2,6-diisopropylnaphthalene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria in vitro (similar OECD 471): negative in S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvr A with and without metabolic activation

Study performed with 2,6-diisopropylnaphthalene (CAS No. 24157-81-1)

Chromosome aberration study in mammalian cells in vitro (OECD 473): negative in Chinese Hamster Ovary (CHO) cells with and without metabolic activation

Read-across from analogue source substance bis(isopropyl)naphthalene (CAS No. 38640-62-9)

Gene mutation study in mammalian cells in vitro (OECD 476): negative in mouse lymphoma L5178Y cells with and without metabolic activation

Read-across from analogue source substance bis(isopropyl)naphthalene (CAS No. 38640-62-9)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 Sep - 04 Nov 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon for S. typhimurium strains and trp operon for E. coli strain

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male Sprague-Dawley rats treated with 500 mg/kg bw Aroclor 1254.
- source of S9 : Molecular Toxicology, Inc., USA, Batch 0745 (33.15 mg of protein per mL) and Batch 0781 (40.7 mg of protein per mL)
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): yes

Test concentrations with justification for top dose:

First and second experiment: 100, 250, 500, 1000, 2500, 5000 µg/plate, with and without metabolic activation
Dose levels selected based on a dose range finding study

Vehicle / solvent:

- Vehicle: Dimethylformamide (DMF)
- Source: Sigma Chemical Co., USA (Lot 125H3633, Lot 37H3649, and Lot 47H3674)
- Justification for choice of solvent/vehicle: The solvent was chosen due to the limited solubility of the test substance

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

other: 2-aminoanthracene, 2.5 µg/plate for S. typhimurium strains, 25 µg/plate for E. coli strain, +S9

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments . 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)
- Exposure duration and conditions: 48 ± 8 h at 37 ± 2 °C

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Inspection of the bacterial background lawn

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Counting of revertant colonies

Evaluation criteria:

The test material may be considered positive in this test system if the following criteria are met: For the test item to be considered mutagenic, two-fold (three-fold for TA 1535 and TA 1537) increases in mean revertant numbers must be observed. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels. All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls.

Statistics:

Mean values and standard deviation were calculated.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

precipitation at and above 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

precipitation at and above 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

precipitation at and above 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

precipitation at and above 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True 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, but tested up to precipitating concentrations

Remarks:

precipitation at and above 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test substance is poorly soluble in aqueous solutions. Therefore, DMF was used as vehicle..
- Precipitation and time of the determination: At a concentration of 1000 µg/mL and above the test material precipitated in the cell culture medium. The precipitation did not interfere with the counting of the colonies.

RANGE-FINDING/SCREENING STUDIES:
In the range finding test, the substance was tested up to 5000 µg/plate in the absence and presence of S9 mix in S. typhimurium TA100 and E. coli WP2uvrA. No reduction of the bacterial lawn and no biologically relevant decrease in the number of revertants were observed. The highest concentration analysed was selected based on the solubility of the test substance in the cell culture medium.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : Refer to pdf document provided under 'Attached background material'.

Detailed result tables, incl. individual plate counts, for the dose range finding experiment and both mutagenicity assays are provided in the pdf document provided under 'Attached background material'. Please note that the registrant is authorised to use the study report for the purpose of the REACH registration despite the 'CONFIDENTIAL' stamp on each page.

Conclusions:

Interpretation of results: negative with and without metabolic activation

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Refer to the analogue approach justification provided in IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: ≥15 µg/ml (without S9); ≥ 60 µg/ml (with S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Source: CAS No. 38640 -62 -9, Brooker, 1988

Conclusions:

Interpretation of results: negative with and without metabolic activation

Executive summary:

The potential to induce chromosome aberration in mammalian cells of the target substance is predicted based on an adequate and reliable in vitro study of a structural analogue source substance. Experiments have been performed both in the presence as well as in the absence of metabolic activation in chinese hamster ovary (CHO) cells. All results obtained are negative, i.e. no chromosome aberration in the cells investigated were observed. There was no evidence of any clastogenic activity under the conditions of the test. Therefore, no hazard with regard to chromosome aberration in mammalian cells is identified for the target substance. As explained in the analogue justification, the differences in molecular structure between the target and the source substances are unlikely to lead to differences in the potential to form chromosome aberrations.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Refer to the analogue approach justification provided in IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: >=25 µg/ml (+/-S9): <= 20% relative survival

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Source: CAS No. 38640 -62 -9, Ransome, 1999

Conclusions:

Interpretation of results: negative with and without metabolic activation

Executive summary:

The potential of the target substance to induce gene mutation in mammalian cells is predicted based on an adequate and reliable in vitro study of a structural analogue source substance. Experiments using mouse

lymphoma L5178Y cells have been performed both in the presence as well as in the absence of metabolic activation. All results obtained are negative, i.e. no gene mutation was observed. There was no evidence of increases in mutant frequency according to the criteria of the test for all test substance concentrations tested without and with metabolic activation. Therefore, no hazard with regard to gene mutation in mammalian cells is identified for the target substance. As explained in the analogue justification, the differences in molecular structure between the target and the source substances are unlikely to lead to differences in gene mutation in mammalian cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Only data on gene mutation in bacteria is available for 2,6-diisopropylnaphthalene (CAS No. 24157-81-1). The genetic toxicity endpoints chromosome aberration and gene mutation in mammalian cells are, therefore, assessed by means of read-across from the analogue source substance bis(isopropyl)naphthalene (CAS No. 38640-62-9).

Gene mutation in bacteria in vitro

The potential of 2,6-diisopropylnaphthalene (CAS No. 24157-81-1) to induce gene mutation in bacteria was tested in a study similar to OECD guideline 471 and observing GLP provisions (Lawlor, 1998). The strains S. typhimurium TA 98, TA 100, TA 1537 and TA 1535 and E. coli WP2 uvr A were tested in two independent experiments according to the plate incorporation procedure in the absence and presence of a metabolic activation system (Aroclor 1254-induced rat liver S9-mix). The experiments were conducted each in 3 replications at concentrations from 100 to 5000 µg/plate. Dimethylformamide (DMF) was used as vehicle. No increase in the number of revertant colonies was noted in any of the bacterial strains, with and without metabolic activation system. No cytotoxicity was observed up to the highest dose tested. The positive and negative controls showed the expected results. Under the conditions of the study, the test substance did not induce mutations in the absence and presence of a metabolic activation system in any of the strains tested.

Chromosome aberration in mammalian cells in vitro

An in vitro mammalian chromosome aberration test was conducted with bis(isopropyl)naphthalene (CAS No. 38640-62-9) in accordance with OECD guideline 473 under GLP conditions (Brooker, 1988). The induction of structural chromosome aberrations was evaluated in vitro in Chinese Hamster Ovary (CHO) cells, incubated for 4 h with and for 20 h without a metabolic activation system (S9-mix from rats treated with Aroclor 1245). In a first main test concentrations of 7.5 - 75 µg/mL without S9 mix were applied. This test was dismissed and not continued for chromosomal analysis due to an unexpected reduction in the mitotic index at 75 µg/mL. Therefore, a second main test was initiated with reduced concentrations. In the second main experiment, concentrations of 0.95 - 9.5 µg/mL (4 h incubation) and 6 - 60 µg/mL (20 h incubation) of the test substance in the vehicle DMSO were applied. The negative as well as the positive controls showed the expected results. In the main experiments, a reduction in the mitotic index was observed from 15 µg/mL without and from 60 µg/mL with metabolic activation. No statistically or a biologically significant increase in the incidence of chromosome aberrations was observed. Therefore, under the conditions of the study, the test substance did not show clastogenic activity with and without metabolic activation performed in Chinese Hamster Ovary (CHO) cells in vitro.

Gene mutation in mammalian cells in vitro

The in vitro mammalian cell gene mutation study of bis(isopropyl)naphthalene (CAS No. 38640-62-9) was carried out according to OECD guideline 476 under GLP conditions (Ransome, 1999). Gene mutations in the thymidine kinase locus TK +/- were investigated in mouse lymphoma L5178Y cells in the presence and absence of a metabolic activation system (S9-mix from Aroclor 1254 induced rat liver). L5178Y cells were incubated with the test material at 5 - 40 µg/mL (experiment 1) and 1 - 25 µg/mL (experiment 2) without S9-mix for 3 and 24 h, respectively. In the experiments with metabolic activation, the test substance was incubated at concentrations of 1 - 60 µg/mL (experiment 1) and 1 - 50 µg/mL (experiment 2) for 3 and 24 h, respectively. The vehicle and positive controls in the study showed the expected results. Mean survival (Day 0) in non-selective medium of the solvent control in the absence of S9-mix was 74% and 68%, and in the presence of S9-mix 72 - 88%. In experiment 1 without S9-mix (3 h treatment), a statistically significant increase in mutant frequency (MF) outside the historical control range was observed at the highest dose (40 µg/mL). Mean Day 0 relative survival was 12% and Day 2 cloning efficiency (CE) was 19%. This effect can be explained by the corresponding decrease in Day 2 CE in non-selective medium. No substantial increase was seen in experiment 2 under equivalent conditions. In experiment 1 with S9-mix (3 h treatment), a statistically significant increase in MF outside the historical control range was observed at 20 µg/mL, but not at 50 µg/mL (survival 14%). But this effect was not reproduced in the second test. Colony sizing demonstrated no difference in the ratios small-to-large colonies from the solvent control, while significantly higher yields of small colonies were found in the positive controls. There was no reproducible increase in MF and no evidence of a dose relationship over at least two dose levels. Based on the results of this test, it can be concluded that the test substance did not demonstrate mutagenic potential in the in-vitro mouse lymphoma mutation assay.

Justification for classification or non-classification

The available data on gene mutation in bacteria for 2,6-diisopropylnaphthalene (CAS No. 24157-81-1) as well as the data on chromosome aberration and gene mutation in mammalian cells with the analogue source substance bis(isopropyl)naphthalene (CAS No. 38640-62-9) do not meet the criteria for classification according to Regulation (EC) No. 1272/2008 (CLP). Data are, therefore, conclusive but not sufficient for classification. Based on an analogue read-across approach, the target substance 2,6-diisopropylnaphthalene (CAS No. 24157-81-1) does not warrant classification for genetic toxicity.