Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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:
May 4, 1984 - May 17, 1984
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-guideline study performed under QAU surveillance. No E. coli WP2/TA 102 included in the test, but with sufficient details and acceptable for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1984
Report Date:
1984

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: the method described by AMES et al. (AMES et al., 1973; AMES et al., 1975)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
no E.coli WP2/TA 102 strain included
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Analytical purity: Commercial grade

Method

Target gene:
his
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced S9 fraction of rat liver
Test concentrations with justification for top dose:
20, 80, 320, 1280 and 5120 µg/0.1 ml in both experiment I and II
Vehicle / solvent:
- Solvent used: acetone
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see "details on test system and conditions"
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours at 37 +/- 1.5 °C in darkness.

NUMBER OF REPLICATIONS: 3 Petri dishes were prepared per strain and per group. In order to confirm the result, the experiment was repeated once.

DETERMINATION OF CYTOTOXICITY
- Method: growth of background lawn

POSITIVE CONTROLS:
- without S9 mix:
TA98: daunorubicin-HCl, 5 and 10 µg/0.1 ml phosphate buffer
TA 100: 4-nitroquinoline-N-oxide, 0.125 and 0.25 µg/0.1 ml phosphate buffer
TA 1535: N-methyl-N'-nitro-N-nitrosoguanidine, 3 and 5 µg/0.1 ml phosphate buffer
TA 1537: 9(5)aminoacridine hydrochloride monohydrate, 50 and 100 µg/0.1 ml DMSO
- with S9 mix:
TA 100: 2-aminoanthracene, 5 µg/0.l ml DMSO.
TA 1535: cyclo-phosphamide, 250 µg/0.1 ml phosphate buffer
Evaluation criteria:
The test substance is generally considered to be non-mutagenic if the colony count in relation to the negative control is not doubled at any concentration.
Statistics:
When the colonies had been counted, the arithmetic mean was calculated.

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:
cytotoxicity
Remarks:
A growth-inhibiting effect was observed at the upper concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At the concentrations of 1280 and 5120 µg/0.l ml the substance precipitated in soft agar


ADDITIONAL INFORMATION ON CYTOTOXICITY: A growth-inhibiting effect of the compound was observed in the experiments without microsomal activation at the upper concentrations. In the experiments with microsomal activation this effect was less pronounced.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

NUMBER OF BACK-MUTANT COLONIES PER PLATE (ARITHMETIC MEAN)

Experiment 1:

  TA98 TA100 TA1535 TA1537
  + S9 -S9 + S9 -S9 + S9 -S9 + S9 -S9
vehicle control 32 28 144 137 13 12 16 11
20 µg/0.1ml 36 20 134 122 10 14 10 11
80 µg/0.1ml 43 27 143 148 16 13 9 8
320 µg/0.1ml 42 19 123 111 9 10 9 7
1280 µg/0.1ml 27 19 102 86 6 8 4 2
5120 µg/0.1ml 15 7 36 27 1 1 2 7
                 
Positive controls*                
vehicle control   25 120 125  12 13 11
concentration 1   318 1798 843 283  - 47
concentration 2   537   1308   2372   965

Experiment II:

  TA98 TA100 TA1535 TA1537
  + S9 -S9 + S9 -S9 + S9 -S9 + S9 -S9
vehicle control 39 30 148 140 16 21 11 5
20 µg/0.1ml 38 19 133 156 19 16 10 5
80 µg/0.1ml 49 22 151 120 16 16 12 6
320 µg/0.1ml 49 21 145 104 15 11 8 7
1280 µg/0.1ml 35 25 111 8 12 4 4 0
5120 µg/0.1ml 13 4 50 2 4 3 0 0
                 
Positive controls*                
vehicle control   28   167 18  13 8
concentration 1   438   1030 561  737 42
concentration 2   816   1809   2403   549

* for details on positive controls, see "details on test system and conditions"

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

No evidence of the induction of point mutations by the test article or by the metabolites of the substance formed as a result of microsomal
activation was detectable in the strains of S. typhimurium used in these experiments.
Executive summary:

The test article was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 with the following concentrations of the trial substance: 20, 80, 320, 1280 and 5120 µg/0.1 ml. In order to confirm the results the experiments were repeated. To ensure that mutagenic effects of metabolites of the test substances formed in mammals would also be detected, experiments were performed in which the cultures were additionally treated with an activation mixture (rat liver microsomes and co-factors). In the experiments performed without and with microsomal activation, comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations of the test substance revealed a reduction in the colony count due to a growth-inhibiting effect of the compound at the upper concentrations. No evidence of the induction of point mutations by the test article or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments. Therefore, the test article is considered to be not mutagenic under the presented experimental conditions.