Isobutyl 2-naphthyl ether

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECDTG 471): negative

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

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

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

- Dose finding study:
all strains tested (with and without S9-mix): 8.19, 20.5, 51.2, 128, 320, 800, 2000 and 5000 µg/plate

- Dose finding study (supplementary study):
TA100, TA1535, TA1537 (without S9-mix): 0.105, 0.262, 0.655, 1.64, 4.10, 10.2, 25.6 µg/plate
TA98 (without S9-mix), TA1535, TA100 (with S9-mix): 0.655, 1.64, 4.10, 10.2, 25.6, 64.0, 160 µg/plate
TA1537 (with S9-mix): 1.64, 4.10, 10.2, 25.6, 64.0, 160, 400 µg/plate

- Final study:
TA100, TA1537 (without S9-mix): 0.305, 0.610, 1.22, 2.44, 4.88, 9.77, 19.5 µg/plate
TA1535 (without S9-mix): 0.610, 1.22, 2.44, 4.88, 9.77, 19.5, 39.1 µg/plate
WP2uvrA (with and without S9-mix): 156, 313, 625, 1250, 2500, 5000 µg/plate
TA98 (without S9-mix), TA100, TA1535, TA1537 (with S9-mix): 4.88, 9.77, 19.5, 39.1, 78.1, 156, 313 µg/plate
TA98 (with S9-mix): 19.5, 39.1, 78.1, 156, 313, 625, 1250 µg/plate

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent: the test substance was found to be soluble in DMSO up to 5000 µg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

furylfuramide

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION:
- Preincubation period: 20 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY:
- Method: on the basis of background bacteria

Evaluation criteria:

STUDY VALIDITY
- That the average number of reverse mutation colonies for both the negative and positive control groups were within the desired ranges as provided by background data.
- That with respect to positive and negative control groups being observed at the same time, the number of colonies in the positive control group were more than double that of the negative control group.

POSITIVE RESULT
Where the average number of reverse mutation colonies increased to more than double than that of the negative control group and that increase was found to be dependent on dosage or reproducible, those results would be deemed to be positive.

As described in OECD guideline 471, 2-Aminoanthracene should not be used as the sole indicater of the efficacy of the S9-mix. If 2-Aminoanthracene is used, each batch of S9 should also be characterised with a mutagen that requires metabolic activation by microsomal enzymes. However, use of such a second indicator is not described in the report.

Key result

Species / strain:

S. typhimurium, other: TA100, TA98, TA1535, TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation in dose range finding study: At the beginning of treatment, in the -S9 treatment group, a dose of 51.2 μg/plate or more resulted in cloudiness in the resulting liquid. Furthermore, transparent oil droplet-like precipitate was observed at doses of 800 μg/plate or more. In the +S9 treatment group, a dose of 320 μg/plate or more resulted in cloudiness in the resulting liquid. Furthermore, white powdery precipitate as well as transparent oil droplet-like precipitate was observed at doses of 800 μg/plate or more. When colonies were being counted, in both the -S9 and +S9 treatment groups, white powdery precipitate was observed at doses of 2000 μg/plate or more. Furthermore, at 5000 μg/plate transparent oil droplet-like precipitate was observed.
- Precipitation in dose range finding study (supplemental study): At the beginning of treatment, a dose of 64.0 μg/plate or more in the -S9 treatment group and a dose of 400 μg/plate in the +S9 treatment group resulted in cloudiness in the resulting liquid. When colonies were being counted, however, there was no observation of any change as a result of precipitate or other phenomena.
- Precipitation in final study: At the beginning of treatment, a dose of 78.1 μg/plate or more in the -S9 treatment group resulted in cloudiness in the resulting liquid. Furthermore, transparent oil droplet-like precipitate was observed at doses of 625 μg/plate or more. In the +S9 treatment group, a dose of 313 μg/plate or more resulted in cloudiness in the resulting liquid. Furthermore, white powdery precipitate was observed at a dose of 625 μg/plate, and transparent oil droplet-like precipitate was observed at doses of 1250 μg/plate or more. When colonies were being counted, in the -S9 treatment group, white powdery precipitate was observed at doses of 1250 μg/plate or more. Furthermore, at 2500 μg/plate or more transparent oil dropletlike precipitate was observed. In the +S9 treatment group, white powdery precipitate was observed at doses of 2500 μg/plate or more. Furthermore, at 5000 μg/plate transparent oil droplet-like precipitate was observed.

RANGE-FINDING/SCREENING STUDIES:
- Dose range finding study: Treatment with the test substance with and without S9-mix did not result in an observation of an increase in reverse mutation. In addition, with respect to all bacterial strains with the exception of Escherichia coli WP2uvrA, low doses, or moderate doses or higher resulted in observations of toxicity. Further, with respect to -S9 treatment group bacterial strain TA100, a dose of 800 μg/plate or more resulted in an observation of strong toxicity such that the colony analyzer could not distinguish between the TA100 strain and a nutrient deficient strain, and colony counts had to be made by the naked eye. In addition, as a result of the effect of precipitate, in the -S9 treatment group at doses of more than 2000 μg/plate or more, and in the +S9 treatment group at doses of 5000 μg/plate, it was judged that the colony analyzer should not be used, and colony counts were made by the naked eye. Each bacterial strain in the positive control group resulted in a striking increase in reverse mutation.
- Dose range finding study (supplemental study): Treatment with the test substance with and without S9-mix did not result in an observation of an increase in reverse mutation. In addition, toxicity was observed in the -S9 treatment group at 10.2 μg/plate or more for strains TA100 and TA1537, at 25.6 μg/plate for TA1535, and at 160 μg/plate for TA1535. With respect to the +S9 treatment group, toxicity was observed at 160 μg/plate for strains TA100 and TA1535, and at 160 μg/plate or more for TA1537. Each bacterial strain in the positive control group resulted in a striking increase in reverse mutation.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
With respect to the -S9 treatment group, a dose of 19.5 μg/plate or more for TA100, 39.1 μg/plate or more for TA1535, 156 μg/plate or more for TA98, and 9.7 μg/plate or more for TA1537 resulted in observations of toxicity. With respect to the +S9 treatment group, a dose of 78.1 μg/plate or more for TA100, 156 μg/plate or more for TA1535 and TA1537, and 1250 μg/plate or more for TA98 resulted in observations of toxicity.
Doses of 1250 μg/plate or more in the -S9 treatment group, and doses of 5000 μg/plate in the +S9 treatment group, resulted in an observation of strong toxicity such that it was judged that the colony analyzer should not be used, and colony counts were made by the naked eye.

Conclusions:

The substance is not mutagenic in the reverse mutation assay performed according to OECD 471 (1997).

Executive summary:

The mutagenic activity of the substance was evaluated in accordance with OECD 471 (1997) and according to GLP principles. The test was performed in three independent pre-incubation experiments, both in the absence and presence of S9-mix. The first two were dose range finding studies. Based on these dose range finding studies mutagenicity was tested up to the following maximum concentrations without growth inhibition or visible precipitation: -S9 mix: 9.77 µg/plate (TA100), 19.5 µg/plate (TA1535), 78.1 µg/plate (TA98), 4.88 µg/plate (TA1537), and 625 µg/plate (WP2 uvrA), +S9 mix: 39.1 µg/plate (TA100), 78.1 µg/plate (TA1535 and TA 1537), 625 µg/plate (TA98), and 1250 µg/plate (WP2 uvrA). Adequate negative and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant colonies in each of the four S. typhimurium tester strains (TA1535, TA1537, TA98, TA100) and E.coli WP2uvrA strain, both in the absence and presence of S9-metabolic activation. Growth inhibition was observed at 19.5 µg/plate in TA100, at 39.1 µg/plate in TA1535, at 156 µg/plate or more in TA98 and at 9.77 µg/plate or more in TA1537 without metabolic activation, and at 78.1 µg/plate or more in TA100, at 156 µg/plate or more in TA1535 and TA1537 and at 1250 μg/plate in TA98 with metabolic activation. With E. coli no growth inhibition was observed up to 5000 µg/plate with or without S9. The positive controls showed the expected positive results. Based on the results of this study it is concluded that the substance is not mutagenic in the reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro

The mutagenic activity of the substance was evaluated in accordance with OECD 471 (1997) and according to GLP principles. The test was performed in three independent pre-incubation experiments, both in the absence and presence of S9-mix. The first two were dose range finding studies. Based on these dose range finding studies mutagenicity was tested up to the following maximum concentrations without growth inhibition or visible precipitation: -S9 mix: 9.77 µg/plate (TA100), 19.5 µg/plate (TA1535), 78.1 µg/plate (TA98), 4.88 µg/plate (TA1537), and 625 µg/plate (WP2 uvrA), +S9 mix: 39.1 µg/plate (TA100), 78.1 µg/plate (TA1535 and TA 1537), 625 µg/plate (TA98), and 1250 µg/plate (WP2 uvrA). Adequate negative and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant colonies in each of the four S. typhimurium tester strains (TA1535, TA1537, TA98, TA100) and E.coli WP2uvrA strain, both in the absence and presence of S9-metabolic activation. Growth inhibition was observed at 19.5 µg/plate in TA100, at 39.1 µg/plate in TA1535, at 156 µg/plate or more in TA98 and at 9.77 µg/plate or more in TA1537 without metabolic activation, and at 78.1 µg/plate or more in TA100, at 156 µg/plate or more in TA1535 and TA1537 and at 1250 μg/plate in TA98 with metabolic activation. With E. coli no growth inhibition was observed up to 5000 µg/plate with or without S9. The positive controls showed the expected positive results. Based on the results of this study it is concluded that the substance is not mutagenic in the reverse mutation assay.

Additional supporting information is available from a limited Ames test (Wild, 1983), using Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 with and without S-9 activation. The substance at concentrations of up to 1 mg/plate did not reveal any mutagenic effect. E. Coli strains were not included in this study. This is not considered to affect the integrity of the study as the substance does not contain peroxides or epoxides.

Furthermore supporting information is available from an in vitro mammalian chromosomal aberration test (MHLW, 2007), performed according to OECD 473 and in compliance with GLP. Under the conditions of the test, the substance was not clastogenic.

In vivo

Additional supporting information is available from a limited micronucleus test (Wild, 1983) in which male and female NMRI mice, 4 per dose, (10-14 weeks old) were treated intraperitoneally with the test substance dissolved in olive oil, once at 800, 1400 and 2000 mg/kg bw. Mice from each dose level were sacrificed at 30 hours, the bone marrow extracted and smear preparations were made and stained. Polychromatic erythrocytes were then scored for the presence of micronuclei.The substance did not induce micronuclei in the bone marrow up to 2000 mg/kg bw/day.

Justification for classification or non-classification

Based on the available information classification and labelling for genetic toxicity is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.