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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:
weight of evidence
Study period:
August 1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1983
Report Date:
1983

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
No E. coli strain tested
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Isolan Rot S-RL - Comparison of two batches

Method

Target gene:
Histidine locus
Species / strain
Species / strain / cell type:
other: TA 1538, TA 1537, TA 100, TA 98
Metabolic activation:
with and without
Metabolic activation system:
rat S9-mix
Test concentrations with justification for top dose:
Concentrations used: 10000, 5000, 1000, 500, 100, 50, 10 and 5 µg/plate
No bacteriotoxic effect up to 10000 µg/plate
Vehicle / solvent:
demineralised water
Controls
Untreated negative controls:
no
Remarks:
because the vehicle was water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Cyclophosphamide, trypaflavine, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation: 0.1 mL TS+0.1 mL bacteria+0.5 mL S9+2 mL soft agar: 30 sec at 45 °C
- Incubation period: 48 hours at 37°C

NUMBER OF REPLICATIONS: 4 plates/strain/concentration

DETERMINATION OF CYTOTOXICITY
- Method: - background growth
- marked and dose-dependent reduction in mutant count compared to negative controls
- titer determination

Acceptance criteria:
a) The negative controls had to be within the expected range, as defined by published data (i.e. Maron and Ames, 1983) and the laboratory's own historical data
b) The positive controls had to show sufficient effects, as defined by the laboratory's experience
c) Titer determinations had to demonstrate sufficient bacterial density in the suspension.

An assay which did not comply with at least one of the above criteria was not used for assessment. Furthermore, the data generated in this assay needed to be confirmed by two additional independent experiments. Even if the criteria for points (a), (b) and (c) were not met, an assay was accepted if it showed mutagenic activity of the test compound.
Evaluation criteria:
A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result; this increase should be about twice the amount of negative controls.
Statistics:
N.A.

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
Remarks:
TA 100: 1.6-fold increase at cytotoxic concentrations
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
There was no indication of a bacteriotoxic effect of the test item at up to 10000 µg per plate. The total bacteria counts consistently produced results comparable to the negative controls, or differed only insignificantly. Nor was any inhibition of growth noted.

All four strains concerned showed a dose related and biologically relevant increase in mutant counts over those of the negative controls. This applied both to both batches with and without S9 mix.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Both batches of Acid Red 414 showed mutagenic effects in the Salmonella/microsome test with and without a metabolic activation.
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA 100, TA 1537, TA 1538 and TA 98 of S. typhimurium were exposed to two batches of Acid Red 414 at concentrations of up to 10000 µg/plate.

Concentrations of up to and including 10000 µg/plate did not cause any bacteriotoxic effects. The total numbers of bacteria remained unchanged. No inhibition of growth was observed.

Evidence of a mutagenic activity of Acid Red 414 was found for both batches. In all Salmonella typhimurium strains used a biologically relevant increase in the mutant count of more than double of the corresponding negative control was found. The lowest effective concentrations for batch Ök.Nr. 1190 and Ök.Nr. 2027 were 10 and 50 µg/plate for Salmonella typhimurium TA 1538, 1000 and 5000 µg/per plate for TA 100, 20 µg/plate for TA 1537 and 500 µg/plate for TA 98, respectively. Both batches of Acid Red 414 showed hence mutagenic effects in the Salmonella/microsome test with and without a metabolic activation.

 

The positive controls Endoxan, trypaflavine and 2-amino-anthracene acted markedly mutagenic, as can be seen from the biologically relevant increase of mutant colonies compared with the corresponding negative control.

 

This study is classified as acceptable. The study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.