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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:
2000/01/30 to 2001/03/09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OCDE guideline with no deviation, GLP conditions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
Mutation in Histidine biosynthesis for Salmonella typhimurium: his D 3052/strain TA98 (frameshift), his G 46/strains TA100 and TA1535 (base-pair substitution) and his C 3076/strain TA1537 (frameshift).
Mutation in Tryptophan biosynthesis for Escherichia coli : WP2uvrA- (deletion in an excision repair gene).
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:
S9
Test concentrations with justification for top dose:
50 - 150 - 500 - 1500 - 5000 µg/plate
Vehicle / solvent:
acetone
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
for TA98 strain without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
for TA100, TA1535 and WPEuvrA strains without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
for TA1537 without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2 aminoanthracene
Remarks:
for TA100, TA1535, TA1537 and WP2uvrA with S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
for TA98 strain with S9
Details on test system and experimental conditions:
(1) Preliminary evaluation of cytotoxicity of the substance
The cytotoxicity was performed of the Salmonella Typhimurium strain TA 100 and in the Escherichia Coli WP2uvrA- with and without metabolic activation (S9 mix). The substance was tested at 10 concentrations : 0.15 ; 0.5 ; 1.5 ; 5 ; 15; 50; 150 ; 500 ; 1500 and 5000 µg/dish. The incubation system contained 0.1 ml of the diluted substance + 2 ml of top agar + 0.1 ml of bacterial culture +0.5 ml of S9 mix or phosphate buffer. The mixture was
plated onto a sterile plates of Vogel-Bonner Minimal agar. The plates were incubated at 37°C for 48 hours.
(2) Main study
4 strains of Salmonella Typhimurium were tested: TA98, TA100, TA1535 and TA1537. 1 strain of Escherichia Coli was tested : WP2uvrA-
As the concentration of 5000 µg/plate was not toxic in the strain TA 100 and UWP2uvrA- with and without metabolic activation during the
preliminary study, the following concentrations were tested during the main study: 50 ; 150 ; 500; 1500 and 5000 µg/dish.
Each concentration was tested 3 times under a constant volume of 0.1 ml with and without metabolic activation (S9 fraction obtained from rat livers pre-treated with ohenobarbitone/b-naphthoflavone at 80/100 mg/kg/day for 3 consecutive days) and used at on each strain. The incubation system contained 0.1 ml of the diluted substance + 2 ml of top agar + 0.1 ml of bacterial culture +0.5 ml of S9 mix or phosphate buffer. The mixture was plated onto the surface of Vogel-Bonner Minimal agar plate and incubated at 37°C for 48 hours.
Acetone and positive controls, 4-Nitroquinoline-1-oxide 0.2 µg/plate for TA-98; N-ethyl-N'-nitro-nitroguanidine at 3 µg/plate for
TA-100, at 5 µg/plate for TA-1535 and at 2 µg/plate for WP2uvrA-; 9-aminoacridine at 80 µg/plate for TA-1537. 2-aminoanthracene was used as
positive reference, in presence of S9 mix, at 1 µg/plate for TA100 ; at 2 µg/plate for TA1535 and TA1537 and at 10 µg/plate for WP2uvrA-.
Benzo(a)pyrene was used, in presence of S9 mix, at 5 µg/plate for TA98.
All the results were confirmed in a second study independent from the first.
Evaluation criteria:
(1) Preliminary cytotoxicity
After incubation, the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn. Manual counts were performed at 5000 µg/plate because of excessive test material precipitation. The signs of
toxicity were noted.
(2) Reverse mutation
At the end of the incubation period, the plates were assessed for numbers of revertant colonies using a Domino colony counter. Manual counts were performed at and above 5000 µg/plate because of excessive test material precipitation.


The test material may considered to be positive in the test system if it should have induced a reproducible, dose-related and statistically significant increase in the revertant count in at least one strain of bacteria. If a greater than twofold increase in revertant count is observed in two experiments
then this is taken as evidence of a positive response.
Statistics:
Dunnett's method of linear regression (p<0.05)

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:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Under experimental conditions employed, no value obtained in the presence of the test article was greater than or equal to twice the value obtained
in the presence of the vehicle with and without metabolic activation on the bacterial strains used.
All the results obtained in presence of positive controls were significant with and without metabolic activation in the used bacterial strains.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
The test article LCE00051, is not mutagenic as it induces no significant increase in the number of revertants with and without metabolic
activation of Salmonella Typhimurium TA98, TA100, TA1535 and TA1537 and in Escherichia Coli WP2uvrA-.
Executive summary:

The substance LCE00051 was tested on 4 strains of salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and in 1 strain of Escherichia Coli (WP2uvrA-), with or without metabolic activation. A range of sub-toxic concentrations was determined in a preliminary study on the strains TA-100 and WP2uvrA- with and without metabolic activation.

The 5 concentrations (50 - 150 - 500 - 1500 and 5000 µg/plate) were tested 3 times on the 5 strains mentioned above with and without metabolic activation. The results were confirmed in a second study, independant of the first.

In each study was included a negative control (vehicle = acetone) and a positive control (specific standard mutagen).

Under the experimental conditions employed, the substance LCE00051 did not show any mutagenic potential for the strain TA98, TA100, TA1535, TA1537 and WP2uvrA- with and without metabolic activation.