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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline study and GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Commission Directive 2000/32/EC, L1362000, Annex 4D,dated May 19, 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Kn 172
Batch No.: Kn-Gi-8634/1
SAT 030768

Method

Target gene:
hisC3076 (frameshift): S. typhimurium TA1537
hisD3052/R-factor (frameshift): S. typhimurium TA98
hisG46 (base-pair substitutions): S. typhimurium TA1535
hisG46/R-factor (base-pair substitutions): S. typhimurium TA100
his G 428; rfa-; uvrB';R-factor(base-pair substitutions): S.typhimurium TA102
Species / strain
Species / strain / cell type:
other: TA 98, TA 100, TA 102, TA 1535, 1537
Additional strain / cell type characteristics:
other: each Salmonella strain, additional mutations: rfa-; uvrB';R-factor
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbitallß-Naphthoflavone induced rat liver S9 is used as the metabolic activation system. The S9 is prepared from 8 - 12 weeks old male Wistar Hanlbm rats.
Test concentrations with justification for top dose:
10 - 5000 ug/plate
Vehicle / solvent:
deionised water
Controls
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
no
Details on test system and experimental conditions:
The experiments were performed to assess the potential of the test item to induce gene
mutations by means of two independent Salmonella typhimurium reverse mutation assays.
Experiment I was performed as a plate incorporation assay. Since a negative result was
obtained in this experiment, experiment II was performed as a pre-incubation assay.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of
revertants exceeding the threshold of twice (strains TA 98, TA 100, and TA 102) or thrice
(strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is
observed (3).
A dose dependent increase is considered biologically relevant if the threshold is exceeded
at more than one concentration (2).
An increase exceeding the threshold at only one concentration is judged as biologically
relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is
regarded as an indication of a mutagenic potential if reproduced in an independent second
experiment. However, whenever the colony counts remain within the historical range of
negative and solvent controls such an increase is not considered biologically relevant.

Results and discussion

Test results
Species / strain:
other: Salmonella typhimurium strains: TA 1535, TA 1537, TA 98, TA 100, TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Toxic effects, evident as a reduction in the number of revertants, were observed at higher concentrations in strains TA 98, TA 100 and TA 102 without metabolic activation in experiment I.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The plates incubated with the test item showed normal background growth up to
5000 mg/plate with and without S9 mix in all strains used with the exception of strain TA
102 where a reduction of the background growth was observed at 5000 mg/plate without
metabolic activation in experiment I.
Remarks on result:
other: strain/cell type: Salmonella typhimurium strains: TA 1535, TA 1537, TA 98, TA 100, TA 102
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Toxic effects, evident as a reduction in the number of revertants, were observed at higher concentrations in strains TA 98, TA 100 and TA 102 without metabolic activation in experiment I.

No substantial increase in revertant colony numbers of any of the five tester strains was

obsewed following treatment with KN 172 at any dose level, neither in the presence nor

absence of metabolic activation (S9 mix) with the exception of strain TA 98 without

metabolic activation in experiment I where a slight increase in revertant colonies was

obsewed at 2500 and 5000 mg/plate. However, this effect could not be reproduced in an

independent experiment. There was also no tendency of higher mutation rates with

increasing concentrations in the range below the generally acknowledged border of

biological relevance.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Therefore, KN 172 is considered to be non-mutagenic in this Salmonella typhimurium
reverse mutation assay.
Executive summary:

This study was performed to investigate the potential of KN 172 to induce gene mutations

according to the plate incorporation test (experiment I) and the pre-incubation test

(experiment II and II A) using the Salmonella typhimurium strains TA 1535, TA 1537,

TA 98, TA 100, and TA 102.

The assay was performed with and without liver microsomal activation. Each

concentration, including the controls, was tested in triplicate. The test item was tested at

the following concentrations:

10, 33; 100; 333; 1000; 2500; and 5000 pglplate

The plates incubated with the test item showed normal background growth up to

5000 pglplate with and without S9 mix in all strains used with the exception of strain TA

102 where a reduction of the background growth was obsewed at 5000 pglplate without

metabolic activation in experiment I.

Toxic effects, evident as a reduction in the number of revertants, were observed at higher

concentrations in strains TA 98, TA 100 and TA 102 without metabolic activation in

experiment I.

No substantial increase in revertant colony numbers of any of the five tester strains was

obsewed following treatment with KN 172 at any dose level, neither in the presence nor

absence of metabolic activation (S9 mix) with the exception of strain TA 98 without

metabolic activation in experiment I where a slight increase in revertant colonies was

obsewed at 2500 and 5000 pglplate. However, this effect could not be reproduced in an

independent experiment. There was also no tendency of higher mutation rates with

increasing concentrations in the range below the generally acknowledged border of

biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase

of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the

experimental conditions reported, the test item did not induce gene mutations by base pair

changes or frameshifts in the genome of the strains used.

Therefore, KN 172 is considered to be non-mutagenic in this Salmonella typhimurium

reverse mutation assay.