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Genetic toxicity in vitro

Link to relevant study records
Reference
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:
8 April 2004 to 17 January 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline Study according to GLP
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
his- (s. typhimurium)
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix
Species / strain:
other: TA97a
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix
Test concentrations with justification for top dose:
5000, 1667, 556, 185, 62, 21 µg/plate
Vehicle:
DMSO was used as solvent for the test substance and for the negative control group.
Details on test system and conditions:
The exposure was performed according to the "Plate Incorporation Assay", in which bacteria, test substance (and microsomes) are in contact on the plate without preceding incubation in liquid state. The number of viable cells in the overnight-cuture is in the range of 200 000 000 cells per mL.
For each sample the following solutions were combined:
• 0.1 mL of the overnight culture of the bacteria,
• 0.5 mL of S9-mix (or phosphate buffered saline for samples without metabolic activation),
• 0.1 mL of the appropriate test- or reference substance solution and
• 2 mL of top agar.
The combined solutions were mixed and spread over a plate with minimal agar (9 cm diameter). After the top agar had solidified, the plates were incubated at 37 °C until the colonies were visible (2 days).

Bacterial strains of S. typhimurium (TA97a, TA98, TA100, TA102 and TA1535) were obtained from Prof. Bruce N. Ames, Berkely, California. The bacteria were stored in small portions in a solutions of 6 % DMSO in PBS in liquid nitrogen.

Counting of colonies: The plates of the strains with a low spontaneous revertant rate, i.e. TA98 and TA1535 were counted visually by marking the colonies with a felt tipped pen. The plates of the other strains were photographed with a video camera and the picture files were scanned for colonies by a computer program.

The results of the first experiment were verified by a second, independent experiment.

Triplicate repetitions were run for each dose group in each of the two separate experiments that were conducted, for the control groups six-fold repetitions were run.

Positive controls:
Strain TA97a: 4-NOPD 10 µg (without S9), DMBA 10 µg (with S9)
Strain TA98: 2-NF 2 µg (without S9), 2-AA 1 µg (with S9)
Strain TA100: Sodium-azide 2 µg (without S9), 2-AA 2ug (with S9)
Strain TA102: t-BHPO 50 µg (without S9), DHA 50 µg (with S9)
Strain TA1535: Sodium-azide 1 µg (without S9), 2-AA 2 µg (with S9)



Evaluation criteria:
Means and standard deviations were calculated for the number of mutants in every concentration group.
The criteria for a positive result were:
A reproducible increase of the number of revertants to more than the following threshold values for at least one of the concentrations:
• For the strains with a low spontaneous revertant rate i.e. TA98 and TA1535: 250 % of the amount of the spontaneous revertants.
• For the strains with a high spontaneous revertant rate i.e. TA97a, TA100 and TA102: 167 % of the amount of the spontaneous revertants.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
other: Toxicity at 5000 µg/plate without metabolisation.
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
other: Toxicity at 5000 µg/plate without metabolisation.
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
other: Toxicity at 5000 µg/plate without metabolisation.
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
other: Toxicity at 5000 µg/plate without metabolisation.
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
other: TA97a
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
other: Toxicity at 5000 µg/plate without metabolisation.
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
PRECIPITATION:
No precipitation of the test substance was seen in any of the concentration groups.

RANGE-FINDING/SCREENING STUDIES:
In a preliminary test the test substance was toxic to the bactera at 5000 µg/plate, resulting in a missing bacterial background lawn. In lower concentrations no toxicity was observed.

COMPARISON WITH HISTORICAL CONTROL DATA:
The numbers of spontaneous revertants were comparable with the historic control data for the negative controls.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the main test the test substance was again toxic to the bacteria at 5000 µg/plate, resulting in a reduced bacterial background in the samples without metabolisation. At 1667 µg/plate and beneath the bacterial background was normal.

Positive control substances:

All positive control substances increased the mutation frequency to more than the threshold values.

Conclusions:
Interpretation of results (migrated information):
negative in strains TA97a, TA98, TA100, TA102, TA1535

According to the results, "N-Chloro Succinimide" is not mutagenic in the Ames test with the strains of Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 up to 5000 µg per plate which is the limit concentration for this kind of test.
Executive summary:

Method

"N-CHLORO SUCCINIMIDE" was tested for mutagenic activity with the "Salmonella typhimurium Reverse Mutation Test" (Ames Test). The study was conducted in accordance with the OECD-guideline 471 and directive 2000/32/EC, part B.13/14.

The test substance, dissolved in DMSO, was tested at concentrations ranging from 21 µg to5000 µg per plate according to the "direct plate incorporation method" without external metabolisation as well as with an external metabolising system (S9-mix). As test system the bacterial strains Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 were used. Negative and positive controls were included. An independent repetition of the experiment was performed.

 

Results

Positive controls:

All positive control groups showed significantly increased mutation frequencies which demonstrate the sensitivity of the test system.

 

Test substance:

Toxicity:

A reduced bacterial background lawn was noted at 5000 µg/plate in the samples without metabolisation.

 

Solubility:

No precipitation of the test substance was seen in any of the concentration groups.

 


Mutagenicity:

In none of the concentrations tested and with none of the strains used an increase of the mutation frequency to more than the threshold values (250 % of the controls for strains TA98 and TA1535, 167 % of the controls for strains TA97a, TA100 and TA102) was obtained. Metabolic activation did not change these results.

 

 

Conclusion

 According to the results obtained in this study, "N-CHLORO SUCCINIMIDE" is non-mutagenic in the Ames test with the strains TA97a, TA98, TA100, TA102 and TA1535 up to 5000 µg/plate, which is the limit concentration for this kind of test.

.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Short description of key information:

N-CHLORO SUCCINIMIDE is non-mutagenic in the Ames test with the strains TA97a, TA98, TA100, TA102 and TA1535 up to 5000 µg/plate.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

No indications for the need of classification were obtained.