Reaction mass of (R*,R*)-7-methoxy-3,7-dimethyl-2-octanol and (R*,S*)-7-methoxy-3,7-dimethyl-2-octanol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 August - 26 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Test guideline 3

Qualifier:

according to guideline

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. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(R*,R*)-7-methoxy-3,7-dimethyl-2-octanol

Cas Number:

87605-57-0

Molecular formula:

C11H24O2

IUPAC Name:

(R*,R*)-7-methoxy-3,7-dimethyl-2-octanol

Constituent 2

Reference substance name:

(R*,S*)-7-methoxy-3,7-dimethyl-2-octanol

Cas Number:

87605-61-6

Molecular formula:

C11H24O2

IUPAC Name:

(R*,S*)-7-methoxy-3,7-dimethyl-2-octanol

impurity 1

Reference substance name:

Non identified impurities

Molecular formula:

Not applicable

IUPAC Name:

Non identified impurities

Test material form:

liquid

Details on test material:

Batch No.: 171894
Purity: 99.7% (sum of the two main constituents)
Name of test material (as cited in study report): 7-METHOXY-3,7-DIMETHYLOCTAN-2-OL MULTICONSTITUENT
Physical state: colourless - slightly yellow liquid
Storage conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry date: 14 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

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
The solubility of 7-methoxy-3,7-dimethyloctan-2-ol multiconstituent 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.

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 results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

pre-incubation method: at 5000 µg/plate with and without S9

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

pre-incubation method: at 5000 µg/plate with and without S9

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

pre-incubation method: at 1500 µg/plate and above without S9 and at 5000 µg/plate with S9

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

pre-incubation method: at 1500 µg/plate and above without S9 and at 5000 µg/plate with S9

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

First Test
No evidence of toxicity was obtained following exposure to 7-methoxy-3,7-dimethyloctan-2-ol multiconstituent in either the absence or presence of S9 mix. No precipitate was observed on any plates containing 7-methoxy-3,7-dimethyloctan-2-ol multiconstituent. A maximum exposure concentration of 5000 µg/plate was, therefore, selected for use in the second test.
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to 7-methoxy-3,7-dimethyloctan-2-ol multiconstituent at any concentration up to and including 5000 µg/plate in either the presence or absence of S9 mix.

Second Test
Toxicity, observed as a thin or absent background lawn of non-revertant colonies and/or a reduction in the number of revertant colonies, was seen in strains TA100, TA1537 and WP2 uvrA (pKM101) at 1500 µg/plate and above and in strains TA98 and TA1535 at 5000 µg/plate in the absence of S9 mix. In the presence of S9 mix, toxicity, observed as a thinning of the background lawn of non-revertant colonies and/or a reduction in revertant colony numbers, was seen in all strains at 5000 µg/plate. No precipitate was observed on any plates containing 7-methoxy-3,7-dimethyloctan-2-ol multiconstituent.
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to 7-methoxy-3,7-dimethyloctan-2-ol multiconstituent at any concentration up to and including 5000 µg/plate in either the presence or absence of S9 mix.

Applicant's summary and conclusion

Conclusions:

7-methoxy-3,7-dimethyloctan-2-ol multiconstituent showed no evidence of mutagenic activity in this bacterial system [strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100) and Escherichia coli, strain WP2 uvrA (pKM101)] at concentrations up to 5000 µg/plate.

Executive summary:

In an in vitro bacterial reverse mutation test performed according to Guideline OECD 471 and in compliance with GLP, strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100) and Escherichia coli, strain WP2 uvrA (pKM101), were exposed to 7-methoxy-3,7-dimethyloctan-2-ol multiconstituent 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 concentrations of test item were used: 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, with and without S9-mix, in experiment 1 and 2.

In the first test, no evidence of toxicity towards the tester strains was observed following exposure to 7-methoxy-3,7-dimethyloctan-2-ol multiconstituent in either the absence or presence of S9 mix.

In the second test, toxicity (observed as a thinning of the background lawn of non-revertant colonies and/or a reduction in revertant colony numbers) was seen in strains TA100, TA1537 and WP2uvrA (pKM101) at 1500 µg/plate and above and in strains TA98 and TA1535 at 5000 µg/plate in the absence of S9 mix.In the presence of S9 mix, toxicity (observed as a thinning of the background lawn of non-revertant colonies and/or a reduction in revertant colony numbers) was seen in all strains at 5000 µg/plate.

No evidence of mutagenic activity was seen at any concentration of 7-methoxy-3,7-dimethyloctan-2-ol multiconstituent in either 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, it was concluded that 7-methoxy-3,7-dimethyloctan-2-ol multiconstituent showed no evidence of mutagenic activity in this bacterial system at concentrations up to 5000 µg/plate.