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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:
12 May 2017 - 26 july 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

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)
Remarks:
d.d. 3 November 2015
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
other: paste
Specific details on test material used for the study:
- Name of test material (as cited in study report): Oleyltrimethylammonium chloride (dried)
- Batch No.: 1439581 (superdry)
- Expiration date of the batch: 09 September 2018
- Appearance: yellow paste
- Storage conditions: At room temperature container flushed with nitrogen, desiccated
- Handlings with test material: in glove box (nitrogen environment)

Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
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:
Rat liver S9-mix induced with Aroclor 1254
Test concentrations with justification for top dose:
Experiment 1
Preliminary test (without and with 5% S9-mix) TA100 and WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
Main study:
TA1535, TA1537 and TA98: without and with 5% S9-mix: 0.18, 0.55, 1.7, 5.4, 17 and 52 µg/plate
Additional:
TA1535 and TA98: with 5% S9-mix: 17, 52, 164 and 512 µg/plate

Experiment 2:
All strains: without S9-mix: 4.7, 8.5, 15, 27, 48 and 86 µg/plate
All strains: with 10% S9-mix: 8.5, 15, 27, 48, 86 and 154 µg/plate
Additional:
TA1535, TA1537, TA98 and TA100: without S9-mix: 0.83, 1.5, 2.6, 4.7, 8.5 and 15 µg/plate
WP2uvrA: with 10% S9-mix: 86, 154, 275 and 492 µg/plate
Vehicle / solvent:
- Vehicle used: Milli-Q Water
- Justification for choice of solvent/vehicle: Test compound was soluble in water and water has been accepted and approved by authorities and international guidelines
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9; 5 µg/plate in saline for TA1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: ICR-191
Remarks:
without S9; 2.5 µg/plate in DMSO for TA1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-nitrofluorene
Remarks:
without S9; 10 µg/plate in DMSO for TA98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9; 650 µg/plate in DMSO for TA100
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9; 10 µg/plate in DMSO for WP2uvrA
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
in DMSO; 2.5 μg/plate for TA1535 (5% + 10% S9-mix) and TA1537 (5% S9-mix); 5 μg/plate for TA1537 (10% S9-mix); 1 μg/plate for TA98 (5% + 10% S9-mix) and TA100 (5% S9-mix); 2 μg/plate for TA100 (10% S9-mix); 15 μg/plate for WP2uvrA (5% + 10% S9-mix).
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 ± 4 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Additional experiments:
Since in the first experiment in the presence of S9-mix, no toxicity and no precipitate was observed at the highest dose levels tested in the tester strains TA1535 and TA98, an additional experiment was performed.
In the second mutation experiment, due to the cytotoxicity, only two to three analyzable dose levels were left for the determination of the mutagenicity of the test item in the tester strains TA1535, TA1537, TA98 and TA100 in the absence of S9-mix. Furthermore, in tester strain WP2uvrA no dose level with toxicity or precipitate on the plates was observed in the presence of S9-mix. Therefore an additional mutation experiment was performed.
Rationale for test conditions:
A dose-range finding test was performed and the highest concentration of the test item used in the subsequent mutation assay was 5000 μg/plate or the level at which the test item inhibited bacterial growth.
Evaluation criteria:
ACCEPTABILITY CRITERIA
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

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

A test substance is considered positive if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a follow up 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.
Statistics:
No formal hypothesis testing was done.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed except in the second experiment where the test item seemed to show slight precipitation at the highest tested concentration of 15 μg/plate in the tester strains TA98 and TA100 in the presence of a reduced bacterial background.

RANGE-FINDING/SCREENING STUDIES:
- In tester strain TA100, toxicity was observed at dose levels of 17 μg/plate and above and 52 μg/plate and above in the absence and presence of S9-mix, respectively. In tester strain WP2uvrA, toxicity was observed at dose levels of 52 μg/plate and above and 164 μg/plate and above in the absence and presence of S9-mix, respectively.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within the laboratory historical control data. The strain-specific positive control values were within the laboratory historical control data ranges, except the response for TA98 in the presence of S9-mix in the second experiment. Since the value (230 ± 69) was more than 8 times greater than the concurrent solvent control values (27 ± 6), this deviation in the mean plate count of the positive control had no effect on the results of the study.

HISTORICAL CONTROL DATA
- Positive historical control data:
TA1535 TA1537 TA98
S9-mix - + - + - +
Range 125 - 1381 78 - 1058 55 – 1324 55 – 1051 410 – 1995 250 - 1977
Mean 839 220 736 382 1369 929
SD 153 112 331 150 310 345
n 2065 1967 1740 1933 1920 2014

TA100 WP2uvrA
S9-mix - + - +
Range 537 – 1848 408 - 2651 93 – 1951 93 - 1359
Mean 908 1330 1128 422
SD 178 324 484 151
n 2007 2020 1679 1728

- Negative (solvent/vehicle) historical control data:
TA1535 TA1537 TA98
S9-mix - + - + - +
Range 3 - 36 3 - 32 3 – 20 3 – 23 8 - 41 9 - 55
Mean 11 11 6 7 16 23
SD 4 4 3 3 5 7
n 2057 2039 1950 1931 2023 2083

TA100 WP2uvrA
S9-mix - + - +
Range 66 - 161 63 - 160 10 – 59 9 - 69
Mean 105 105 25 31
SD 19 20 7 8
n 2027 2033 1739 1745


ADDITIONAL INFORMATION ON CYTOTOXICITY:
TA1535: without S9: 27 µg/plate and above; with 5% S9: 164 µg/plate and above; with 10% S9: 154 µg/plate and above
TA1537: without S9: 8.5 µg/plate and above; with 5%S9: 52 µg/plate and above; with 10% S9: 154 µg/plate and above
TA98: without S9: 15 µg/plate and above; with 5% S9: 164 µg/plate and above; with 10% S9: 86 µg/plate and above
TA100: without S9: 15 µg/plate and above; with 5% S9: 52 µg/plate and above; with 10% S9: 86 µg/plate and above
WP2uvrA: without S9: 48 µg/plate and above; with 5% S9: 164 µg/plate and above; with 10% S9: 275 µg/plate and above

Applicant's summary and conclusion

Conclusions:
Under the study conditions, the test substance was not mutagenic in Salmonella typhimurium strain TA98, TA100, TA1535 or TA 1537 and E.Coli WP2uvrA, with or without metabolic activation.
Executive summary:

An in vitro study was conducted to determine the mutagenic potential of test substance, Oleyl TMAC, according to OECD Guideline 471 and EU Method B.13/B.14, in compliance with GLP. Based on the results of the dose-range finding test, the test substance was tested in the first mutation assay at a concentration range of 0.18 to 52 μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test substance 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 tester strains in the absence and presence of S9-mix, except for the tester strains TA1535 and TA98 in the presence of S9. So, an additional experiment was performed with these two tester strains TA1535 and TA98, using concentration range of 17 to 512 μg/plate of the test substance. No precipitation on the plates was observed at this dose level, however, cytotoxicity was observed in both tester strains. In a follow-up experiment of the assay with additional parameters, the test substance was tested at concentration ranges of 4.7 to 86 μg/plate and 8.5 to 154 μg/plate in the absence and presence of 10% (v/v) S9-mix, respectively, in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test substance did not precipitate on the plates at this dose level. Cytotoxicity, was observed in all tester strains in the absence and presence of S9-mix. Except for tester strain WP2uvrA, where no cytotoxicity was observed in the presence of S9 mix. In the second mutation experiment, due to the cytotoxicity, only two to three analyzable dose levels were left for the determination of the mutagenicity of the test substance in the tester strains TA1535, TA1537, TA98 and TA100 in the absence of S9-mix. Furthermore, in tester strain WP2uvrA no dose level with toxicity or precipitate on the plates was observed in the presence of S9-mix. Therefore an additional mutation experiment was performed. In this additional experiment, the test substance was tested at concentration ranges of 0.83 to 15 μg/plate in strains TA1535, TA1537, TA98 and TA100 in the absence of S9 mix and at 86 to 492 μg/plate in tester strain WP2uvrA in the presence of S9 mix. The test substance seemed to show slight precipitation at the highest tested concentration of 15 μg/plate in the tester strains TA98 and TA100 in the presence of a reduced bacterial background. Cytotoxicity, was observed in all tester strains in the absence or presence of S9-mix. Except for tester strain TA1535, where no cytotoxicity was observed in the absence of S9 mix. The test substance 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 the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment. In this study, acceptable responses were obtained for the negative and strain-specific positive control substances, indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Under the study conditions, it was concluded that the test substance was not mutagenic in Salmonella typhimurium strain TA98, TA100, TA1535 or TA 1537 and E.Coli WP2uvrA, with or without metabolic activation (Gijsbrechts, 2017).