. Furthermore, the individual revertant colony counts at 50 IJg/plate were
within the in-house historical untreated/vehicle control range for the tester strain and the
fold increase was only 1.26 times the concurrent vehicle control.
Conclusion. The test item, F-2200HM was considered to be non-mutagenic under the
conditions of this test.