Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

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:
other: Study performed according to OECD Guideline 471. Study in Japanese (original study report available) without complete English translation.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Principles of method if other than guideline:
Pre-incubation method; Study performed according to Guidelines for Screening Toxicity Testing of Chemicals (Japan) and OECD Guidelines No. 471
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Diisopropylbenzene
EC Number:
246-835-6
EC Name:
Diisopropylbenzene
Cas Number:
25321-09-9
Molecular formula:
C12H18
IUPAC Name:
1,2-bis(propan-2-yl)benzene; 1,3-bis(propan-2-yl)benzene; 1,4-bis(propan-2-yl)benzene
Details on test material:
Lot/batch No.: CAF5267
Purity: 99.8 %

Method

Target gene:
histidine (S. typhimurium), tryptophan (E. coli)
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: from rat liver (induction with phenobarbital and 5,6-benzoflavone)
- method of preparation of S9 mix :
S9 0.1 mL
magnesium chloride 8 μmol
potassium chloride 33 μmol
glucose-6-phosphate 5 μmol
NADH 4 μmol
NADPH 4 μmol
Sodium-phosphate buffer (pH 7.4) 100 μmol

- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): metabolic capability is proven with specific positive control (2-Aminoanthracene)
Test concentrations with justification for top dose:
-S9 mix:
0, 0.195, 0.391, 0.781, 1.56, 3.13 and 6.25 ug/plate (TA 1537)
0, 0.781 - 50.0 µg/plate (TA 100, TA 1535, TA 98 [Test 1])
0, 0.391 - 12.5 µg/plate (TA 1535 [Test 2])
0, 0.781 - 25.0 µg/plate (TA 100, TA98 [Test 2])
0, 156 - 5000 µg/plate (E. coli WP2 uvrA)

+S9 mix:
0, 6.25 - 200 µg/plate (TA 100, TA 1535, TA 98, TA 1537)
0, 19.5 - 625 µg/plate (E. coli WP2 uvrA)

Diisopropylbenzene was tested with a ratio of about 3 in the range of 50.0 to 5000 μg/plate. As a result, in the S9 mix-free test, antimicrobial activity was observed at 5000 μg/plate for WP2 uvrA and at all doses for the other test bacteria. In the S9 mix addition test, antimicrobial properties were observed for WP2 uvrA at 500 μg/plate or more and for the other test bacteria at more than 150 μg/plate.
Therefore, the highest dose in this study was 5000 μg/plate for WP2 uvrA and 50.0 μg/plate for other test bacteria for S9 mix-free test. In the S9 mix addition test, WP2 uvrA was tested up to 625 μg/plate, and for the other test bacteria, 200 μg/plate was used.
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
see below
Positive control substance:
9-aminoacridine
sodium azide
other: 2-Aminoantracene, 2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:

- Test substance added in preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 min with test substance
- Exposure duration/duration of treatment: 48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition
Evaluation criteria:
Among the five test bacteria used, in the S9 mix-free test or S9 mix addition test of one or more test bacteria, when the average number of mutant colonies on a flat plate containing the test substance increased more than twice the solvent control value, and reproducibility and dose dependence were observed in the increase, the test substance is determined to be mutagenic (positive) in this test system.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES (if applicable):
Diisopropylbenzene was tested with a ratio of about 3 in the range of 50.0 to 5000 μg/plate. As a result, in the S9 mix-free test, antimicrobial activity was observed at 5000 μg/plate for WP2 uvrA and at all doses for the other test bacteria. In the S9 mix addition test, antimicrobial properties were observed for WP2 uvrA at 500 μg/plate or more and for the other test bacteria at more than 150 μg/plate.
Therefore, the highest dose in this study was 5000 μg/plate for WP2 uvrA and 50.0 μg/plate for other test bacteria for S9 mix-free test. In the S9 mix addition test, WP2 uvrA was tested up to 625 μg/plate, and for the other test bacteria, 200 μg/plate was used.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: Please refer to attached tables

Ames test:
- Signs of toxicity: Please refer to attached tables
- Individual plate counts: Please refer to attached tables
- Mean number of revertant colonies per plate and standard deviation: Please refer to attached tables

Applicant's summary and conclusion

Conclusions:
The test substance was not mutagenic in Salmonella typhimurium TA 100, TA 1535, TA 98, TA 1537 and Escherichia coli WP2 uvrA, with or without an exogenous metabolic activation system.
Executive summary:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 as well as E. coli WP2 uvr A were tested in a pre-incubation assay with and without metabolic activation (S9) performed according to OECD Guidelines No 471.

The test concentrations were given as follows:

-S9 mix:

0, 0.195, 0.391, 0.781, 1.56, 3.13 and 6.25 µg/plate (TA 1537)

0, 0.781 - 50.0 µg/plate (TA 100, TA 1535, TA 98 [Test 1])

0, 0.391 - 12.5 µg/plate (TA 1535 [Test 2])

0, 0.781 - 25.0 µg/plate (TA 100, TA98 [Test 2])

0, 156 - 5000 µg/plate (E. coli WP2 uvrA)

+S9 mix:

0, 6.25 - 200 µg/plate (TA 100, TA 1535, TA 98, TA 1537)

0, 19.5 - 625 µg/plate (E. coli WP2 uvrA).

The test substance was not mutagenic in Salmonella typhimurium TA 100, TA 1535, TA 98, TA 1537 and Escherichia coli WP2 uvrA, with or without an exogenous metabolic activation system.