Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Neryl acetate showed no evidence of mutagenic activity in an Ames test at doses up to 5000 μg/plate conducted under GLP according to Guideline OECD 471.

Neryl acetate showed no evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes in a study conducted under GLP according to Guideline OECD 487.

Neryl acetate did not demonstrate mutagenic potential in an in vitro HPRT cell mutation assay conducted under GLP according to Guideline OECD 476.

Link to relevant study records

Referenceopen allclose all

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
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
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
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.
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
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.
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.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
04-25 July 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD 487 Guideline without any deviation.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
15 February 2016
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Les Dérivés Résiniques et Terpéniques (DRT) / 171893
- Appearance: Colourless to slightly amber liquid
- Date received: 05 May 2016
- Expiration date of the lot/batch: 09 December 2017
- Purity test date: 11 January 2016

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 2-8 °C, in the dark, under nitrogen
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
10% (v/v) S9 fraction; S9 fraction was obtained from the liver homogenates of male Sprague-Dawley rats induced with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
Preliminary Toxicity Test:
3 h treatment in the absence and presence of S9 mix: 3.83, 7.67, 15.34, 30.67, 61.34, 122.68, 245.36, 490.73, 981.45 and 1962.9 µg/mL
20 h treatment in the absence of S9 mix: 3.83, 7.67, 15.34, 30.67, 61.34, 122.68, 245.36, 490.73, 981.45 and 1962.9 µg/mL

Main Experiment
3 h treatment in the absence and presence of S9 mix: 15, 30, 60, 120 and 250 μg/mL
20 h treatment in the absence of S9 mix: 15, 30, 60, 70, 80, 90, 100, 110 and 120 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The solubility of the test substance in a vehicle compatible with this test system was assessed. Test substance was found to be soluble at 392.58 mg/mL in DMSO which resulted in a final concentration of 1962.9 μg/mL (10 mM) when dosed at 0.5% v/v.
- Test substance was dissolved and diluted in DMSO, shortly before dosing. The final volume of DMSO added to the cultures was 0.5% v/v. All concentrations cited in this report are expressed in terms of test substance as received and containers of the neat test material were used within 7 days of opening for the first time.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
other: Colchicine
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Details on test system and experimental conditions:
CULTURE OF LYMPHOCYTES: Human blood was collected aseptically from two healthy, non-smoking, adult (between 18-35 years of age) donors, pooled (in equal volumes from each donor) and diluted with HML media. As lymphocytes do not normally undergo cell division, they were stimulated to do so by the addition of phytohaemagglutinin (PHA), a naturally occurring mitogen. Cultures were established from the prepared (pooled) sample and dispensed as 5 mL aliquots (in sterile universal containers) so that each culture contained blood (0.4 mL), HML media (4.5 mL) and PHA solution (0.1 mL). All cultures were then incubated at 37 °C, and the cells were resuspended (twice daily) by gentle inversion.

METHOD OF APPLICATION: in medium
HML Media: RPMI 1640, supplemented with 10% fetal calf serum, 0.2 IU/mL sodium heparin, 20 IU/mL penicillin / 20 μg/mL streptomycin and 2.0 mM L-glutamine.

DURATION
- Exposure duration: 3 h (± S9) and 20 h continuous exposure (-S9) in preliminary toxicity test; 3 h (± S9) and 20 h continuous exposure (-S9) in main experiments
- Fixation time (start of exposure up to fixation or harvest of cells): 20 h

SPINDLE INHIBITOR (cytogenetic assays): Prior to the mitosis (after exposure of the test substance) the chemical cytochalasin B (6 μg/mL) was added to the cultures.

STAIN (for cytogenetic assays): Acridine orange solution (0.0125 mg/mL using purified water) for 4 minutes

NUMBER OF REPLICATIONS:
- Preliminary toxicity test: Single culture for test item and vehicle control
- Main test: Quadruplicate cultures for vehicle controls and duplicate cultures for test item and positive controls

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Harvesting and Fixation: The cells were harvested by centrifugation at 500 g for 5 minutes. The supernatant was removed and the cell pellet re-suspended and treated with a 4 mL hypotonic solution (0.075M KCl) at 37 °C, cultures were then incubated for 3 minutes at 37 °C to cause swelling. Cultures were agitated, 4 mL of ice-cold fixative (3:1 v/v methanol: acetic acid) was added slowly onto the culture surface and the cultures were slowly inverted to mix. The cultures were centrifuged at 500 g for five minutes. The supernatant was removed, and the cell pellet re-suspended. A further 4 mL of fresh fixative was then added and the cells stored at 4 °C until slide preparation.
Slide Preparation: The cultures were centrifuged at 500 g for 5 minutes and the supernatant removed. A homogeneous cell suspension was prepared. Pre-cleaned microscope slides were prepared for each culture by aliquoting the re-suspended cells onto the slides, and allowing the slides to air-dry. One slide was prepared from each culture. The remaining cell cultures were stored at approximately 4 °C until slide analysis was complete.
Slide Staining: The prepared slides were stained in acridine orange solution (0.0125 mg/mL using purified water). The prepared slides were examined by fluorescence microscopy.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):
Interphase cells were examined by fluorescence microscopy and the incidence of micronucleated cells per 1000 binucleate cells per culture were scored where possible.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity of test item in the lymphocyte cultures was determined using the cytokinesis-block proliferation index (CBPI index).
Cytotoxicity = 100-100{(CBPIT – 1)/(CBPIC –1)}
CBPI = [(No. mononucleate cells) + (2 x No. binucleate cells) + (3 x No. multinucleate cells)] / [Total number of cells]
T = test substance treatment culture
C = vehicle control culture
Thus, a CBPI of 1 (all cells are mononucleate) is equivalent to 100% cytotoxicity.
The highest concentration was intended to be that which caused a depression in the cytokinesis-block proliferative index (CBPI) equivalent to 55 ± 5% cytotoxicity (approximately) when compared with the concurrent vehicle control or, where no cytotoxicity was observed, the maximum concentration as recommended in the test guidelines or the limit of solubility.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
The analysis for micronucleated cells was based on the following criteria (Fenech and Morley 1985 and Fenech, 1993):
Cells were included in the analysis provided the cytoplasm remained essentially intact and any micronuclei present were separate in the cytoplasm or only just touching the main nucleus (not connected to the nucleus by a nucleoplasmic bridge). Micronuclei should lie in the same focal plane as the cell, and should possess a generally rounded shape with a clearly defined outline. The main nuclei of the binucleate cells scored for micronuclei should be of approximately equal size. The diameter of the micronucleus should be between 1/16 and 1/3 that of the main nucleus. The color of the micronuclei should be the same or lighter than the main nucleus. There should be no micronucleus-like debris in the surrounding area.
Evaluation criteria:
Providing that all of the acceptance criteria have been met, the test item was considered to be clearly positive if, in any of the experimental conditions examined:
- At least one of the test concentrations exhibits a statistically significant increase in the frequency of micronucleated cells compared with the concurrent negative control.
- The increase in the frequency of micronucleated cells is dose-related when evaluated with an appropriate trend test.
- Any of the results are outside the distribution of the historical negative control data.
If all of these criteria are met, the test item was considered able to induce chromosome breaks and/or gain or loss in the test system.
Providing that all of the acceptance criteria have been met, a negative response will be claimed if, in all of the experimental conditions examined:
- None of the test concentrations exhibits a statistically significant increase in the frequency of micronucleated cells compared with the concurrent negative control.
- There is no concentration-related increase when evaluated with an appropriate trend test.
- All results are inside the distribution of the historical negative control data.
If all of these criteria are met, the test item was considered unable to induce chromosome breaks and/or gain or loss in the test system.
Statistics:
The analysis assumed that the replicate was the experimental unit. An arcsine square-root transformation was used to transform the data. Neryl acetate treated groups were then compared to control using Williams’ tests (Williams 1971, 1972). Positive controls were compared to control using t-tests. Trend tests have also been carried out using linear contrasts by group number. These were repeated, removing the top dose group, until there were only 3 groups.
Statistical significance was declared at the 5% level for all tests.
Data were analyzed using SAS (SAS Institute 2002) and Quasar (Quasar 1.4.1 2016)
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No fluctuations in pH of the medium were observed at 1962.9 μg/mL of more than 1.0 unit compared with the vehicle control.
- Effects of osmolality: The osmolality of the test substance in medium was tested at 1962.9 μg/mL; no fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed compared with the vehicle control.
- Other confounding effects: None

PRELIMINARY TOXICITY TEST:
- In all treatment conditions the highest concentration tested was 1962.9 μg/mL (10 mM) and precipitate, assessed by eye at the end of treatment, was observed at 245.36 μg/mL and above. Cultures treated at 490.73 μg/mL and above were, therefore, discarded at the end of treatment.
- In the absence of S9 mix following 3 h treatment, a reduction in CBPI compared with vehicle control values, equivalent to 33.4% cytotoxicity, was obtained with test substance at 245.36 μg/mL. In the presence of S9 mix following 3 h treatment, there were no reductions in CBPI at any concentration analysed. In the absence of S9 mix following 20 h treatment, a reduction in CBPI compared with vehicle control values, equivalent to 73.1% cytotoxicity, was obtained with test substance at 122.68 μg/mL. The concentrations selected for the main test were based upon these data.

MAIN TEST:
3 h treatment in the absence of S9 mix
- Cytotoxicity: Precipitate was observed by eye at the end of treatment at 250 μg/mL. A reduction in CBPI compared with vehicle control values, equivalent to 51.2% cytotoxicity, was obtained with test substance at 250 μg/mL. Concentrations of test substance selected for micronucleus analysis were 60, 120 and 250 μg/mL.
3 h treatment in the presence of S9 mix
- Cytotoxicity: Precipitate was observed by eye at the end of treatment at 250 μg/mL. There were no reductions in CBPI at any concentration tested. Concentrations of test substance selected for micronucleus analysis were 60, 120 and 250 μg/mL.
20 h treatment in the absence of S9 mix
- Cytotoxicity: No precipitate was observed at the end of treatment. A reduction in CBPI compared with vehicle control values, equivalent to 56.3% cytotoxicity, was obtained with test substance at 80 μg/mL. Concentrations of test substance selected for micronucleus analysis were 30, 70 and 80 μg/mL.

NUMBER OF CELLS WITH MICRONUCLEI
- Test substance did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared with the vehicle controls.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation)
- Positive historical control data:
Binucleate Individual MN/1000 cells (3 h treatment in the absence of S9 mix): 16-67 (31.9 ± 10.6) - Mitomycin C; 15-43 (24.1 ± 6.5) - Colchicine
Binucleate Group MN (3 h treatment in the absence of S9 mix): 18-64.5 (31.9 ± 10.3) - Mitomycin C; 17.5-38 (24.1 ± 5.7) - Colchicine
Binucleate Individual MN/1000 cells (3 h treatment in the presence of S9 mix): 12-29 (18.6 ± 4.1) - Cyclophosphamide
Binucleate Group MN (3 h treatment in the presence of S9 mix): 13-27 (18.6 ± 3.8) - Cyclophosphamide

- Negative (solvent/vehicle) historical control data:
Binucleate Individual MN/1000 cells (3 h treatment in the absence of S9 mix): 1-12 (6.2 ± 2.4)
Binucleate Group MN (3 h treatment in the absence of S9 mix): 2.5-8.8 (6.2 ± 1.5)
Binucleate Individual MN/1000 cells (3 h treatment in the presence of S9 mix): 0-13 (6.2 ± 2.7)
Binucleate Group MN (3 h treatment in the presence of S9 mix): 2.5-10.5 (6.2 ± 2.0)

Table 7.6.1/1: Main test results

Treatment/Concentration

(μg/mL)

CBPI

Mean CBPI

Mean Cytotoxicity (%)

Binucleated cells containing micronuclei

per 1000 cells

Mean

p-valueb

Trend test p-valuec

3 h treatment in the absence of S9 mix

Vehiclea

1.94

1.93

0

8

7.5

-

-

1.95

7

1.93

8

1.90

7

15

1.90

1.91

2.7

NA

-

-

-

1.91

NA

30

1.88

1.91

2.7

NA

-

-

-

1.93

NA

60

1.95

1.95

-2.4

9

8.0

1.000

-

1.96

7

120

1.53

1.56

39.7

7

6.5

0.542

0.454

1.59

6

250

1.46

1.45

51.2

8

6.5

0.535

0.283

1.45

5

Mitomycin C 0.3

1.65

1.62

33.9

43

48.5

<0.001***

-

1.58

54

Colchicine 0.06

1.78

1.73

21.9

30

30.0

<0.001***

-

1.68

30

3 h treatment in the presence of S9 mix

Vehiclea

1.84

1.87

0

9

7.3

-

-

1.89

5

1.90

6

1.88

9

15

1.90

1.91

-3.6

NA

-

-

-

1.91

NA

30

1.90

1.92

-4.9

NA

-

-

-

1.93

NA

60

1.90

1.89

-1.3

5

6.5

1.000

-

1.87

8

120

1.92

1.91

-4.1

5

5.0

1.000

0.143

1.90

5

250

1.83

1.85

2.9

8

8.0

0.727

0.843

1.87

8

Cyclophosphamide 5

1.53

1.53

39.1

27

28.0

<0.001***

-

1.54

29

20 h treatment in the absence of S9 mix

Vehiclea

1.95

1.96

0

10

8.5

-

-

1.90

8

1.99

10

2.01

6

15

1.96

1.97

-0.7

NA

-

-

-

1.98

NA

30

1.97

1.95

1.4

7

8.0

0.809

-

1.93

9

60

1.80

1.78

18.7

NA

-

-

-

1.76

NA

70

1.67

1.69

27.7

6

7.5

0.701

0.588

1.72

9

80

1.40

1.42

56.3

5

7.5

0.684

0.503

1.44

10

90

1.37

1.30

69.2

NA

-

-

-

1.23

NA

100

1.22

1.18

81.3

NA

-

-

-

1.14

NA

110

1.06

1.05

94.5

NA

-

-

-

1.04

NA

120

1.02

1.02

97.8

NA

-

-

-

1.02

NA

Mitomycin C 0.1

1.76

1.74

22.7

29

30.0

<0.001***

-

1.73

31

Colchicine 0.015

1.63

1.59

38.6

18

17.0

0.004**

-

1.55

16

 

a. Vehicle control = DMSO (0.5% v/v)

b.p-values are for comparisons to control using Williams' test for test substance and the t-test otherwise

c. Trend testp-values are for the linear contrast including the control group and lower concentrations of the same compound

**p<0.01, ***p<0.001

CBPI: Cytokinesis block proliferative index

NA: Not analysed for micronucleus frequency

Conclusions:
The test substance did not show any evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes.
Executive summary:

In an in vitro micronucleus test performed according to Guideline OECD 487 and in compliance with GLP, cultured peripheral human lymphocytes were exposed to the test substance in the presence and absence of a metabolic activation system. Metabolic activation system used in this test was 10% (v/v) S9 fraction; S9 fraction was obtained from the liver homogenates of male Sprague-Dawley rats induced with phenobarbital and 5,6-benzoflavone.

Preliminary Toxicity Test

3 h treatment in the absence and presence of S9 mix: 3.83, 7.67, 15.34, 30.67, 61.34, 122.68, 245.36, 490.73, 981.45 and 1962.9 µg/mL

20 h treatment in the absence of S9 mix: 3.83, 7.67, 15.34, 30.67, 61.34, 122.68, 245.36, 490.73, 981.45 and 1962.9 µg/mL

Main Experiment

3 h treatment in the absence and presence of S9 mix: 15, 30, 60, 120 and 250 μg/mL

20 h treatment in the absence of S9 mix: 15, 30, 60, 70, 80, 90, 100, 110 and 120 μg/mL

Cytokinesis was blocked following mitosis using Cytochalasin B. Then the cells were harvested and slides prepared, so that binucleate cells could be examined for micronucleus induction.

 

In order to assess the cytotoxicity of the test substance to cultured human lymphocytes, the cytokinesis-block proliferative index (CBPI) was calculated for all cultures. Three test substance concentrations were assessed for determination of induction of micronuclei. The highest concentration selected was either that which caused a reduction in CBPI equivalent to 55 ± 5% cytotoxicity or that showing a visible precipitate at the end of the treatment period. Following 3 h treatment in the absence of S9 mix, reductions in CBPI equivalent to 51.2% cytotoxicity were obtained with test substance at 250 μg/mL. Concentrations of test substance selected for micronucleus analysis were 60, 120 and 250 μg/mL. Following 3 h treatment in the presence of S9 mix, test substance caused no reductions in CBPI at any concentration tested up to 250 μg/mL, where precipitate was observed at the end of treatment. Concentrations of test substance selected for micronucleus analysis were 60, 120 and 250 μg/mL. In the absence of S9 mix following 20 h treatment, a reduction in CBPI equivalent to 56.3% cytotoxicity was obtained with test substance at 80 μg/mL. Concentrations of test substance selected for micronucleus analysis were 30, 70 and 80 μg/mL.

 

In both the absence and presence of S9 mix, following 3 h treatment, and in the absence of S9 mix, following 20 h treatment, test substance did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared to the vehicle controls.

 

The positive control compounds (mitomycin C, colchicine and cyclophosphamide) caused statistically significant increases in the number of binucleate cells containing micronuclei under appropriate conditions, demonstrating the efficacy of the S9 mix and the sensitivity of the test system.

 

Therefore, the test substance did not show any evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes, in this in vitro test system under the experimental conditions described.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
July-August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
hemizygous hypoxanthine phosphoribosyl transferase (HPRT) gene
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Source: European Collection of Cell Cultures
- CHO-KI cells are functionally hemizygous at the HPRT locus.
- Type and identity of media: Ham’s Nutrient Mixture F12 medium
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: No; karyotype was assumed to be stable.
- Other details: Prior to exposure to test item, spontaneous mutants were eliminated from the stock cultures by incubating the cells in H10 containing 15 μg/mL hypoxanthine, 0.3 μg/mL amethopterin and 4 μg/mL thymidine for three days. All cell cultures were maintained at 37 °C in an atmosphere of 5 % CO2 in air.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction was prepared from liver homogenates of male Sprague Dawley rats treated with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
Preliminary toxicity test: 15.34, 30.67, 61.34, 122.68, 245.36, 490.73, 981.45 and 1962.9 μg/mL.
Mutation tests: The upper concentration levels were selected based on cytotoxicity.
-S9 mix Test (3 hours) 5, 25, 50, 75, 100, 125 and 150 μg/mL.
+S9 mix Test (3 hours) 5, 25, 75, 125 and 250 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Neryl acetate was found to be soluble at 392.58 mg/mL in dimethyl sulphoxide (DMSO).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
250 μg/mL; without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
Remarks:
5 μg/mL; with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: Ham’s Nutrient Mixture F12 medium
- Ham’s Nutrient Mixture F12, supplemented with 2 mM L-glutamine and 50 μg/mL gentamicin. The resulting medium is referred to as H0.
- H0 medium supplemented with 10 % HiFCS referred to as H10, is used for general cell culture, e.g. when growing cells up from frozen stocks.

DURATION
- Exposure duration: 3 h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7 days
- All incubations were performed at 37 °C in a humidified atmosphere of 5 % CO2 in air.

SELECTION AGENT (mutation assays): Selective medium, in which only HPRT deficient cells will grow, consisted of H10 supplemented with 6-thioguanine (6-TG) at a final concentration of 10 μg/mL.

NUMBER OF REPLICATIONS:
- Preliminary toxicity test: Single culture/dose for test item and 2 cultures for vehicle control
- Main test: , 2 cultures/dose for test item

NUMBER OF CELLS EVALUATED: 200 cells/plate were seeded for cloning efficiency and 10^6 cells were analyzed for mutant frequencies.

DETERMINATION OF CYTOTOXICITY
- Method: Cloning efficiency, Survival and Relative Survival
Cloning efficiency: Total no of colonies for each culture / (Number of plates scored for colony formation x 200)
Survival: Cloning efficiency x Cell count Correction Factor
Relative Survival (RS): (Individual survival value x100) / Mean control survival value
Following the expression period, three plates were scored for the presence of colonies from each culture and the CE was calculated.
Relative Cloning Efficiency (RCE): (Individual CE x100) / Mean control CE

OTHER:
Mutant Frequency (MF) per 10^6 viable cells for each set of plates was calculated as: (Total no. of mutant colonies x 5) / (CE x no. of uncontaminated plates)
Rationale for test conditions:
Mutation tests: The upper concentration levels were selected based on cytotoxicity.
Evaluation criteria:
The criteria for a positive response will be:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control
- the increase is concentration-related when evaluated with an appropriate trend test
- any of the results are outside the distribution of the historical vehicle control data

The criteria for a negative response will be:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control
- there is no concentration-related increase when evaluated with an appropriate trend test
- all results are inside the distribution of the historical vehicle control data.
Statistics:
The statistical significance of the data was analysed by weighted analysis of variance, weighting assuming a Poisson distribution following the methods described by Arlett et al. (1989). Tests were conducted for a linear concentration-response relationship of the test substance, for non-linearity and for the comparison of positive control and treated groups to solvent control. Data was analysed using SAS (SAS Institute Inc., 2002).
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No fluctuations in pH of the medium of more than 1.0 unit compared with the vehicle control were observed at 1962.9 μg/mL.
- Effects of osmolality: No fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed when compared with the vehicle control at 1962.9 µg/mL.

PRELIMINARY TOXICITY TEST:
- Neryl acetate was initially dosed at concentrations up to 1962.9 µg/mL (10 mM). Precipitate was observed by eye at the end of treatment at concentrations of 245.36 µg/mL and above and this was, therefore, the highest concentration plated for determination of relative survival (RS) in both the absence and presence of S9 mix. Exposure to Neryl acetate for 3 hours at concentrations from 15.34 to 245.36 µg/mL in both the absence and presence of S9 mix resulted in RS values from 87 to 1% and from 103 to 40%, respectively.
Concentrations for the main test were based upon these data and toxicity was the primary determinant for the dose selection.

MAIN MUTATION TEST 1
3-hour treatment in the absence of S9 mix: Cultures were exposed to Neryl acetate at concentrations from 5 to 150 µg/mL. No precipitate was seen by eye at the end of treatment. Exposure to Neryl acetate resulted in RS values from 109 to 4%. All cultures were plated out for determination of cloning efficiency and mutant frequency. Cultures treated at 150 µg/mL were not analysed for mutant frequency as RS was below 10% at this concentration. No significant increases in mutant frequency were observed after exposure to Neryl acetate at any concentration analysed.
3-hour treatment in the presence of S9 mix: Cultures were exposed to Neryl acetate at concentrations from 5 to 250 µg/mL. Precipitate was seen by eye at the end of treatment at 250 µg/mL. Exposure to Neryl acetate resulted in RS values from 113 to 62%. All cultures were plated out for determination of cloning efficiency and mutant frequency and no significant increases in mutant frequency were observed at any concentration analysed.

Table 7.6.1/1: Summary results

Main Test: 3-hour treatment in the absence of S9 mix

Day 1 relative survival

Day 8 cloning efficiency

mutant frequency

Concn.of test item   (µg/mL)

Cell Count Day 1 (x106/mL)

No. of colonies on plate

Total no. of Colonies

Cloning Efficiency (%)

Adjusted Cloning Efficiency

(%)

RS (%)

Mean RS (%)

No. of colonies on plate

Total no. of Colonies

Cloning Efficiency in non‑selective medium (%)

No. of colonies on plate

Total no. of Colonies

Cloning Efficiency in selective medium (%)

Mutant Frequencya

Mean Mutant Frequencya

Plate 1

Plate 2

Plate 3

Plate 1

Plate 2

Plate 3

Plate 1

Plate 2

Plate 3

Plate 4

Plate 5

 

 

0b

 

1.48

125

153

163

441

74

93

100

100

129

133

158

420

70

0

0

1

0

0

1

0.00004

0.57

2.82

1.49

147

133

131

411

142

137

127

406

68

6

2

0

1

2

11

0.00044

6.50

1.50

168

138

165

471

145

139

161

445

74

0

1

0

0

0

1

0.00004

0.54

1.51

151

146

151

448

126

138

128

392

65

0

1

2

2

1

6

0.00024

3.67

5

1.43

135

134

136

405

68

82

88

85

123

143

137

403

67

0

3

1

0

1

5

0.00020

2.98

1.75

1.38

113

164

120

397

66

77

83

186

128

150

464

77

0

0

0

1

0

1

0.00004

0.52

25

1.57

175

164

169

508

85

112

120

109

150

136

147

433

72

0

0

0

0

0

0

0.00000

0.00

2.01

1.42

143

159

152

454

76

91

97

129

127

161

417

70

2

0

1

2

2

7

0.00028

4.03

50

1.41

126

157

170

453

76

90

96

89

105

121

105

331

55

1

0

2

0

0

3

0.00012

2.18

2.37

1.61

112

119

107

338

56

76

82

125

111

138

374

62

0

3

1

0

0

4

0.00016

2.57

75

1.38

161

131

128

420

70

81

87

86

145

121

137

403

67

0

1

0

0

0

1

0.00004

0.60

0.87

1.40

156

128

122

406

68

80

85

131

138

151

420

70

0

0

0

2

0

2

0.00008

1.14

100

1.14

120

121

89

330

55

53

57

48

114

117

113

344

57

0

0

0

0

1

1

0.00004

0.70

0.62

0.93

92

85

97

274

46

36

38

135

150

160

445

74

1

0

0

0

0

1

0.00004

0.54

125

0.58

58

50

51

159

27

13

14

14

 

113

136

132

381

64

1

0

0

2

0

3

0.00012

1.89

1.32

1.61

0.64

57

44

50

151

25

14

15

106

143

114

363

61

1

0

1

0

0

2

0.00008

150

0.41

17

19

14

50

8

3

3

4

Culture not analysed as day 1 relative survival <10%

Culture not analysed as day 1 relative survival <10%

 

 

0.50

27

19

18

64

11

4

5

EMS – positive control

250

1.41

129

114

130

373

62

74

79

75

127

130

122

379

63

19

20

16

19

19

93

0.00372

58.89

84.24

1.38

120

121

99

340

57

66

71

90

86

89

265

44

42

19

24

18

18

121

0.00484

109.58

**

a. Mutant frequencies expressed per 106viable cells

b. Vehicle control = DMSO 1% (v/v)

**p<0.01, statistically significant increase over concurrent vehicle control mutant frequency

EMS: Ethyl methanesulphonate

Main Test: 3-hour treatment in the presence of S9 mix

Day 1 relative survival

Day 8 cloning efficiency

mutant frequency

Concn.of test item   (µg/mL)

Cell Count Day 1 (x106/mL)

No. of colonies on plate

Total no. of Colonies

Cloning Efficiency (%)

Adjusted Cloning Efficiency

(%)

RS (%)

Mean RS (%)

No. of colonies on plate

Total no. of Colonies

Cloning Efficiency in non‑selective medium (%)

No. of colonies on plate

Total no. of Colonies

Cloning Efficiency in selective medium (%)

Mutant Frequencya

Mean Mutant Frequencya

Plate 1

Plate 2

Plate 3

Plate 1

Plate 2

Plate 3

Plate 1

Plate 2

Plate 3

Plate 4

Plate 5

 

 

0b

 

1.58

189

167

139

495

80

86

100

100

163

136

138

437

73

1

0

2

1

2

6

0.00024

3.30

3.60

1.50

167

160

146

473

147

142

130

419

70

1

1

4

3

2

11

0.00044

6.30

1.38

183

131

140

454

147

130

134

411

69

0

0

0

2

0

2

0.00008

1.17

1.38

165

176

153

494

120

137

138

395

66

2

2

1

0

1

6

0.00024

3.65

5

1.67

143

166

129

438

73

90

105

113

124

110

129

363

61

1

1

3

2

3

10

0.00040

6.61

3.99

1.84

159

164

137

460

77

104

121

118

103

131

352

59

1

0

1

0

0

2

0.00008

1.36

25

1.74

145

148

131

424

71

90

105

111

152

131

141

424

71

4

0

0

2

4

10

0.00040

5.66

4.52

1.86

159

154

123

436

73

99

116

129

138

160

427

71

0

2

1

1

2

6

0.00024

3.37

75

1.26

130

123

124

377

63

58

68

69

114

117

130

361

60

1

1

0

0

0

2

0.00008

1.33

1.44

1.25

138

131

129

398

66

61

71

140

156

170

466

78

0

0

0

2

1

3

0.00012

1.55

125

1.28

155

129

128

412

69

64

75

69

126

121

85

332

55

4

0

0

0

0

4

0.00016

2.89

1.84

2.36

1.02

175

129

122

426

71

53

62

150

133

109

392

65

0

0

0

1

2

3

0.00012

250c

1.31

108

119

113

340

57

55

64

62

132

127

127

386

64

3

1

2

0

1

7

0.00028

4.35

2.08

3.22

1.30

113

108

109

330

55

52

61

145

142

175

462

77

0

0

1

1

2

4

0.00016

3MC – positive control

5

1.68

145

136

140

421

70

87

101

107

162

122

133

417

70

23

23

23

19

19

107

0.00428

61.58

63.35

1.55

199

151

161

511

85

97

113

121

132

145

398

66

21

19

28

19

21

108

0.00432

65.13

***

a. Mutant frequencies expressed per 106viable cells

b. Vehicle control = DMSO 1% (v/v)

c. Precipitate observed by eye at the end of treatment

***p<0.001, statistically significant increase over concurrent vehicle control mutant frequency

3MC: 3-Methylcholanthrene

Conclusions:
Neryl acetate did not demonstrate mutagenic potential in this in vitro HPRT cell mutation assay.
Executive summary:

In an in vitro mammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP, Chinese hamster Ovary (CHO-K1) cells were exposed to the test item for 3 hours, with and without metabolic activation (S9 [v/v] fraction of male Sprague Dawley rats liver induced with phenobarbital and 5,6-benzoflavone), at the concentrations below. 

Preliminary toxicity test: 15.34, 30.67, 61.34, 122.68, 245.36, 490.73, 981.45 and 1962.9 μg/mL

Mutation tests:

-S9 mix Test 1 (3 hours) 5, 25, 50, 75, 100, 125 and 150 μg/mL

+S9 mix Test 1 (3 hours) 5, 25, 75, 125 and 250 μg/mL

In the main mutation test in the absence of S9 mix, cells were exposed to neryl acetate at concentrations from 5 to 150 µg/mL. No precipitate was observed by eye at the end of treatment. RS values ranged from 109 to 4% when compared with the vehicle control. Neryl acetate did not induce any statistically significant increases in mutant frequency at concentrations up to 125 µg/mL where RS was reduced to 14%. The positive control substance, ethyl methanesulphonate, induced a significant increase in mutant frequency.

In the main mutation test in the presence of S9 mix, cells were exposed to neryl acetate at concentrations from 5 to 250 µg/mL of neryl acetate. Precipitate, observed by eye at the end of treatment, was seen at 250 µg/mL. Day 1 survival values ranged from 113 to 62% when compared with the vehicle control. Neryl acetate did not induce any statistically significant increases in mutant frequency at concentrations up to 250 µg/mL. The positive control substance, 3‑methylcholanthrene, induced a significant increase in mutant frequency.

Therefore, neryl acetate did not demonstrate mutagenic potential in this in vitro HPRT cell mutation assay, under the experimental conditions described.


Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

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.

In an in vitro micronucleus test performed according to Guideline OECD 487 and in compliance with GLP, cultured peripheral human lymphocytes were exposed to the test substance in the presence and absence of a metabolic activation system. Metabolic activation system used in this test was 10% (v/v) S9 fraction; S9 fraction was obtained from the liver homogenates of male Sprague-Dawley rats induced with phenobarbital and 5,6-benzoflavone.

Preliminary Toxicity Test

3 h treatment in the absence and presence of S9 mix: 3.83, 7.67, 15.34, 30.67, 61.34, 122.68, 245.36, 490.73, 981.45 and 1962.9 µg/mL

20 h treatment in the absence of S9 mix: 3.83, 7.67, 15.34, 30.67, 61.34, 122.68, 245.36, 490.73, 981.45 and 1962.9 µg/mL

Main Experiment

3 h treatment in the absence and presence of S9 mix: 15, 30, 60, 120 and 250 μg/mL

20 h treatment in the absence of S9 mix: 15, 30, 60, 70, 80, 90, 100, 110 and 120 μg/mL

Cytokinesis was blocked following mitosis using Cytochalasin B. Then the cells were harvested and slides prepared, so that binucleate cells could be examined for micronucleus induction.

In order to assess the cytotoxicity of the test substance to cultured human lymphocytes, the cytokinesis-block proliferative index (CBPI) was calculated for all cultures. Three test substance concentrations were assessed for determination of induction of micronuclei. The highest concentration selected was either that which caused a reduction in CBPI equivalent to 55 ± 5% cytotoxicity or that showing a visible precipitate at the end of the treatment period. Following 3 h treatment in the absence of S9 mix, reductions in CBPI equivalent to 51.2% cytotoxicity were obtained with test substance at 250 μg/mL. Concentrations of test substance selected for micronucleus analysis were 60, 120 and 250 μg/mL. Following 3 h treatment in the presence of S9 mix, test substance caused no reductions in CBPI at any concentration tested up to 250 μg/mL, where precipitate was observed at the end of treatment. Concentrations of test substance selected for micronucleus analysis were 60, 120 and 250 μg/mL. In the absence of S9 mix following 20 h treatment, a reduction in CBPI equivalent to 56.3% cytotoxicity was obtained with test substance at 80 μg/mL. Concentrations of test substance selected for micronucleus analysis were 30, 70 and 80 μg/mL.

In both the absence and presence of S9 mix, following 3 h treatment, and in the absence of S9 mix, following 20 h treatment, test substance did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared to the vehicle controls.

The positive control compounds (mitomycin C, colchicine and cyclophosphamide) caused statistically significant increases in the number of binucleate cells containing micronuclei under appropriate conditions, demonstrating the efficacy of the S9 mix and the sensitivity of the test system.

In an in vitro mammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP, Chinese hamster Ovary (CHO-K1) cells were exposed to the test item for 3 hours, with and without metabolic activation (S9 [v/v] fraction of male Sprague Dawley rats liver induced with phenobarbital and 5,6-benzoflavone), at the concentrations below. 

Preliminary toxicity test:15.34, 30.67, 61.34, 122.68, 245.36, 490.73, 981.45 and 1962.9 μg/mL

Mutation tests:

-S9 mix Test 1 (3 hours)5,25,50,75,100,125 and 150  μg/mL

+S9 mix Test 1 (3 hours)5,25,75,125 and 250  μg/mL

In the main mutation test in the absence of S9 mix, cells were exposed to neryl acetate at concentrations from 5 to 150 µg/mL. No precipitate was observed by eye at the end of treatment. RS values ranged from 109 to 4% when compared with the vehicle control. Neryl acetate did not induce any statistically significant increases in mutant frequency at concentrations up to 125 µg/mL where RS was reduced to 14%. The positive control substance, ethyl methanesulphonate, induced a significant increase in mutant frequency.

In the main mutation test in the presence of S9 mix, cells were exposed to neryl acetate at concentrations from 5 to 250 µg/mL of neryl acetate. Precipitate, observed by eye at the end of treatment, was seen at 250 µg/mL. Day 1 survival values ranged from 113 to 62% when compared with the vehicle control. Neryl acetate did not induce any statistically significant increases in mutant frequency at concentrations up to 250 µg/mL. The positive control substance, 3‑methylcholanthrene, induced a significant increase in mutant frequency.

Justification for classification or non-classification

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

Neryl acetate did not show any evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes.

Neryl acetate did not demonstrate mutagenic potential in an in vitro HPRT cell mutation assay.

Therefore, the substance is not classified according to CLP (Regulation (EC) No 1272/2008) and GHS regulations.

As the results obtained with neryl acetate were negative in an Ames test, in a micronuclei test and in a HPRT cell mutation assay, the test item is not classified according to CLP Regulation (EC) No 1272/2008 and to GHS Regulation.