Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 March - 28 September 2018
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
31 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Test item information
Identification: Photomer 4250
Appearance: Colourless to pale yellow liquid
Batch: 17C13003
Purity/Composition: UVCB
Test item storage: At room temperature protected from light
Stable under storage conditions until: 14 March 2019 (expiry date)

Additional information
Test Facility test item number: 209236/A
Purity/Composition correction factor: No correction factor required
Test item handling: Use amber glassware or wrap container in aluminum-foil
CAS number 73003-78-8

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
mammalian liver post-mitochondrial fraction (S-9)
Test concentrations with justification for top dose:
Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and WP2uvrA, both with and without S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate were tested in triplicate.
The highest concentration of Photomer 4250 used in the subsequent mutation assays was 5000 µg/plate or the level at which the test item inhibited bacterial growth. At least five different doses (increasing with approximately half-log steps) of the test item were tested in triplicate in each strain in the absence and presence of S9-mix. The first experiment was a direct plate assay and the second experiment was a pre-incubation assay.
The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.
Vehicle / solvent:
dimethyl sulfoxide
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
mitomycin C
other: 2-aminoanthracene
Evaluation criteria:
No formal hypothesis testing was done.
In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
First Experiment: Direct Plate Assay
Photomer 4250 was initially tested in the tester strains TA100 and WP2uvrA as a dose-range finding test with concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate in the absence and presence of S9-mix. Based on the results of the dose-range finding test, the following dose-range was selected forthe mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 5.4, 17, 52, 164, 512 and 1600 μg/plate. The results are shown in Table 1 and Table 2. The individual data are presented in Appendix 3.

Precipitate
Precipitation of Photomer 4250 on the plates was observed at the start of the incubation period at the concentration of 5000 µg/plate and no precipitate was observed at the end of the incubation period.

Toxicity
To determine the toxicity of the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed. The definitions are stated in Appendix 2. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester
strains in the absence and presence of S9-mix.

Mutagenicity
In the direct plate test, no increase in the number of revertants was observed upon treatment with Photomer 4250 under all conditions tested.

Second Experiment: Pre-Incubation Assay
To obtain more information about the possible mutagenicity of the test item, a pre-incubation experiment was performed in the absence and presence of S9-mix. Based on the results of the first mutation assay, Photomer 4250 was tested up to the dose level of 1600 µg/plate in the tester strains TA1535, TA1537, TA98 and TA100 and up to 5000 µg/plate in tester strain WP2uvrA. The results are shown in Table 3, the individual data are presented in Appendix 3.

Precipitate
Precipitation of Photomer 4250 on the plates was observed at the start of the incubation period at the concentration of 5000 µg/plate and no precipitate was observed at the end of the incubation period.

Toxicity
Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.

Mutagenicity
In the pre-incubation test, no increase in the number of revertants was observed upon treatment with the test item under all conditions tested

Applicant's summary and conclusion

Conclusions:
In conclusion, based on the results of this study it is concluded that Photomer 4250 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

The objective of this study was to determine the potential of Photomer 4250 and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophanlocus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9).

The test was performed in two independent experiments, at first a direct plate assay was performed and the second a pre-incubation assay.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch 17C13003 of Photomer 4250 was a colourless to pale yellow liquid. The vehicle of the test item was dimethyl sulfoxide.

In the dose-range finding study, the test item was initially tested up to concentrations of 5000 µg/plate in the tester strains TA100 and WP2uvrA in the direct plate assay. The test item did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in tester strain TA100 at dose levels of 1600 and 5000 μg/plate in the absence and presence of S9-mix and in tester strain WP2uvrA at 5000 μg/plate in the absence and presence of S9-mix. Results of this dose-range finding test were reported as part of the first mutation assay.

In the first mutation experiment, the test item was tested up to concentrations of 1600 µg/plate in the tester strains TA1535, TA1537 and TA98. Photomer 4250 did not

precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all three tester strains.

In the second mutation experiment, the test item was tested up to concentrations of 1600 µg/plate in the tester strains TA1535, TA1537, TA98 and TA100 and up to

5000 μg/plate in tester strain WP2uvrA in the pre-incubation assay. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

In conclusion, based on the results of this study it is concluded that Photomer 4250 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.