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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 07, 2014 to January 12, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD test Guideline No. 471 without any deviation.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
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
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Program (inspected on March 12 to 14, 2014 / Signed on May 12, 2014)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Name of test material (as cited in study report): CISTUS CONCRETE
- Physical state: Dark brown solid at room temperature
- CAS No.: 89997-74-0
- EINECS-No.: 289-711-7
- Analytical purity: 100%UVCB
- Lot/batch No.: 14822 (réf. 101412)
- Production date: 13 May 2013
- Date of Receipt: 11 February 2014
- Expiration date of the lot/batch: 13 May 2015
- Composition: Paraffins, labdanolic derivated
- Storage condition of test material: Room temperature 20 ± 5 °C, keep away from light and humidity

Method

Target gene:
Histidine and tryptophan.
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
Metabolic activation:
with and without
Metabolic activation system:
10% S9: S9-mix from the livers of male rats treated with phenobarbitone/β-naphthoflavone (80/100 mg/kg bw/day by oral route).
Test concentrations with justification for top dose:
Test for Mutagenicity (Experiment 1) – Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in all strains with and without S9-mix

Test for Mutagenicity (Experiment 2) – Pre-Incubation Method: 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15 and 50 μg/plate in TA 100, TA 1535 and TA 1537 strains without S9-mix; 0.15, 0.5, 1.5, 5, 15, 50 and 150 μg/plate in TA 98 and WP2uvrA strains without S9-mix
Test for Mutagenicity (Experiment 2) – Pre-Incubation Method: 0.15, 0.5, 1.5, 5, 15, 50, 150 and 500 μg/plate in TA 100, TA 1535 and TA 1537 strains with S9-mix; 15, 50, 150, 500, 1500 and 5000 μg/plate in TA 98 and WP2uvrA strains with S9-mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Tetrahydrofuran
- Justification for choice of solvent/vehicle: The test item was insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL in solubility checks performed in–house. The test item formed the best doseable suspension in tetrahydrofuran, therefore, this solvent was selected as the vehicle.
- Preparation of test materials: The test item was accurately weighed and approximate half-log dilutions prepared in tetrahydrofuran by mixing on a vortex mixer and sonication for 30 minutes at 40 °C on the day of each experiment. Tetrahydrofuran is toxic to the bacterial cells at and above 50 μL (0.05 mL), therefore all of the formulations were prepared at concentrations four times greater than required on Vogel-Bonner agar plates. To compensate, each formulation was dosed using 25 μL (0.025 mL) aliquots. Tetrahydrofuran is considered an acceptable vehicle for use in this test system (Maron et al., 1981). All formulations were used within four hours of preparation and were assumed to be stable for this period.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Tetrahydrofuran
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Tetrahydrofuran
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-Aminoanthracene
Remarks:
With S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Exposure duration: Plates were incubated at 37 °C ± 3 °C for approximately 48 hours

NUMBER OF REPLICATIONS: Triplicate plates per dose level.

DETERMINATION OF CYTOTOXICITY
- Method: The plates were viewed microscopically for evidence of thinning (toxicity).

OTHERS:
After incubation, the plates were assessed for numbers of revertant colonies using an automated colony counting system. Manual counts were performed at and above 500 μg/plate because of test item precipitation.
Evaluation criteria:
There are several criteria for determining a positive result. 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 (De Serres and Shelby, 1979).
- A reproducible increase at one or more concentrations.
- Biological relevance against in-house historical control ranges.
- Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
- Fold increases 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:
Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not applicable
- Effects of osmolality: Not applicable
- Evaporation from medium: No data
- Water solubility: None
- Precipitation: A test item precipitate (greasy and particulate in appearance) was noted at and above 500 μg/plate; this observation did not prevent the scoring of revertant colonies.
- Other confounding effects: None

COMPARISON WITH HISTORICAL CONTROL DATA: All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and positive controls. The comparison was made with the historical control ranges for 2013 and 2014 of the corresponding Testing Laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- There was no visible reduction in the growth of the bacterial background lawns at any dose level, either in the presence or absence of S9-mix, in the first mutation test (plate incorporation method) and consequently the same maximum dose level was originally selected for the second mutation test. In the initial second experiment, the toxicity of the test item yielded results that differed from Experiment 1 (due to a change in test methodology from plate incorporation to pre-incubation) and consequently, there were an insufficient number of non-toxic dose levels for the majority strains in both the absence and presence of S9-mix (the data is not given in this report). Therefore, a repeat second experiment was performed employing additional dose levels and an expanded dose range. In the second mutation test (pre-incubation method) the test item induced a stronger toxic response with weakened bacterial background lawns initially noted in the absence of S9-mix from 5 μg/plate (TA1535), 15 μg/plate (TA1537), 50 μg/plate (TA100) and 150 μg/plate (TA98 and WP2uvrA). In the presence of S9-mix weakened lawns were initially noted at 50 μg/plate (TA100 and TA1537) and 500 μg/plate (TA1535). No toxicity was noted to TA98 or WP2uvrA at any test item dose level in the presence of S9-mix. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology.

OTHERS:
- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The test material formulation, amino acid supplemented top agar and S9-mix used in this experiment were shown to be sterile.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

See the attached document for information on tables of results

Applicant's summary and conclusion

Conclusions:
Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA.
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli strain WP2 uvrA- were exposed to test material both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors).

Test for Mutagenicity (Experiment 1) – Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in all strains with and without S9-mix

Test for Mutagenicity (Experiment 2) – Pre-Incubation Method: 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15 and 50 μg/plate in TA 100, TA 1535 and TA 1537 strains without S9-mix; 0.15, 0.5, 1.5, 5, 15, 50 and 150 μg/plate in TA 98 and WP2uvrA strains without S9-mix

Test for Mutagenicity (Experiment 2) – Pre-Incubation Method: 0.15, 0.5, 1.5, 5, 15, 50, 150 and 500 μg/plate in TA 100, TA 1535 and TA 1537 strains with S9-mix; 15, 50, 150, 500, 1500 and 5000 μg/plate in TA 98 and WP2uvrA strains with S9-mix

Negative, vehicle (tetrahydrofuran) and positive control groups were also included in mutagenicity tests.

The vehicle control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

There was no visible reduction in the growth of the bacterial background lawns at any dose level, either in the presence or absence of S9-mix, in the first mutation test (plate incorporation method) and consequently the same maximum dose level was originally selected for the second mutation test. In the second mutation test (pre-incubation method) the test item induced a stronger toxic response with weakened bacterial background lawns initially noted in the absence of S9-mix from 5 μg/plate (TA1535), 15 μg/plate (TA1537), 50 μg/plate (TA100) and 150 μg/plate (TA98 and WP2uvrA). In the presence of S9-mix weakened lawns were initially noted at 50 μg/plate (TA100 and TA1537) and 500 μg/plate (TA1535). No toxicity was noted to TA98 or WP2uvrA at any test item dose level in the presence of S9-mix. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. A test item precipitate (greasy and particulate in appearance) was noted at and above 500 μg/plate; this observation did not prevent the scoring of revertant colonies.

 

There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 1 (plate incorporation method). Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in the second mutation test (pre-incubation method). Small, statistically significant increases in revertant colony frequency were observed in the first Experiment at 15 μg/plate (TA98 dosed in the absence of S9-mix) and in the second experiment at 15 μg/plate (TA100 dosed in the presence of S9-mix). These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for each tester strain and the maximum fold increase was only 1.5 times the concurrent vehicle controls.

 

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.