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Genetic toxicity in vitro

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The mutagenicity of the test substance, Terpenes and terpenoids, turpentine oil, beta-pinene fraction polymerised, was determined in Salmonella Typhimurium (S. Typhimurium) TA98, TA100, TA1535 and TA1537 strains and E.coli Wp2uvrA strain, in a method compatible with the following guidelines: OECD No. 471 'Bacterial Reverse Mutation Test', Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008, the USA EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test and the Japanese Regulatory Authorities including METI, MHLW and MAFF.

The bacteria were treated with the test substance using both the Ames plate incorporation (Experiment 1) and the pre-incubation method (Experiment 2) at eight dose levels, testing in triplicate with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was pre-determined to be 1.5 -5000 µg/plate, based on the General Study Plan. The result of Experiment 1 was deemed negative and so the test was repeated using the pre-incubation method over the dose range of 15 -5000 µg/plate, based on results from Experiment 1. Six test substance concentrations were chosen in Experiment 2 in order to achieve four non-toxic dose levels and the potential toxic limit of the test substance following the change in methodology. The vehicle control used was acetone. The negative untreated controls were employed in order to assess the spontaneous revertant colony rate. The positive controls used were N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), 9 -Aminoacridine (9AA), 4 -Nitroquinoline-1 -oxide (4NQO), 2 -Aminoanthracene (2AA) and benzo(a)pyrene (BP).

The maximum dose levels of the test substance in Experiment 1 was selected as the maximum recommended dose level of 5000 µg/plate. In both Experiment 1 and 2, there was no visible reduction in the growth of the bacterial background lawn in any of the doses tested in the presence and absence of the S9 -mix. A test substance precipitate (globular in appearance) was observed under a low power microscope at 1500 µg/plate and by eye at 5000 µg/plate, but these precipitates did not prevent the scoring of revertant colonies. In Experiment 2, a small statistically significant increase in WP2uvrA revertant colony frequency was seen in the presence of the S9-mix at 5000µg/plate, but it was not considered to be biologically relevant, since there was no evidence of a dose-response relationship or reproducibility. The individual revertant colony counts at 5000 µg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.4 times the concurrent vehicle control.

The vehicle (acetone) control produced counts of revertant colonies falling within the normal range and all of the positive control chemicals induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. All of the evaluation and acceptability criteria were, therefore this validated the sensitivity of the assay and the efficacy of the S9 -mix.

Based on the results of the study and in accordance with the evaluation criteria, the test substance, Terpenes and terpenoids, turpentine oil, beta-pinene fraction polymersied, was considered to be non-mutagenic to the bacterial strains testsed under the conditions of the test.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 January - 28 February 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
Japanese Ministry of Economy, Trafe and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Physical state/appearance: amber coloured liquid
- Batch No.of test material: AN-0400-112
- Expiration date of the lot/batch: 01 January 2019
- Purity test date: UVCB (treated as 100%)
> no correction for purity was required

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: approximately -20°C in the dark under nitrogen
- Stability under test conditions: all test substance formulations were used within 4 hours of preparation and were assumed to be stable during this time.
Target gene:
The histidine or tryptophan locus
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Remarks:
TA 1537: his C 3076; rfa-; uvrB-; frame-shift mutation TA98: his D 3052; rfa-; uvrB-; R-factor TA1535: his G 46; rfa-, uvrB-, R-factor; base-pair substitution TA100: his G 46; rfa-;uvrB-; R-factor
Species / strain / cell type:
E. coli WP2 uvr A
Remarks:
trp-; uvrA-; base-pair substitution
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Experiment 1 - Plate incorporation method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2 - Pre-Incubation method: 15, 50, 150, 500, 1500 and 5000 µg/plate - determined by the results of Experiment 1 with the plate incorporation
Vehicle / solvent:
Vehicle/solvent control used: acetone
- Tested in triplicate
- Justification for choice of solvent/vehicle: test substance was fully soluble in acetone at 100 mg/L in solubility checks performed in-house.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-aminoanthracene (2AA)
Details on test system and experimental conditions:
BACTERIAL STRAINS
Source: University of California, Berkeley, on culture discs (04/08/95) and British Industrial Biological Research Association, on nutrient agar plate (17/08/87)
> Salmonella strains
- histidine dependent due to a mutation through the histidine operon and are derived from Salmonella typhimurium (S. typhimurium) strain LT2 via mutations in the histidine locus.
- possess a 'deep-rough' (rfa-) mutation, which means they have a faulty liposaccharide coat to the bacterial cell surface thus increasing permeability to larger molecules.
- deletion of uvrB- bio gene, thus inactivating the excision repair system and a dependence on exogenous biotin
- in the TA98 and TA100 strains, the R-factor plasmid pKM101 enchances chemical and UV-induced mutagenesis via increasing the error-prone repair pathway and confers ampicillin resistance

>E.coli strain
- mutation in tryptophan operon
- a uvrA- DNA repair deficiency that increases its sensitivity to some mutagenic compounds, allowing the strain to show enahnced mutability as the uvrA repair system would normally act to remove and repair te damage section of the DNA molecule.

Storage: stored at approximately -196°C in a Statebourne liquid nitrogen freezer

TEST SUBSTANCE PREPARATION
- the test substance was accurately weighed and approximate half-log dilutions prepared in acetone by mixing on a vortex micer and sonication for 10 minutes at 40°C on the day of each experiment.
- in Experiment 2 (plate incorporation method), since acetone is toxic to the bacterial cells at 0.1 mL (100 µL), the formulations were prepared at concentrations two times greater than required on the Vogel-Bonner agar plates. Each formulation was dosed using 0.05 mL (50 µL) aliquots and the solvent was dried prior to use, in order to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x10^-4 microns.

METHOD OF APPLICATION: onto Vogel-Bonner agar plates; pre-incubation

DURATION
- Preincubation period: Overnight sub-cultures of appropriate-coded stock cultures were prepared in nutrient broth and incubated at 37°C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.
- Exposure duration: approximately 48 hours at 37 ± 3°C

OBSERVATIONS AND SCORING
- plates were viewed microscopically for evidence of thinning (toxicity) and the presence of revertant colonies were scored via an automated colony counting system

POSITIVE CONTROL CONCENTRATIONS: performed in triplicate
1) ENNG: 2 µg/mL for WP2 uvrA; 3 µg/plate for TA100; 5 µg/plate for TA1535
2) 9AA: 80 µg/plate for TA1537
3) 4NQO: 0.2 µg/plate for TA98
4) 2AA: 1 µg/plate for TA100; 2 µg/plate for TA1535 and TA1537; 10 µg/plate for WP2uvrA
5) BP: 5 µg/plate for TA98

STERILITY CONTROLS: performed in triplicate
1) Top agar and histidine/biotin or tryptothan in the absence of the S9-mix
2) Top agar and histidine/biotin or tryptophan in the presence of the S9-mix
3) The maximum dosing solution of the test substance in the absence of the S9-mix (test in singular only)

Evaluation criteria:
Any, one, or all of the following can be used to determine the overall result of the study:
1) A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2) A reproducible increase at one or more concentrations.
3) Biological relevance against in-house historical control ranges.
4) Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5) Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.
Statistics:
Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
EXPERIMENT 1:
- The maximum dose level of the test substance was selected as the maximum recommended dose level of 5000 µg/plate.

EXPERIMENT 1 and 2:
- There was no visible reduction in the growth of the bacterial background lawn at any dose level either in the absence or presence of metabolic activation.
- No toxicologically signficant increases in the frequency of revertant colonies were recorded for any of the bacterial strains at any dose of the test susbtance and with or without the S9-mix.
- A test substance precipitate (globular in appearance) was observed under a low power microscope at 1500 µg/plate and by eye at 5000 µg/plate, but these precipitates did not prevent the scoring of revertant colonies.

EXPERIMENT 2
- A small statistically significant increase in WP2uvrA revertant colony frequency was seen in the presence of the S9-mix at 5000µg/plate, but it was not considered to be biologically relevant, since there was no evidence of a dose-response relationship or reproducibility.
- The individual revertant colony counts at 5000 µg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.4 times the concurrent vehicle control.

CONTROLS:
Vehicle control (Acetone) plates: the counts of revertant colonies within the normal range.
Positive control chemicals: induced marked increases in the frequency of revertant colonies, both with and without metabolic activation.
> therefore the sensitivity of the assay and efficiacy of the S9-mix were validated.
Negative (untreated) controls: considered to be acceptable

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%) :
- please see Figure 1 in the 'attached background material' sectiom.

Plate incorporation method

- Manual counts were performed at 5000 µg/plate due to the presence of a test film and also on other plates where there were spreading of revertant colonies, thus distorting the actual plate count.

- The result was deemed negative.

- Master strains were checked for characteristics, viability and spontaneous reversion rate and were all found to be satisfactory.

- Animo acid supplemented top agar, the S9 -mix and test substance formulation were shown to be sterile.

Conclusions:
Based on the results of the study, the test substance, Terpenes and terpenoids, turpentine oil, beta-pinene fraction polymerised, was considered to be non-mutagenic in S. typhimurium TA1537, TA1535, TA98 and TA100 strains and E.coli WP2uvrA strain, under the conditions of test.

Executive summary:

The mutagenicity of the test substance, Terpenes and terpenoids, turpentine oil, beta-pinene fraction polymerised, was determined in Salmonella Typhimurium (S. Typhimurium) TA98, TA100, TA1535 and TA1537 strains and E.coli Wp2uvrA strain, in a method compatible with the following guidelines: OECD No. 471 'Bacterial Reverse Mutation Test', Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008, the USA EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test and the Japanese Regulatory Authorities including METI, MHLW and MAFF.

The bacteria were treated with the test substance using both the Ames plate incorporation (Experiment 1) and the pre-incubation method (Experiment 2) at eight dose levels, testing in triplicate with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was pre-determined to be 1.5 -5000 µg/plate, based on the General Study Plan. The result of Experiment 1 was deemed negative and so the test was repeated using the pre-incubation method over the dose range of 15 -5000 µg/plate, based on results from Experiment 1. Six test substance concentrations were chosen in Experiment 2 in order to achieve four non-toxic dose levels and the potential toxic limit of the test substance following the change in methodology. The vehicle control used was acetone. The negative untreated controls were employed in order to assess the spontaneous revertant colony rate. The positive controls used were N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), 9 -Aminoacridine (9AA), 4 -Nitroquinoline-1 -oxide (4NQO), 2 -Aminoanthracene (2AA) and benzo(a)pyrene (BP).

The maximum dose levels of the test substance in Experiment 1 was selected as the maximum recommended dose level of 5000 µg/plate. In both Experiment 1 and 2, there was no visible reduction in the growth of the bacterial background lawn in any of the doses tested in the presence and absence of the S9 -mix. A test substance precipitate (globular in appearance) was observed under a low power microscope at 1500 µg/plate and by eye at 5000 µg/plate, but these precipitates did not prevent the scoring of revertant colonies. In Experiment 2, a small statistically significant increase in WP2uvrA revertant colony frequency was seen in the presence of the S9-mix at 5000µg/plate, but it was not considered to be biologically relevant, since there was no evidence of a dose-response relationship or reproducibility. The individual revertant colony counts at 5000 µg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.4 times the concurrent vehicle control.

The vehicle (acetone) control produced counts of revertant colonies falling within the normal range and all of the positive control chemicals induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. All of the evaluation and acceptability criteria were, therefore this validated the sensitivity of the assay and the efficacy of the S9 -mix.

Based on the results of the study and in accordance with the evaluation criteria, the test substance, Terpenes and terpenoids, turpentine oil, beta-pinene fraction polymersied, was considered to be non-mutagenic to the bacterial strains testsed under the conditions of the test.

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

Additional information

Justification for classification or non-classification