Custom Yellow #2

Genetic toxicity: in vitro

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:

May 11 - August 8, 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Principles of method if other than guideline:

In the modified repeat experiment according to Prival and Mitchell, only 3 tester strains were used instead of the 5 strains required in the OECD Guidance document.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

Color: Yellow
Physical state: solid
Odour: odourless
Chemical family: azo pigment
Storage conditions: room temperature (15-30°C)

Method

Target gene:

Histidine gene in S. typhimurium
Tryptophan gene in E. coli

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain 3

Species / strain / cell type:

other: S. typhimurium TA 98, TA 100 and TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Standard Aroclor 1254-induced rat liver S9 mix, 10%

Test concentrations with justification for top dose:

Experiment 1 - 5: 0, 21, 62, 190, 560, 1670, 5000 µg/plate
Experiment 6: 0, 21, 41, 62, 130, 190 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMF
- Justification for choice of solvent/vehicle: it was the only solvent completely dissolving the test substance.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

DMF

Controls 2

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9; TA100 and TA1535 (5 µg/plate)

Migrated to IUCLID6: in water

Controls 3

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9; E-coli (1 µg/plate)

Migrated to IUCLID6: in water

Controls 4

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9; TA98 (5 µg/plate)

Migrated to IUCLID6: in DMSO

Controls 5

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9; TA1537 (100 µg/plate)

Migrated to IUCLID6: HCl form in DMSO

Controls 6

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9; TA98, TA100 and TA1537 (5 µg/plate)

Migrated to IUCLID6: in DMSO

Controls 7

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene in DMSO

Remarks:

with S9; E-coli (100 µg/plate)

Controls 8

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9; TA1535 (100 µg/plate)

Migrated to IUCLID6: monohydrate form in water

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) for experiment 1 and preincubation for experiments 2-6

DURATION
- Preincubation period: 30 minutes
- Exposure duration: 48-72 hours

NUMBER OF REPLICATIONS: triplicate testing

DETERMINATION OF CYTOTOXICITY
- Method: bacterial background lawn evaluation

Evaluation criteria:

Positive result:
a) a concentration-related increase over the range tested
b) a reproducible increase in at least one or more concentrations in the number of revertant colonies per plate in at least one strain with or without S9.
Negative result: if a result does not meet the above criteria, it is concluded as negative.
Equivocal result: if no definite judgement can be made to fit the above criteria, even after repeated experiments.

Statistics:

Statistical analysis may be applied to numbers suspected to be abnormally high or to have a dose-related increase in revertant counts (Mahon et al, 1989).

Results and discussion

Test results

Test results 1

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: observed at 560, 1670 and 5000 mg/plate. At the highest dose the precipitate may have interfered with revertant colony counting.

The number of revertants per plate of TA100 in Experiment 5 was slightly out of the range of the solvent control at one concentration, 0.062 mg/plate. Although it showed statistical significance when compared with its solvent control by Dunnett's analysis, this data series did not show any trend of dose-response relationship, indicating that it was unlikely due to treatment with the substance. Secondly, the result was not reproduced in experiment 6. In this confirmatory experiment, the exposure concentrations covered nearly one order of magnitude, centering around 0.062 mg per plate. The interval between doses was smaller than what was used in the previous experiments in order to demonstrate a possible dose-response relationship. The number of revertants from plates treated with the substance were all within range of the solvent control. This result supports the assumption that the observation of TA100 in Experiment 5 at 0.062 mg/plate was probably not a manifestation of mutagenicity of the test substance.

COMPARISON WITH HISTORICAL CONTROL DATA:
The negative and strain-specific positive control values were within the laboratory historical control data
ranges indicating that the test conditions were adequate and that the metabolic activation system functioned
properly.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The bacterial reverse mutation assay as presented includes the metabolic activation experiment as recommended by Pival and Mitchell - for addressing the potential mutagenic effects of azo compounds such as the test substance. The three tester strains used, instead of the recommended five, in the modified activation experiment cover both the frame shift and the base-pair substitution reverse mutations.
It was concluded that the test substance was not mutagenic to S. typhimurium strains, TA98, TA100, TA1535, TA1537 and TA1538 and E. coli WP2 uvrA in the absence and presence of standard and modified S9 mixtures.