Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene

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:

24 April 2012 - 22 June 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Test guideline 3

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

The major Japanese Regulatory Authorities including METI, MHLW and MAFF

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: The USA EPA (TSCA) OPPTS harmonised guidelines

GLP compliance:

yes (incl. QA statement)

Remarks:

No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. This exception is considered not to affect the purpose or integrity of the study.

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(Z)-3,7-dimethylocta-1,3,6,-triene

EC Number:

222-081-3

EC Name:

(Z)-3,7-dimethylocta-1,3,6,-triene

Cas Number:

3338-55-4

Molecular formula:

C10H16

IUPAC Name:

3,7-dimethylocta-1,3,6-triene

Constituent 2

Reference substance name:

Dipentene

EC Number:

205-341-0

EC Name:

Dipentene

Cas Number:

138-86-3

Molecular formula:

C10H16

IUPAC Name:

1-methyl-4-(prop-1-en-2-yl)cyclohexene

impurity 1

Reference substance name:

2,6-dimethylocta-2,4,6-triene

EC Number:

211-614-5

EC Name:

2,6-dimethylocta-2,4,6-triene

Cas Number:

673-84-7

Molecular formula:

C10H16

IUPAC Name:

2,6-dimethylocta-2,4,6-triene

impurity 2

Reference substance name:

(E)-3,7-dimethylocta-1,3,6-triene

EC Number:

223-241-5

EC Name:

(E)-3,7-dimethylocta-1,3,6-triene

Cas Number:

3779-61-1

Molecular formula:

C10H16

IUPAC Name:

3,7-dimethylocta-1,3,6-triene

Test material form:

liquid

Details on test material:

Name: [Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene]
Public name: Ocimene PQ
EC numbers: 933-779-9 (pre-registration 222-081-3)
CAS numbers: n/a (pre-registration 3338-55-4)
Batch/Lot number: A170524D
Description: Colourless liquid
Expiry date: 06 June 2019
Purity: Considered as 100%

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Sponsor's identification: Ocimene PQ
- Description: colourless liquid
- Composition: 73.1% Cis-beta-Ocimene 73.1%; Dipentene 21%
- Batch No.of test material: A120304A
- Expiration date of the lot/batch: 04 March 2014

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark
- Stability under test conditions: all formulations were used within four hours of preparation and were assumed to be stable fo rthis period

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- the test material was accurately weighed and approximate half-log dilutions were prepared in acetone by mixing on a vortex mixer on the day of each experiment
- formulated concentrations were adjusted to allow for the stated water/impurity content (26.9%) of the test substance
- prior to use, the solvent was dried to remove water, using molecular sieves (2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x10^-4 microns)

Method

Target gene:

Histidine or tryptophan locus

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-mix

Test concentrations with justification for top dose:

Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.

Mutation test 1:
- Salmonella strains TA98 and TA1537 (absence and presence of S9-mix): 5, 15, 50, 150, 500, 1500, 5000 µg/plate.
- Salmonella strains TA1535 and TA100 (absence and presence of S9-mix): 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate.
- Escherichia coli strain WP2uvrA (absence and presence of S9-mix): 50, 150, 500, 1500, 5000 µg/plate.

Mutation test 2:
- Salmonella strains TA1537 and TA98 (absence of S9-mix): 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate
- Salmonella strains TA1535 and TA100 (absence and presence of S9-mix): 0.05, 0.15, 0.5, 1.5, 5, 15, 50 µg/plate
- Salmonella strain TA1537 (presence of S9-mix): 1.5, 5, 15, 50, 150, 500, 1500 µg/plate
- Salmonella strain TA98 (presence of S9-mix): 5, 15, 50, 150, 500, 1500, 5000 µg/plate
- Escherichia coli strain WP2uvrA (absence and presence of S9-mix): 15, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test substance was immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/mL, but was fully miscible in acetone at 100 mg/mL in solubility checks performed in-house.

Controls

Untreated negative controls:

yes

Remarks:

2-Aminoanthracene; Benzo(a)pyrene

Negative solvent / vehicle controls:

yes

Remarks:

Vehicle control

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth and incubated at 37°C for approximately 10 hours. The cultures were monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.
- Exposure duration: approximately 48-hour incubation at 37°C

MEASUREMENTS
- After the incubation period, the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.

PRELIMINARY TOXICITY TEST
- A preliminary test was conducted in order to select appropriate dose levels for use in the main test.
- 0.1 mL bacterial culture (TA100 or WP2uvrA), 2 mL of molten, trace histidine or tryptophan supplemented top agar, 0.1 mL of test substance formulation and 0.5 mL S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30mL/plate).
- 10 concentrations of the test substance formulation and a vehicle control (acetone) were tested.
- 0.1 mL of the maximum concentration of the test substance and 2 mL molten trace histidine or tryptophan supplemented top agar were overlaid onto a sterile nutrient agar plate in order to assess the sterility of the test substance.

MUTATION TEST 1
- 0.1 mL of one of the bacterial cultures were dispensed into sets of test tubes with 2mL molten, trace histidine or tryptophane supplemented top agar, 0.1 mL vehicle, test material formulation or postiive control and 0.5 mL of S9-mix or phosphate buffer.
- The test tubes were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate)
- The procedure was repeated in triplicate for each bacterial strain and for each concentration of test material both with and without S9-mix.

MUTATION TEST 2
- Performed using fresh bacterial cultures, test substance and control solutions.
- Based on results from Experiment 1 and the change in test methodology led to a change in the test substance dose range.

CONTROLS
1) Vehicle control - solvent treatment group

2) Positive controls - used in a series of plates without S9-mix:
- N-ethyl-N'-nitro-N-nitrosguanidine (ENNG): 2µg/plate for WP2uvrA
- N-ethyl-N'-nitro-N-nitrosguanidine (ENNG): 3µg/plate for TA100
- N-ethyl-N'-nitro-N-nitrosguanidine (ENNG): 5µg/plate for TA1535
- 9-Aminoacridine (9AA): 80 µg/plate for TA1537

3) Negative controls - non-mutagenic substances in the absence of metabolising enzymes were used in the series of plates with S9-mix:
- 2-Aminoanthracene (2AA): 2 µg/plate for TA100
- 2-Aminoanthracene (2AA): 2 µg/plate for TA1535 and TA1537
- 2-Aminoanthracene (2AA): 10 µg/plate for WP2uvrA
- Benzo(a)pyrene (BP): 5µg/plate for TA98

Evaluation criteria:

Any one or all of the following can be used to determine the overall result of the study:
- A dose-related increase in mutant frequency over the dose range tested.
- A reproducible increase at one or more concentrations.
- Biological relevance against in-house historical control ranges.
- Statistical analysis of data as determined by UKEMS.
- Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).

A test substance will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Results will be reported as equivocal when data generated will prohibit making a definite judgement about test substance activity.

Statistics:

Not specified

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

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary test:
- The test substance was toxic to TA100 at 5000 µg/plate and non-toxic to WP2uvrA.
- The test substance formulation and S9-mix used in this experiment were both shown to be sterile.

Controls:
- The results for the negative controls (spontaneous mutation rates) were considered to be acceptable.
- All positive control chemicals induced marked increases in the frequency of revertant colonies thus confirming the activity of the S0-mix and the sensitivity of the bacterial strains.

Mutation test 1:
- The test substance (from the 15 µg/plate) caused a visible reduction in the growth of the Salmonella bacterial background lawns (except for TA98 in the presence of the S9-mix)
- No toxicity was seen to E.coli WP2uvrA

Mutation test 2:
- The test substance caused a slightly stronger toxic response with weakened bacterial background lawns initially from 5µg/plate in the absence of S9-mix and 15 µg/plate in the presence of the S9-mix.
- Weakened lawns in the E.coli WP2uvrA strain were noted at 1500 µg/plate only in the absence of metabolic activation.

Acceptance criteria met: yes

Any other information on results incl. tables

Main experiment

- The master strains were checked prior to use for characteristics, viability and spontaneous reversion rate, which were all found to be satisfactory.

- The amino acid supplemented top agar and S9 -mix were shown to be sterile.

- The culture density for each bacterial strain was checked and considered to be acceptable.

- The sensitivity of the bacterial tester strains to the toxicity of the test substance varied slightly between strain type, the presence and absence of metabolic activation and the experimental methodology.

- The test substance was tested up to the toxic limit or maximum recommended dose level of 5000 µg/plate depending on the bacterial strain type, the presence or absence of the S9 -mix and the experimental methodology.

- A test substance precipitate (oily in appearance) was seen under an inverted microscope on the 5000 µg/plate, but it did not prevent the scoring of the revertant colonies.

Acceptance criteria - the reverse mutation assay may be considered valid if the following criteria are met:

- All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks.

- All tester strain cultures should show a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.

- All tester strain cultures should be in the range of 0.9-9 x10^9 bacterial per mL.

- Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control cehmicals used in the study should induce marked increases in the frequency of revertant colonise, both in the presence and absence of metabolic activation.

- There should be a minimum of four non-toxic test substance dose levels.

- There should be no evidence of excessive contamination.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study and based on the results, the test substance, Ocimene PQ, was considered to be non-mutagenic to Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and E.coli strain WP2uvrA in the presence and absence of metabolic activation (S9-mix).

Executive summary:

This study was conducted to evaluate the ability of the test substance, Ocimene PQ, to induce reverse mutations, either directly or after metabolic activation, at the histidine or tryptopjhan locus in the genome of five strains of bacteria. The study was performed in accordance with OECD 471, EU Method B.13/14, the major Japanese Regulatory Authorities including METI, MHLW and MAFF, and the USA EPA (TSCA) OPPTS harmonised guidelines.

The bacterial strains employed in this study were Salmonella typhimurium TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA. The bacterial strains wree treated with the test substance using the Ames plate incorporation (Mutation test 1) and pre-incubation (Mutation test 2) methods at up to seven dose levels, tested in triplicate with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). A preliminary toxicity assay was initially performed in order to select appropriate dose levels for the main experiment. The test ubstance was found to be toxic to S. typhimuriumTA100 at the 5000 µg/plate and non-toxic to E.coli WP2uvrA.

The dose range for Mutation test 1 was determined by the preliminary test and ranged from 0.15 -5000 µg/plate, depending on the bacterial strain and the presence or absence of the S9 -mix. Mutation test 2 was repeated on a separate dat (pre-incubation), using an amended dose range of 0.05 -5000 µg/plate, fresh cultures of bacterial strains and fresh test substance preparations. Additional dose levels and an expanded dose range were selected where applicable, in order to achieve the four non-toxic dose levels and the toxic limit of the test substance.

In Mutation test 1, the test substance caused a visible reduction in the growth of the Salmonella bacterial background lawns from the 15 µg/plate (except for TA98 in the presence of the S9-mix), but no toxicity was seen to E.coli WP2uvrA. Mutation test 2, the test substance caused a slightly stronger toxic response with weakened bacterial background lawns initially from 5µg/plate in the absence of S9-mix and 15 µg/plate in the presence of the S9-mix. Weakened lawns in the E.coli WP2uvrA strain were noted at 1500 µg/plate only in the absence of metabolic activation. The sensitivity of the bacterial tester strains to the toxicity of the test substance varied slightly between strain type, the presence and absence of metabolic activation and the experimental methodology. The test substance was tested up to the toxic limit or maximum recommended dose level of 5000 µg/plate depending on the bacterial strain type, the presence or absence of the S9 -mix and the experimental methodology. There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains with any dose of the test material, either in the presence or absence of the S9 -mix or in either exposure method employed.

There was a test substance precipitate (oily in appearance) seen under an inverted microscope on the 5000 µg/plate, but it did not prevent the scoring of the revertant colonies.The vehicle (acetone) control plates gave revertant colony counts within the normal range and all positive chemicals induced marked increases in the frequency of revertant colonies, both in the presence and absence of metabolic activation. The acceptance criteria were met and therefore the sensitivity of the assay and effiicacy of the S9 -mix were validated.

Under the conditions of the study and based on the results, it can be conluded that the test substance, Ocimene PQ, was considered to be non-mutagenic to Salmonella strains TA98, TA100, TA1535 and TA1537 and E.coli strain WP2uvrA in the presence and absence of metabolic activation (S9-mix).