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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:
07 March - 03 June 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted and well described study in accordance with GLP and OECD Guideline 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:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Camphor white oil
- Physical state: clear clear colorless liquid
- Analytical purity: Conforms to Standard
- Lot/batch No.: 160341
- Storage condition of test material: Room temperature in the dark

Method

Target gene:
None
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:
other: see table 7.6.1/1
Metabolic activation:
with and without
Metabolic activation system:
10 % (v/v) S9 mix; S9 fraction prepared from liver homogenates of rats induced with Phenobarbitone/β-Naphthoflavone at 80/100 mg/kg bw/day by oral route
Test concentrations with justification for top dose:
Preliminary Toxicity Test: 50, 150, 500, 1500 and 5000 μg/plate in TA100 or WP2uvrA strains, with and without S9- mix using preincubation method.

Mutation Test:
Experiment 1 (preincubation method):
- All strains (absence of S9-mix): 0.05, 0.15, 0.5, 1.5, 5, 15, 50, and 150 µg/plate.
- All Salmonella strains (presence of S9-mix): 0.15, 0.5, 1.5, 5, 15, 50, 150 and 500 µg/plate.
- E.coli strain WP2uvrA (presence of S9-mix): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.

Experiment 2 (preincubation method):
- All strains (absence of S9-mix): 0.05, 0.15, 0.5, 1.5, 5, 15 and 50 µg/plate.
- All Salmonella strains (presence of S9-mix): 0.15, 0.5, 1.5, 5, 15, 50, 150 and 500 µg/plate.
- E.coli strain WP2uvrA (presence of S9-mix): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water, dimethyl formamide and acetonitrile at 50 mg/mL and acetone at 100 mg/mL but was fully miscible in dimethyl sulphoxide at 50 mg/mL and tetrahydrofuran at 200 mg/mL in solubility checks. Dimethyl sulphoxide was selected as the vehicle..
- Formulation preparation: Test item was accurately weighed and approximate half-log dilutions prepared in DMSO by mixing on a vortex mixer and 5 minutes sonication at 40 °C on the day of each experiment. All formulations were used within four hours of preparation and were assumed to be stable for this period. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino silicate pellets with a nominal pore diameter of 4 x 10-4 microns.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
See Table 7.6.1/2
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
See Table 7.6.1/2
Positive control substance:
benzo(a)pyrene
other: 2-Aminoanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
SOURCE OF TEST SYSTEM: All strains of bacteria used in the test were obtained from the University of California, Berkeley, on culture discs, on 04 August 1995 or from the British Industrial Biological Research Association, on nutrient agar plates, on 17 August 1987.

METHOD OF APPLICATION: Preincubation (test item, vehicle and positive controls) and plate incorporation (negative control) methods

DURATION
- Preincubation period: 20 minutes at 37 ± 3 °C
- Incubation period: Plates were placed in anaerobic jars or bags (one jar/bag for each concentration of test item/vehicle) and incubated at 37 ± 3 °C for approximately 48 h.

NUMBER OF REPLICATIONS:
-1 plate/dose for preliminary toxicity test and 3 plates/dose for treatment, vehicle and positive controls in mutation test (Experiment 1 & 2).

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity was determined on the basis of growth of the bacterial background lawn.

OTHER: After approximately 48 h incubation at 37 °C the plates were assessed for numbers of revertant colonies using a Domino colony counter.
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:
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 judgement 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
Remarks:
initially from 15 µg/plate (in the absence of S9-mix) and 50 µg/plate (in the presence of S9-mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

CYTOTOXICITY:
Preliminary Toxicity Test: The test item was initially toxic to TA100 and WP2uvrA from 50 µg/plate (absence of S9-mix), to TA100 from 150 µg/plate (presence of S9-mix) and WP2uvrA from 1500 µg/plate (presence of S9-mix).
Mutation Test: The test item caused a visible reduction in the growth of the bacterial background lawns or a substantial decrease in the number of revertants present in all of the tester strains, initially from 15 µg/plate (in the absence of S9-mix) and 50 µg/plate (in the presence of S9 mix). The sensitivity of the tester strains to the toxicity of the test item varied slightly between strain type, experiment number and exposures with or without S9 mix. The test item was tested up to either the toxic limit or the maximum dose level, depending on bacterial strain type and presence or absence of S9-mix. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Results were compared with historical negative, solvent and positive control data (2012 and 2013).

OTHERS:
- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and S9-mix used in both experiments was shown to be sterile.
- The culture density for each bacterial strain was also checked and considered acceptable.
- Test item formulation and S9-mix used in this experiment were both 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 attached Document for Tables of Results

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under the test conditions, Camphor white oil is not considered as mutagenic in S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and E. coli (WP2uvrA) strains.
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and E.coli strain WP2 uvrA- were exposed the test material diluted in dimethyl sulfoxide both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors) using the pre-incubation method (under anaerobic conditions). The dose range for the first experiment was determined in a preliminary toxicity assay and ranged between 0.05 and 5000 µg/plate depending on bacterial strain type and presence or absence of metabolic activation. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test material formulations. The dose range was based on the results of Experiment 1 and ranged between 0.05 and 5000 µg/plate, depending on bacterial strain type and presence or absence of S9 -mix. Additional dose levels and an expanded dose range were selected in both experiments in order to achieve both four non-toxic dose levels and the toxic limit of the test item.

The vehicle (dimethyl sulfoxide) 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.

The test item caused a visible reduction in the growth of the bacterial background lawns or a substantial decrease in the number of revertants present in all of the tester strains, initially from 15 µg/plate (in the absence of S9-mix) and 50 µg/plate (in the presence of S9‑mix). The sensitivity of the tester strains to the toxicity of the test item varied slightly between strain type, experiment number and exposures with or without S9‑mix. The test item was tested up to either the toxic limit or the maximum dose level, depending on bacterial strain type and presence or absence of S9-mix. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

No 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.

Under the test condition, Camphor white oil was not mutagenic to S. thyphimurium strains TA1535, TA1537 TA98, TA100, and E.coli WP2 uvrA-, in the presence and absence of metabolic activation.

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