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Toxicological information

Genetic toxicity: in vitro

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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:
1996-03-14 to 1996-04-26
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1983)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
(1992)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
2,2,4(or 2,4,4)-trimethyl-1,6-hexanediol of Hüls AG
purity 94.6 % (GC-FID area)
batch No. Pt. 15570/3033 of August 1995; sample ID 0637/81 770

Method

Target gene:
mutated gene loci responsible for histidine auxotrophy
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Test concentrations with justification for top dose:
30 to 5000 µg/plate
Vehicle / solvent:
The solvent controls quantify the spontaneous mutation frequency of each bacterial strain in the presence of acetone.
Test concentration: 2.5 µg/plate (plate incorporation and preincubation test)
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Aceton
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
other: with metabolic activation, 2-aminoanthracene, 2.5 µg/plate
Details on test system and experimental conditions:
SYSTEM OF TESTING
- Metabolic activation system:   Aroclor 1254-induced rat liver S9 fraction, obtained from Cytotest Cell  Research GmbH & Co. KG (Rossdorf, Germany), lots No. 170795 and  050296,  prepared from male Wistar rats

ADMINISTRATION: 
- Dosing:   
 Plate incorporation test: 50/160/500/1600/5000 µg/plate (+/- metabolic  activation) repeated for TA 1537 (- metabolic activation) due to absence of 
 background lawn in all plates   Pre-incubation test: same concentrations for TA 100 and TA 1537; for TA  98 and TA 1535 only 30/100/300/1000/3000 µg/plate (+/- metabolic  activation)   repeated for TA 1535 (- metabolic activation) due to exceptionally low  revertant frequency in control (2 +/- 2; published background level 3 to  37) - Number of replicates: 3
- Application: solvent acetone (CAS No. 67-64-1)
- Positive and negative control groups and treatment:   
 positive without metabolic activation:   
TA 98: 2-nitrofluorene (2.5 µg/plate in dimethyl sulfoxide)   
TA 100: sodium azide (5.0 µg/plate in twice distilled water)   
TA 1535: sodium azide (2.5 µg/plate in twice distilled water)   
TA 1537: 9-aminoacridine (25 µg/plate in dimethyl sulfoxide)   
positive with metabolic activation and activity of metabolic system:   
all strains: 2-aminoanthracene (2.5 µg/plate in dimethyl sulfoxide)   
negative: solvent control, 25 µl/plate
- Pre-incubation: 30 minutes at 30 °C   incubation 72 hours at 37 °C

CRITERIA FOR EVALUATING RESULTS:    ratio of revertant rates treated/control >= 2 with generally positive  dose-response relationship in any strain in two independent experiments
Rationale for test conditions:
OECD Guideline 471
Evaluation criteria:
For a test compound to be considered positive, it must (in two independent experiments) cause at least a doubling in the mean revertants per plate of at least one tester strain. This increase must be accompanied by a dose response towards increasing concentrations of the test article. A test article that does not meet these criteria will be called non-mutagenic in bacteria. Single increases in revertant frequencies, which are not dose-related and not reproducible in two independent tests are considered non-relevant. If however these increases do occur in both tests, this will be taken as an indication of a mutagenic effect.
Statistics:
The stated means, standard deviations and factors were calculated through a computer program written by Messrs BIOSYS. The usual statistical methods were used for the calculations.

Results and discussion

Test results
Key result
Species / strain:
other: TA 98, TA 100, TA 1535, TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: 5000 µg/plate in some cases, only with metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
GENOTOXIC EFFECTS: 
- With metabolic activation: None
- Without metabolic activation: None   
The positive controls were functional.
PRECIPITATION CONCENTRATION: No precipitation was observed.
CYTOTOXIC CONCENTRATION (including effects on background lawn): 
- With metabolic activation only:   
 TA 98, TA 1535 in plate incorporation assay  
 TA 1537 in pre-incubation assay

Any other information on results incl. tables

no other results

Applicant's summary and conclusion

Conclusions:
The test item did not induce a mutagenic effect in S. typhimurium TA 98, TA 100, TA 1535 and TA 1537, both in the presence and in the absence of Arochlor-induced liver microsomes . It is therefore not considered to be a bacterial mutagen.
Executive summary:

The test item was tested in the Ames Salmonella mutagenicity test for any mutagenic activity.

Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 were treated with the test compound by the Ames test plate incorporation ss well as the preincubation method.

Dose levels covering the range between 30 and 5000 fLg/plate, in triplicate both with and without the addition of a metabolising system

( Aroclor 1254 ioduced rat liver S9 mix) were employed. All four bacterial strains exhibited mutagenic responses to the appropriate positive control substances.

Solvent controls were also tested with each strain and the mean numbers of spontaneous revertants were in an acceptable range.

Mutagenic activity of the test compound to one or more of the tester strains was not observed in either experiment with and without metabolic activation. It is therefore concluded, that the test item is not a bacterial mutagen.