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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:
weight of evidence
Study period:
12-28 July 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Type:
impurity
Test material form:
liquid
Details on test material:
Batch No.: 171893
Purity: 90.9%
Name of test material (as cited in study report): neryl acetate
Physical state: colourless to slightly amber liquid
Storage conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry date: 9 December 2017

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction prepared from liver homogenates of male Sprague Dawley rats
Test concentrations with justification for top dose:
Experiment 1 (plate-incorporation method):
-TA1535, TA1537, TA98, TA100 and WP2 uvra 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, with and without S9-mix

Experiment 2 (preincubation method with and without S9 mix):
- 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, with and without S9-mix

Additional experiment 2 (pre-incubation method) without S9:
- 0.05, 0.15, 1.5, 5, 15, 50, 150, 500 μg/plate without S9-mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
The solubility of Neryl acetatet was assessed at 50 mg/mL in dimethyl sulphoxide (DMSO) in which it dissolved. DMSO (ACS reagent grade) was, therefore, used as the vehicle for this study. The highest concentration of neryl acetate tested in this study was 50 mg/mL in the chosen vehicle, which provided a final concentration of 5000 µg/plate.
Controls
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: Benzo[a]pyrene
Details on test system and experimental conditions:
SOURCE OF TEST SYSTEM:
The strains of S. typhimurium and E. coli were obtained from Moltox Inc.

METHOD OF APPLICATION: In agar (plate incorporation); preincubation

DURATION
- Preincubation period: 30 minutes at 37 ± 1 °C, with shaking
- Incubation period: Plates were inverted and incubated at 37 ± 1 °C in the dark for 3 days in both direct
plate and preincubation methods.

NUMBER OF REPLICATIONS:
- Vehicle, treatment (test item) and positive controls were included in triplicate plates,
Evaluation criteria:
Criteria for Assessing Mutagenic Potential:

If exposure to a test item produces a reproducible increase in mean revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, it is considered to exhibit mutagenic activity in this test system.
If exposure to a test item does not produce a reproducible increase in mean revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis. Biological importance will be considered along with statistical significance. In general, treatment-associated increases in mean revertant colony numbers below two or three times those of the vehicle controls (as described above) are not considered biologically important. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained.
Occasionally, these criteria may not be appropriate to the test data and, in such cases, the Study Director would use his/her scientific judgment.
Statistics:
The mean number and standard deviation of revertant colonies will be calculated for all groups. The means for all treatment groups will be compared with those obtained for the vehicle control groups

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
Remarks:
plate incorporation method: at 1500 µg/plate and above with S9 and in pre-incubation method: at 50 µg/plate and above without S9 and 500 µg/plate and above with S9
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
Remarks:
plate incubation method: at 1500 µg/plate and above without S9 and at 5000µg/plate with S9, in pre-incubation method: at 150 µg/plate and above without and at 500 µg/plate without S9
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
Remarks:
plate incorporation method: at 1500 µg/plate and above with and without S9, in pre-incubation method: at 50 µg/plate and above without S9 and 500 µg/plate and above with S9
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
Remarks:
plate incorporation method: at 1500 µg/plate and above with and without S9, in pre-incubation method: at 150 µg/plate and above without S9 and at 500 µg/plate with S9
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in pre-incubation method at 1500 µg/plate with S9
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
First Test
In the absence of S9 mix, toxicity (observed as thinning of the background lawn of non-revertant colonies and/or a reduction in revertant colony numbers) was seen in strains TA98, TA100 and TA1537 at 1500 µg/plate and above. In the presence of S9 mix, toxicity (observed as thinning or complete absence of the background lawn of non-revertant colonies and/or a reduction in revertant colony numbers) was seen in strains TA98 at 5000 µg/plate and in strains TA100, TA1535 and TA1537 at 1500 µg/plate and above.
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to Neryl acetate at any concentration up to and including 5000 µg/plate in either the presence or absence of S9 mix.

Second Test
In the absence of S9 mix, toxicity (observed as thinning of the background lawn of non-revertant colonies and/or a reduction in revertant colony numbers) was seen in strains TA98 and TA1537 at 150 µg/plate and above, and in strains TA100 and TA1535 at 50 µg/plate and above. In the presence of S9 mix, toxicity (observed as thinning or complete absence of the background lawn of non-revertant colonies and/or a reduction in revertant colony numbers) was seen in strains TA98, TA100, TA1535 and TA1537 at 500 µg/plate and above, and in strain WP2 uvrA (pKM101) at 1500 µg/plate and above.
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to Neryl acetate at any concentration up to and including 5000 µg/plate in either the presence or absence of S9 mix.
As there were an insufficient number of non-toxic concentrations in strains TA98, TA100, TA1535 and TA1537 in the absence of S9 mix these were repeated in an additional test using modified dose concentrations.

Additional Second Test
In the additional second test, toxicity (observed as thinning or complete absence of the background lawn of non-revertant colonies and/or a reduction in revertant colony numbers) was seen in all strains at 50 µg/plate and above.
Toxicity (observed as a reduction in revertant colony numbers 0.5 x fold those of the concurrent vehicle control values) was also seen in strain TA98 at 0.5 µg/plate. These reductions in colony numbers would, however, appear to be anomalous when considered with the remaining cultures in the exposure condition and were likely an effect of experimental variation.
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to Neryl acetate at any concentration up to and including 500 µg/plate.

Applicant's summary and conclusion

Conclusions:
Neryl acetate showed no evidence of mutagenic activity in this bacterial system at doses up to 5000 μg/plate.
Executive summary:

In an in vitro bacterial reverse mutation test performed according to OECD Guideline 471 and in compliance with GLP, strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100) and Escherichia coli, strain WP2 uvrA (pKM101), were exposed to neryl acetate diluted in dimethyl sulphoxide (DMSO). 

Two independent mutation tests were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. The first test was a standard plate incorporation assay; the second included a pre-incubation stage.

The following concentration of the test item was used: 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, with and without S9-mix, in experiment 1 and 0.05, 0.15, 1.5, 5, 15, 50, 150, 500  μg/plate without S9-mix in experiment 2 .

In the first test, in the absence of S9 mix, toxicity (observed as thinning of the background lawn of non-revertant colonies and/or a reduction in revertant colony numbers) was seen in strains TA98, TA100 and TA1537 at 1500 µg/plate and above. In the presence of S9 mix, toxicity (observed as thinning or complete absence of the background lawn of non-revertant colonies and/or a reduction in revertant colony numbers) was seen in strains TA98 at 5000 µg/plate and in strains TA100, TA1535 and TA1537 at 1500 µg/plate and above.

In the second test, in the absence of S9 mix, toxicity (observed as thinning of the background lawn of non-revertant colonies and/or a reduction in revertant colony numbers) was seen in strains TA98 and TA1537 at 150 µg/plate and above, and in strains TA100 and TA1535 at 50 µg/plate and above. In the presence of S9 mix, toxicity (observed as thinning or complete absence of the background lawn of non-revertant colonies and/or a reduction in revertant colony numbers) was seen in strains TA98, TA100, TA1535 and TA1537 at 500 µg/plate and above, and in strain WP2uvrA (pKM101) at 1500 µg/plate and above. As there were an insufficient number of non-toxic concentrations in strains TA98, TA100, TA1535 and TA1537 in the absence of S9 mix these were repeated in an additional test using modified dose concentrations.

In the additional test, toxicity (observed as thinning or complete absence of the background lawn of non-revertant colonies and/or a reduction in revertant colony numbers) was seen in all strains at 50 µg/plate and above. 

No evidence of mutagenic activity was seen at any concentration of neryl acetate in any mutation test.

The concurrent positive controls verified the sensitivity of the assay and the metabolizing activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the current historical control range for the laboratory.

Therefore, neryl acetate showed no evidence of mutagenic activity in this bacterial system with and without metabolic activation at doses up to 5000 μg/plate.