Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 November 2017 - 05 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
31 May 2008
Deviations:
no
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:
Physical appearance: pink powder
Storage conditions: at room temperature
Specific details on test material used for the study:
pH: 3.90 – 3.89

Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix from rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).
Test concentrations with justification for top dose:
Experiment 1
Dose-range finding test (without and with S9) TA100 and WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 17, 52, 164, 512, 1600 and 5000 µg/plate
Experiment 2:
Without and with S9-mix (all strains): 17, 52, 164, 512, 1600 and 5000 µg/plate
Vehicle / solvent:
- Vehicle used: dimethyl sulfoxide
- Justification for vehicle: the vehicle is according to OECD guideline 471 and a solubility test was performed based on visual assessment. Dimethyl sulfoxide was used as vehicle in this study. Due to the colour of the test item it could not be observed if the test item was dissolved or suspended in dimethyl sulfoxide at the concentration of 53.7 mg/ml. At concentrations of 17.2 mg/ml and lower the test item was dissolved in dimethyl sulfoxide.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR 191; 2-aminoanthracene
Remarks:
For more details on positive controls, see 'additional details on methods'
Details on test system and experimental conditions:
Two individual experiments were performed. The first experiment was a direct plate assay and included a dose-range finding test, the second experiment was a pre-incubation assay.

METHOD OF APPLICATION: In the first experiment top agar in top agar tubes was melted by heating to 45 ± 2°C. Bacterial culture, one of the tester strains, a dilution of test item in Milli-Q water and either S9 mix or phosphate buffer were successively added to 3 mL molten top agar. The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. The plates were inverted incubated after solidification of the top agar.
In the second experiment a mixture of either S9 mix or phosphate buffer, bacterial culture, one of the tester strains and test item diluted in Milli-Q water were pre-incubated for 30 minutes by 70 rpm at 37°C. After the pre-incubation period the solutions were added to 3 mL molten top agar. The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. The plates were inverted and incubated after solidification of the top agar.

DURATION
- Exposure duration: 48 ± 4 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain.

NUMBER OF CELLS EVALUATED: 10E9 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were determined. Revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

ACCEPTABILITY CRITERIA:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at the test facility.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the pre-incubation assay, in the presence of S9, at 512 μg/plate and upwards
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: in the dose-range finding test and the direct plate assay precipitation was oberved in all tester strains, in the absence and presence of S9 at and above 1600 μg/plate. In the pre-incubation assay, precipitation was observed in all tester strains at and above 164 and 512 μg/plate in the absence and presence of S9, respectively.

RANGE-FINDING/SCREENING STUDIES: no cytotoxicity and/or mutagenic effects were observed in the dose-range finding study in both strains tested.

HISTORICAL CONTROL DATA: the solvent and strain-specific positive control values were within the laboratory historical control data ranges

Applicant's summary and conclusion

Conclusions:
In an Ames test, performed according to OECD guideline 471 and GLP principles, C.I. Pigment Red 81:2 was found not to be mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay, in the absence and in the presence of metabolic activation.
Executive summary:

An Ames test was performed according to OECD guideline 471 and GLP principles. The test was performed in two independent experiments (one direct plate assay and one pre-incubation assay) in presence and absence of S9-mix.

C.I. Pigment Red 81:2 was tested up to a dose level of 5000 μg/plate in both experiments. Precipitation was oberved in the direct plate assay in all tester strains, in the absence and presence of S9 at and above 1600 μg/plate. In the pre-incubation assay, precipitation was observed in all tester strains at and above 164 and 512 μg/plate in the absence and presence of S9, respectively. In the first experiment, no cytotoxicity and/or mutagenicity was observed at any of the strains. In the second experiment, a decrease in the number of revertant was observed in tester strain TA1537 only, in the presence of S9 -mix at and above a dose level of 512 μg/plate. In the other strains, no cytotoxicity and/or mutagenicity was observed. Precipitation of the test substance was observed at the start of the incubation period at the highest concentration. 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.

Based on the results of this study it is concluded that C.I. Pigment Red 81:2 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or without metabolic activation.