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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 Aug 2001 - 29 Nov 2001 (experimental)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
- Name of test material (as cited in study report): Tert.-Butylacrylat
- Physical state: colorless liquid
- Analytical purity: 99.6%
- Purity test date: 2001-07-16
- Lot/batch No.: Tank B 601
- Stability under test conditions: verified by reanalysis
- Storage condition of test material: Refrigerator, protected from light

Method

Target gene:
his- (S. typhimurium), trp- (E. coli)
Species / strainopen allclose all
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:
Aroclor 1254 induced rat liver S-9 mix
Test concentrations with justification for top dose:
20, 100, 500, 2500 and 5000 µg/plate both, in the standard plate test (SPT) and in the preincubation test (PIT)
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
other: N-methyl-N'-nitro-N-nitrosoguanidine (4-MNNG), 4-nitrophenylenediamine (4-NPD), 2-aminoanthracene (2-AA)
Details on test system and experimental conditions:
STANDARD PLATE TEST
Test tubes containing 2 ml soft agar kept in a water bath at 45°C, and remaining components added in the following order:
0.1 mL test solution or vehicle
0.1 mL bacterial suspension
0.5 mL S-9 mix (in tests with metabolic activation) or 0.5 mL phosphate buffer (in tests without metabolic activation).
After mixing, the samples are poured onto Vogel-Bonner agar plates (S. typhimurium) or minimal agar plates (E.coli)

PREINCUBATION TEST
0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S-9 mix are incubated at 37°C for the duration of 20 minutes. Subsequently, 2 mL of soft agar is added and, after mixing, the samples are poured onto the agar plates.

BOTH TESTS
In each experiment 3 test plates per dose or per control used; after incubation at 37°C for 48 hours in the dark, the bacterial colonies ( his+/trp+ revertants) are counted. The titer was determined and in regularly measurements the strain characteristics were checked. Sterility control was performed.
Evaluation criteria:
POSITIVE RESULTS
- A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adding a metabolizing system.
- A test substance is generally considered nonmutagenic in this test if: the number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

ACCEPTANCE CRITERIA
- The number of revertant colonies in the negative controls was within the normal range of the historical control data for each tester strain.
- The sterility controls revealed no indication of bacterial contamination.
- The positive control articles both with and without S-9 mix induced a significant increase in the number of revertant colonies within the range of the historical control data.
- The titer of viable bacteria was >= E+08/mL

Results and discussion

Test resultsopen allclose all
Key result
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:
cytotoxicity
Remarks:
A slight decrease in the number of revertants was occasionally observed in the standard plate test. In the preincubation test bacteriotoxicity was found from about 500 µg/plate onward.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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:
cytotoxicity
Remarks:
A slight decrease in the number of revertants was occasionally observed in the standard plate test. In the preincubation test bacteriotoxicity was found from about 500 µg/plate onward.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No test substance precipitation was found.

Any other information on results incl. tables

Genotoxicity

Standard plate test (20 - 5000 µg/plate)

Strain

Metabolic activation system

Replicates

maximum revertant factor*

dose dependency

Assessment

TA 98

no

3

0.9

no

negative

 

yes

3

0.7

no

negative

TA 100

no

3

1.0

no

negative

 

yes

3

1.0

no

negative

TA 1535

no

3

0.9

no

negative

 

yes

3

1.0

no

negative

TA 1537

no

3

1.0

no

negative

 

yes

3

1.0

no

negative

WP2 uvr A

no

3

1.0

no

negative

 

yes

3

0.8

no

negative

Preincubation test (20 - 5000 µg/plate)

Strain

Metabolic activation system

Replicates

maximum revertant factor*

dose dependency

Assessment

TA 98

no

3

0.9

no

negative

 

yes

3

0.9

no

negative

TA 100

no

3

1.0

no

negative

 

yes

3

0.9

no

negative

TA 1535

no

3

1.0

no

negative

 

yes

3

1.0

no

negative

TA 1537

no

3

1.0

no

negative

 

yes

3

0.9

no

negative

WP2 uvr A

no

3

1.0

no

negative

 

yes

3

1.3

no

negative

* in non-toxic doses

Applicant's summary and conclusion