2,2'-(vinylenedi-p-phenylene)bis[5-methylbenzoxazole]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Remarks:

source of read across

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1982

Report date:

1982

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Similar Substance 01

IUPAC Name:

Similar Substance 01

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium, other: TA 100, TA 1535, TA 1537, TA 1538, TA 98

Details on mammalian cell type (if applicable):

Bacteria were grown overnight in nutrient broth (25 g oxid Nurrient Broth No 2/ litre) at 37 °C. The suitable amount of bacteria in the cell suspension was checked by nephelometry. For incubation, stock cultures which were stored at - 80 °C were used.

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Bacteria were grown overnight in nutrient broth (25 g oxid Nurrient Broth No 2/ litre) at 37 °C. The suitable amount of bacteria in the cell suspension was checked by nephelometry. For incubation, stock cultures which were stored at - 80 °C were used.

Metabolic activation:

with and without

Metabolic activation system:

rat Iiver homogenate fraction (S9)

Test concentrations with justification for top dose:

Main experiment: 4, 20, 100, 500, 2500 and 5000 µg/plate
Toxicity experiment and dose range finding: 4, 20, 100, 500, 2500 and 10000 µg/plate

Vehicle / solvent:

- Solvent: DMSO

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: N-Methyl-N'-nitro-N-nitrosoguanidine // 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION
Top Agar was prepared for the salmonella strains by mixing 100 ml agar (0.6 % NaCl) with 5 ml of a 1.0 mM histidine and 5 ml of 1.0 mM biotin solution.
In the case of E. coli histidine was replaced by tryptophan (5 ml, 0.5 mM).
The following ingredients were added (in order) to 2 ml of molten top agar at 45 °C: 0.1 ml test compound solution; 0.1 ml of an overnight broth culture of the bacterial tester strain; 0.5 ml S9 Mix (if required) or buffer.
After mixing, the liquid was poured into a petri dish with minimal agar (1.5 % agar, Vogel-Bonner E medium with 2 % glucose).

INCUBATION
Incubation for 48 to 72 hours at 30 °C in the dark.

NUMBER OF REPLICATIONS
3 plates per dose.

METABOLIC ACTIVATION
The requirement for metabolic activation was investigated by incorporating into the test an activation system by nicotinamide-adenine dinucleotia phosphate (NADP+)-cytochrome P450 dependent mixed function oxidase enzymes of the liver. The 9000 g supernatant of rat liver homogenate has been shown to be very useful in metabolic activation of foreign compounds. The animals were pretreaied with Aroclor 1254 as an inducer of several drug metabolizing enzymes.

Preparation and storage of Iiver homogenate fraction (S9)
Male Sprague Dawley rats (200 - 300 g) received a single intraperitoneal injection of Aroclor 1254 (500 mg/kg bw) 5 days before sacrifice. Preparation was performed at 0 to 4 °C using cold sterile solutions and glassware. The liver from at least 5 - 6 animals were removed and pooled, washed in 150 mM KCl (approximately 1 ml/g wet livers). The washed livers were cut into small pieces and homogenised in three volumes of KCl. The homogenate was centrifuged at 9000 g for 10 minutes. The supernatant was the S-9 fraction. It was divided into small portions, rapidly frozen and stored at -80 °C for not longer than 3 months.

Preparation of S9 Mix and concentration of cofactors
Sufficient S9 fraction was thawed immediately before each test at room temperature. One volume of the S9 fraction were miixed with 9 volumes of the S9 co-factor solution and kept on ice until used. The concentrations of the different compounds in the S9 Mix were:
8 mM MgCl
33 mM KCl
5 mM glucose-6-phosphate
4 mM NADP+
100 mM phosphate buffer pH 7 ,4

TOXICITY EXPERIMENT
- Preparation: 0.1 ml of the different dilutions of the test compound were thoroughtly mixed with 0.1 ml 10^-6 dilutions of the overnight culture of TA 100 and plated with histidine and biotin rich top agar.
- Replicates: 3 plates per dose.

DOSE RANGE FINDING
- Stratins: preliminary toxicity tests were performed with all tester strains.
- Indication of toxicity: a reduced rate of spontaneously occurring colonies as well as visible thinning of the bacterial lawn were used as indicator for toxicity. Thinning of the bacterial lawn was controlled microscopicaly.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium, other: TA 100, TA 1535, TA 1537, TA 1538, TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

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

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Test compound did not cause a significant increase in the number of colonies with any of the tester strains in the absense or presence of S9 mix. No dose dependent effect was obtained.

TOXICITY TEST
Test compound was tested at doses of 4 to 10000 µg/plate and proved to be not toxic. Visible precipitation on the plates has been observed at 500 µg/plate. Thinning of the bacterial lawn or a reduction in the number of colonies have not been observed.
For mutagenicity test, 5000 µg/plate was chosen as the highest dose.

STERILITY CHECKS AND CONTROL PLATES
Sterility of S9 mix and the test compound was indicated by the absence of contamination on test material and S9 mix sterility check plates. Control plates (background control and positive controls) gave the expected number of colonies.

Applicant's summary and conclusion

Conclusions:

The substance resulted to be not mutagenic in bacterial test systems neither with nor without exogenous metabolic activation.

Executive summary:

The substance was tested for mutagenicity with strains TA 100, TA 1535, TA 1537, TA 1538, TA 98 of Salmonella typhimurium and Escherichia coli WP2uvrA.

The mutagenicity studies were conducted in the absence and in the presence of a metabolizing system derived from rat liver homogenate. A dose ränge of 6 different doses from 4 µg/plate to 5000 µg/plate was used.

Control plates without mutagen showed that the number of spontaneous revertant colonies was similiar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies.

The test compound proved to be not toxic to the bacteria. 5000 µg/plate was chosen -as top dose level for the mutagenicity study.

In the absence of the metabolic activation system the test compound did not show a dose dependent influence on the number of revertants in any of the bacterial strains. Also in the presence of metabolic activation system, treatnent of the cells with test item did not result in relevant increases in the number of revertant colonies.

Conclusion

The substance resulted to be not mutagenic in bacterial test systems neither with nor without exogenous metabolic activation at the dose levels investigated.