Cajuput, ext.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames test: non mutagenic (OECD 471, GLP, K, rel. 1).

Link to relevant study records

Reference

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:

27 February - 28 March 2013

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.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

None

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):
- WP2uvrA (absence and presence of S9-mix): 0.5, 1.5, 5, 15, 50, 150, 500 μg/plate.
- All Salmonella strains (presence of S9-mix): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 μg/plate.
- All Salmonella strains (absence of S9-mix): 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 μg/plate.

Experiment 2 (preincubation method):
- All tester strains (presence of S9-mix): 1.5, 5, 15, 50, 150, 500, 1500 μg/plate.
- All tester strains (absence of S9-mix): 0.15, 0.5, 1.5, 5, 15, 50, 150 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: Test item was immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/mL but was fully miscible in acetone at 100 mg/mL. Acetone was therefore selected as the vehicle.
- Formulation preparation: Test item was accurately weighed and approximate half-log dilutions prepared in acetone by mixing on a vortex mixer on the day of each experiment. Acetone is toxic to the bacterial cells at 100 μL after employing the pre-incubation modification, therefore all of the formulations for each experiment were prepared at concentrations two times greater than required on Vogel-Bonner agar plates. To compensate, each formulation was dosed using 50 μL aliquots. All formulations were used within four hours of preparation and were assumed to be stable for this period.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

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:

Acetone

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 °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 °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 an automated 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).
- 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)).

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 50 µg/plate in the absence of S9-mix, and 150 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.
- Evaporation from medium: Evaporation was prevented by the use of anaerobic conditions.

CYTOTOXICITY:
Preliminary Toxicity Test: Test item initially exhibited toxicity to TA100 in the absence and presence of S9-mix from 50 and 500 μg/plate respectively. Weakened bacterial background lawns were noted for WP2uvrA from 150 μg/plate in both the absence and presence of S9-mix.
Mutation Test: Test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 50 μg/plate in the absence of S9-mix and 150 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 and exposures with or without S9-mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Results were compared with historical negative, solvent and positive control data (2010 and 2011).

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'.

See attached Document for Tables of Results

Conclusions:

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

Under the test conditions, Cajeput green 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 acetone both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors) using the plate 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 1500 µg/plate depending on bacterial strain type and presence or absence of metabolic activation. The experiment was repeated on a separate day using and amended range to Experiment 1 (0.15 to 500 µg/plate), fresh cultures of the bacterial strains and fresh test material formulations. 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 (acetone) 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 of all of the tester strains, initially from 50 μg/plate in the absence of S9-mix and 150 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 and exposures with or without S9-mix. The test item was tested up to the toxic limit. 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 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. A small, statistically significant increase in TA1535 revertant colony frequency was observed in the presence of S9-mix at 0.5 μg/plate in Experiment 1. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at 0.5 μg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.6 times the concurrent vehicle control.

 

Under the test condition, Cajuput green 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Table 7.6/1: Summary of genotoxicity test

Test n°	Test / Guideline Reliability	Focus	Strains tested	Metabolic activation	Test concentration	Statement
1   Thompson, 2013	Ames Test (OECD 471) K, rel. 1	Gene mutation	TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA-	-S9 +S9	Up to cytotoxic concentration	-S9 : non mutagenic +S9 : non mutagenic

 

 

A Bacterial Reverse mutation Assay (Ames test) was performed according to OECD test guideline No 471 with Cajuput oil (Test No. 1). No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose, either in the presence or absence of metabolic activation. Cajuput oil does not induce gene mutations in bacteria under the test conditions whereas the positive control chemical (with and without metabolic activation) induced significant increase of colonies. Cajuput oil is therefore considered as non-mutagenic according to the Ames test.

Justification for selection of genetic toxicity endpoint
Only one study is available. The key study is GLP-compliant and of high quality (Klimisch score =1). The result being negative, no further tests are required for this REACH Annex VII substance.

Justification for classification or non-classification

Harmonized classification:

Cajuput oil has no harmonized classification according to the Regulation (EC) No. 1272/2008.

Self-classification:

Based on the available information, no additional classification is proposed.