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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: Well performed GLP and OECD guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
his- for S. typhimurium strains TA 1537, TA 1535, TA 100, TA 98
trp- for E. coli WP2 uvr A
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital/ß-naphthoflavone-induced rat liver S9-mix
Test concentrations with justification for top dose:
Experiment I (plate incorporation) and Experiment II (pre-incubation): 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate both with and without metabolic activation
Vehicle / solvent:
DMSO, purity >99% (Merck, Darmstadt, Germany). The solvent was chosen because of its solubility properties and its relative non-toxicity to bacteria
Controls
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without metabolic activation: sodium azide, 4-nitro-o-phenylene-diamine, methyl methane sulfonate / With metabolic activation: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION:
plate incorporation (experiment I); preincubation (experiment II). Since experiment I gave a negative result, experiment II was performed as a
preincubation assay.

DURATION
- Preincubation period: 60 minutes at 37°C
- Exposure duration: 48 hours at 37°C

NUMBER OF REPLICATIONS: Two Experiments (Exp. I and II). Each concentration, including the controls, was tested in triplicate.

DETERMINATION OF CYTOTOXICITY: yes
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 WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

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.
Statistics:
Not mandatory according to OECD guideline 471

Results and discussion

Test resultsopen allclose all
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:
TA 1535: 5000 µg/plate without metabolic activation; TA 1537: 2500 - 5000 µg/plate with metabolic activation; TA 98: 5000 µg/plate with metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
Pre-experiment was reported as experiment I because the criterion (evaluable plates (>0 colonies) at five concentrations or more in all strains are used) was met.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

The plates incubated with the test item showed normal background growth in all strains with and without metabolic activation in both independent experiments.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups at the following concentrations (µg/plate):



Strain

Experiment I

Experiment II

 

without S9 mix

with S9 mix

without S9 mix

with S9 mix

TA 1535

/

/

5000

/

TA 1537

/

2500 -5000

/

/

TA 98

/

5000

/

/

TA 100

/

/

/

/

WP2 uvrA

/

/

/

/

/ = no toxic effects observed

Precipitation of the test item was observed in the overlay agar in the test tubes from 2500 µg/plate up to 5000 µg/plate in both experiments. Precipitation on the incubated agar plates was observed from 1000 µg/plate up to 5000 µg/plates in experiment I and II in the absence of metabolic activation. In the presence of metabolic activation precipitation was observed from 333 µg/plate up to 5000 µg/plates in experiment I and from 2500 µg/plate up to 5000 µg/plates in experiment II. The undissolved particles had no influence on the data recording.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There is also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknoledged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative both with and without metabolic activation

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, Pigment Red 254 is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:
This study was performed to investigate the potential of Pigment Red 254 to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and the Escherichia coli strain WP2 uvrA.

 

The assay was performed according to OECD TG 471 with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

 

Pre-Experiment (Experiment I) and Experiment II: 3; 10; 33; 100; 333; 1000; 2500; and 5000μg/plate.

 

The plates incubated with the test item showed normal background growth up to 5000μg/plate with and without metabolic activation in both independent experiments.

 

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in nearly all strains with and without metabolic activation. Only in strains TA 1535, TA 1537 and TA 98 a minor reduction in the number of revertants (below the indication factor of 0.5) was observed at 2500 µg/plate and/or 5000 µg/plate.

 

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Pigment Red 254 at any dose level, neither in the presence nor absence of metabolic activation (S9 mix).

 

 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, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.