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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:
2000-01-25 to 2000-03-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21st July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
29 Decembre 1992
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Remarks:
Escherichia coli WP2 uvrA pkM101
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
- source of S9
Male (Wistar) rats (aged 6-8 weeks) were given a single intraperitoneal injection of Aroclor 1254 (500 mg/kg body weight) diluted in Miglyol 812 oil (MERCK, Article-No. 6175). Five to seven days after application of Aroclor, the rats were sacrificed and the livers collected in ice-cooled, sterilized beakers containing 0.15 M KCl. The animals were prepared unfasted.

- method of preparation of S9 mix
The livers were homogenized in a sterile glass potter homogenizer'-containing 3 mL of 0.15 M KCl per gram of liver wet-weight. The homogenate was spun at 9000 x g for 10 minutes at about +4°C and the supernatant fluid was decanted and transferred into sterilized and precooled plastic tubes. The S9 was then frozen in liquid nitrogen and stored at -196 °C.

- concentration or volume of S9 mix in the final culture medium : 0.5 mL/plate (S9 homogenate 0.1 mL (1st series); 0.3 mL (2nd series))

- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)
Every S9-batch is tested for its metabolic activity by the use of specific substrates, requiring different enzymes of the P450-isoenzyme family. The mutagenicity of 2-aminoanthracene, benzo[a]pyrene, and 3-methylcholanthrene is thus determined once for every S9-batch.
Test concentrations with justification for top dose:
1st series: 5.00, 15.8, 50.0, 158, 500, 1580 and 5000 µg/plate (± S9 mix)
2nd series: 50.0, 158, 500, 1580 and 5000 µg/plate (± S9 mix)
Vehicle / solvent:
Solvent: DMSO
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
DAUN
Positive control substance:
other: Daunomycin
Remarks:
TA 98 (without S9 mix)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
ENNG
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
TA 100, 1535, WP2 (without S9 mix)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
CUM
Positive control substance:
cumene hydroperoxide
Remarks:
TA 102 (without S9 mix)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
9-AA
Positive control substance:
9-aminoacridine
Remarks:
TA 1537 (without S9 mix)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
2-AA
Positive control substance:
other: 2-Aminoanthracene
Remarks:
TA 98, TA 100, TA 1535, TA 1537, WP 2 (with S9 mix)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
B(a)P
Positive control substance:
benzo(a)pyrene
Remarks:
TA 102 (with S9 mix)
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; in agar (plate incorporation)

TREATMENT SCHEDULE:
- Exposure duration/duration of treatment: 48 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition
Evaluation criteria:
A test material is defined as non-mutagenic in this assay if
• "no" or "weak increases" occur in the first and second series of the main experiment. ("Weak increases" randomly occur due to experimental variation.)
A test material is defined as mutagenic in this assay if
• a dose-related (over at least two test material concentrations) increase in the number of revertants is induced, the maximal effect is a "clear increase", and the effects are reproduced at similar concentration levels in the same test system;
• "clear increases" occur at least at one test material concentration, higher concentrations show strong precipitation or cytotoxicity, and the effects are reproduced at the same concentration level in the same test system.

In all further cases, a third test series with the bacterial strain in question should be performed. If the criteria for a positive test result are not fulfilled in at least two out of the three series, the test material is defined as being non-mutagenic in this test system.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no 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:
TEST-SPECIFIC CONFOUNDING FACTORS
None observed.

STUDY RESULTS
Ames test:
- Signs of toxicity : No cytotoxicity was observed.
- Mean number of revertant colonies per plate and standard deviation : Refer to attached background material

Applicant's summary and conclusion

Conclusions:
With and without addition of S9 mix as external metabolising system, the test substance was not mutagenic under the experimental conditions described.
Executive summary:

The investigations for mutagenic potential were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA pkM 101. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed. The test substance was dissolved in dimethyl sulfoxide and tested at the following concentrations:

 

1st series: 5.00, 15.8, 50.0, 158, 500, 1580 and 5000 µg/plate (± S9 mix)

2nd series: 50.0, 158, 500, 1580 and 5000 µg/plate (± S9 mix)

 

Precipitation of the test material on the agar plates and toxicity to the bacteria was not observed. Daunomycin, N-ethyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine and cumene hydroperoxide served as strain specific positive control compounds in the absence of S9 mix. 2-Aminoanthracene and benzo[a]pyrene were used for testing the bacteria and the activity of the S9 mix. Each treatment with the substances used as positive controls led to a clear increase in revertant colonies, thus showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.

With and without addition of S9 mix as the external metabolizing system, the test substance was not mutagenic under the experimental conditions described.