Oligomerisation products of alpha-pinene and beta-pinene

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:

27 August 2014 - 06 March 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 471 and in compliance with GLP.

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:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OJ L 142/248

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EPA 712-C-98-247

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK Department of Health (inspected on 01 - 03 July 2014 / signed on 16 September 2014)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

alpha-pinene and beta-pinene dimers

IUPAC Name:

alpha-pinene and beta-pinene dimers

Constituent 2

Reference substance name:

alpha-pinene and beta-pinene trimers

IUPAC Name:

alpha-pinene and beta-pinene trimers

Constituent 3

Reference substance name:

alpha-pinene and beta-pinene tetramers

IUPAC Name:

alpha-pinene and beta-pinene tetramers

Test material form:

liquid: viscous

Details on test material:

Batch No. 14-02 SI
Purity: 100% (UVCB substance)
Name of the test item (as cited in the study report): TERPENIC OLIGOMERS
Physical state: amber viscous liquid
Storage Conditions: +2°C to +8°C, under nitrogen and protected from light
Reassay date: 14 November 2014

Method

Target gene:

S. typhimurium: Histidine gene
E. coli: Tryptophan gene

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

No

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix (Test 1: 10% v/v and Test 2: 20% v/v)

Test concentrations with justification for top dose:

First Test: 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Second test: 50, 150, 500, 1500 and 5000 µg/plate

The highest dose, 5000 µg/plate, is the standard limit concentration recommended in the regulatory guidelines that this assay follows.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The solubility of Terpenic Oligomers was assessed at 50 mg/mL in water, dimethylsulphoxide (DMSO), acetone and ethanol. It was found to be insoluble in water and DMSO, but dissolved in acetone and ethanol. Acetone (analytical reagent grade) was used as the vehicle for this study.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without S9 mix, 2 µg/plate in DMSO for strains TA100 and TA1535

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without S9 mix, 50 µg/plate in DMSO for strain TA1537

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Without S9 mix, 2 µg/plate in DMSO for strain TA98

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without S9 mix, 2 µg/plate in DMSO for strain WP2uvrA (pKM101)

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With S9 mix, 5 µg/plate in DMSO for strains TA100 and TA1535

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With S9 mix, 10 µg/plate in DMSO for strain WP2uvrA (pKM101)

Controls 7

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9 mix, 5 µg/plate in DMSO for strains TA98 and TA1537

Details on test system and experimental conditions:

TEST SYSTEM: The strains of S. typhimurium and E. coli were obtained from Moltox Inc. Batches of the strains were stored at ca -80°C as aliquots of nutrient broth cultures. Dimethylsulphoxide (DMSO) was added to the cultures at 8% v/v as a cryopreservative. Each batch of frozen strain was tested for amino acid requirement and, where applicable, for cell membrane permeability (rfa mutation), sensitivity to UV light, and the pKM101 plasmid, which confers resistance to ampicillin. The responses of the strains to a series of reference mutagens were also assessed.

METHOD OF APPLICATION: plate incorporation in agar (two independent tests)
- Cell density at seeding (if applicable): at least 10^9 per mL

DURATION :
- Preincubation period: 10 hours at 37°C with shaking
- Exposure duration: ca. 72 hours at approximately 37°C

NUMBER OF REPLICATIONS: Triplicate plates per dose level.

DETERMINATION OF CYTOTOXICITY
- Method: After this period, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter (Perceptive Instruments Sorcerer).

ACCEPTANCE CRITERIA:
For a test to be considered valid:
- The mean of the vehicle control revertant colony numbers for each strain should lie within or close to the current historical control range for the laboratory unless otherwise justified by the Study Director. The historical range is maintained as a rolling record over a maximum of five years.
- Also, the positive control compounds must induce an increase in mean revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537, which have relatively low spontaneous reversion rates) that of the concurrent vehicle controls.
- Mean viable cell counts in the 10-hour bacterial cultures must be at least 10^9/mL.

Evaluation criteria:

- If exposure to a test substance produces a reproducible increase in 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 substance does not produce a reproducible increase in 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 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 judgement.

Statistics:

None

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

HISTORICAL CONTROL DATA
- The concurrent positive controls verified the sensitivity of the assay and the metabolising 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.

ADDITIONAL INFORMATION ON CYTOTOXICITY
- Toxicity (observed as a reduction in revertant colony numbers) was seen in strain TA1537 following exposure to Terpenic Oligomers at 5000 μg/plate in the absence of S9 mix in the second test. No signs of toxicity towards any other tester strains were observed in either mutation test.
- Precipitate was observed on all plates containing Terpenic Oligomers at 5000 μg/plate in the presence of S9 mix in both tests and an oily residue was observed on all plates containing Terpenic Oligomers at 5000 μg/plate in the absence of S9 mix in both tests.

MUTAGENICITY RESULTS
No evidence of mutagenic activity was seen at any concentration of Terpenic Oligomers in either mutation test.

Remarks on result:

other: No evidence of mutagenic activity

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

Terpenic Oligomers did not induce mutation in four histidine-requiring Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and one tryptophan-dependent Escherichia coli strain, WP2 uvrA (pKM101).

Executive summary:

In a reverse gene mutation assay performed according to OECD Guideline No. 471, EU Method B.13/14, US EPA Health Test Guideline OPPTS 870.5100 and in compliance with GLP, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA(pKM101) were treated with the test item using the Ames plate incorporation method. Up to seven dose levels were evaluated in triplicate with and without the addition of a rat liver homogenate metabolizing system (10% v/v liver S9 in standard co-factors) in a first test. As a clear negative response was obtained in the first test, a variation to the test procedure was used for the second test. The pre-incubation procedure is not suitable for acetone, which is toxic under such conditions. The variation used was, therefore, an increase in the S9 content of the S9 mix from 10% v/v to 20% v/v. The maximum concentration chosen was again 5000 μg/plate, but only five concentrations were used. These dose levels ranged from 50 to 5000 μg/plate.

Results of the first experiment did not show any evidence of toxicity following exposure to Terpenic Oligomers. Precipitate was observed on all plates containing Terpenic Oligomers at 5000 μg/plate in the presence of S9 mix and an oily residue was observed on all plates containing Terpenic Oligomers at 5000 μg/plate in the absence of S9 mix. 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 Terpenic Oligomers at any concentration up to and including 5000 μg/plate in either the presence or absence of S9 mix.

After the exposure period of the second experiment, toxicity (observed as a reduction in revertant colony numbers) was seen in strain TA1537 following exposure to Terpenic Oligomers at 5000 μg/plate in the absence of S9 mix. No signs of toxicity towards any other tester strains were observed. Precipitate was observed on all plates containing Terpenic Oligomers at 5000 μg/plate in the presence of S9 mix and an oily residue was observed on all plates containing Terpenic Oligomers at 5000 μg/plate in the absence of S9 mix.

No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to Terpenic Oligomers at any concentration up to and including 5000 μg/plate in either the presence or absence of S9 mix.

The concurrent positive controls verified the sensitivity of the assay and the metabolising 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, Terpenic Oligomers did not induce mutation in four histidine-requiring Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and one tryptophan-dependent Escherichia coli strain, WP2 uvrA (pKM101), when tested at concentrations up to 5000 µg/plate (the maximum recommended concentration according to current regulatory guidelines) in the absence and presence of a rat liver metabolic activation system (S-9).