Benzene, ethylenated, residues, distn. lights

Genetic toxicity: in vitro

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:

8 July 2002 to 24 July 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was carried out according to OECD guideline 471 and Eu Method B13/14 nad it is GLP compliant.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Remarks:

.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

ethylbenzene

EC Number:

700-371-5

Cas Number:

178535-25-6

IUPAC Name:

ethylbenzene

Details on test material:

- Name of test material (as cited in study report): Ethylbenzene, manuf. of, distn. residues, distn. lights.
- Physical state: Clear, liquid yellow liquid
- Purity test date: 100%
- Lot/batch No.: P0204-1936
- Stability under test conditions: Stable for the duration of the test
- Storage condition of test material: Room temperature

Method

Target gene:

Reversion to histidine independence in Salmonella typhimurium and reversion to tryptophan independence in Escherichia coli.

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Type and identity of media: The batches are aliquots of nutrient broth cultures stored at -80C.
- Properly maintained: yes

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Details on mammalian cell type (if applicable):

- Type and identity of media: The batches were aliquots of nutrient broth cultures and were stored at -80C.
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Dose range-finding: 5, 15, 50, 150, 500, 1500 and 5000 ug/plate
Definitive test: 50, 150, 500, 1500 and 5000 ug/plate

Vehicle / solvent:

DMSO

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Test media

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Remarks:

5 µg/plate

Positive control substance:

benzo(a)pyrene

Remarks:

With S9 mix for strains TA1537, TA98 and TA100

Migrated to IUCLID6: 5 µg/plate

Controls 2

Untreated negative controls:

yes

Remarks:

Test media

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Remarks:

1 µg/plate

Positive control substance:

2-nitrofluorene

Remarks:

Without S9 mix for strain TA98

Migrated to IUCLID6: 1 µg/plate

Controls 3

Untreated negative controls:

yes

Remarks:

Test media

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Remarks:

0.5 µg/plate

Positive control substance:

sodium azide

Remarks:

Without S9 mix for strainsTA 1535 and TA100

Migrated to IUCLID6: 0.5 µg/plate

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

2 µg/plate

Positive control substance:

other: 2-aminoanthracene

Remarks:

With S9 mix for strain WP2uvrA/pKM101

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

50 µg/plate

Positive control substance:

9-aminoacridine

Remarks:

Without S9 mix for strain TA1537

Migrated to IUCLID6: 50 µg/plate

Controls 6

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide

Remarks:

Without S9 mix for strain WP2 uvrA/pKM101

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar plate (pre-incubation method)

DURATION
- Preincubation period: 1st test 10 hours, 2nd test: 30 minutse
- Exposure duration: 72 hours

NUMBER OF CELLS EVALUATED: 1000000000 cells/mL

Three replicates were used at each concentration.

Evaluation criteria:

If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice the concurrent solvent/vehicle controls, with some evidence of a positive dose-relationship (increased revertant colony counts at concentrations below that at which the maximal increase is obtained., it will be considered to show evidence of mutagenic activity in this system.

If exposure to a test substance does not produce an increase in revertant colony numbers in two separate experiments, with any bacterial strain either in the presence or absence of S9 mix, it will be considered to show no evidence of mutagenic activity in this test system.

If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response, even after the additional testing outlined in the mutation test procedure, the test data may be subjected to analysis to determine the statistical significance of any increase in revertant colony numbers. The statistical procedures used will be those described by Mahon et al (1989) and will usually be analysis of variance followed by Dunnett`s test. Biological significance should always be considered along with statistical significance.

Statistics:

Statistical analysis is not performed unless the results faild to identify a clear positive or negative response, the test data may be subjected to analysis to determine the statistical significance of any increase in revertant colony numbers. The statistical procedures used will be those described by Mahon et al (1989) and will usually be analysis of variance followed by Dunnett`s test.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: The range-finding test was carried out in the presence and absence of S9 mix up to 5000 µg/plate. No substantial increases in revertant colony numbers were reported at any concentration tested.

COMPARISON WITH HISTORICAL CONTROL DATA: The mean revertant colony counts for the solvent controls were within the 99% confidence limits of the current historical control range of the laboratory

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions of this study, Ethylbenzene, manuf. of, distn. residues, distn. lights. is not considered to be mutagenic.

Executive summary:

An in vitro gene mutation assay was carried out on Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and a tryptophan dependent mutant of Escherichia coli, strain WP2uvrA/pKM101. The strains were exposed to Ethylbenzene, manuf. of, distn. residues, distn. lights. at concentration up to 5000 µg/plate. The test 1 was tested at 5, 15, 50, 150, 500, 1500 and 5000 ug/plate and the test 2 was performed at

50, 150, 500, 1500 and 5000 ug/plate. L

Ethylbenzene, manuf. of, distn. residues, distn. lights. was diluted in DMSO which was used as negative control. No signs of toxicity were observed towards the tester strains in test 1. Toxicity (thinning of the background lawn of non-revertant cells, together with a reduction in revertant colony numbers) was seen in all strains except CM891 following to Ethylbenzene, manuf. of, distn. residues, distn. lights. at 5000 µg/plate. No evidence of mutagenic activity was seen at any concentration of Ethylbenzene, manuf. of, distn. residues, distn. lights. in either mutation test. The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. It can be concluded that, under the test conditions employed, Ethylbenzene, manuf. of, distn. residues, distn. lights. is not considered to be mutagenic.