Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Nov - Dec 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1997)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Test material form:
solid
Specific details on test material used for the study:
Physical state/Appearance: Dark brown solid
Sponsor’s description: Solid, dark blue
EC Number: 916-899-6
Empirical formula: UVCB substance
Expiry Date: 28 April 2017
Storage Conditions: Room temperature over silica gel in the dark
Stability under test conditions: The stability of the substance in the vehicle used is analytically verified for a period of at least 24 hours.
Formulated concentrations were adjusted to allow for the stated water/impurity content of the test item.

Method

Target gene:
Histidine locus
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:
S9 preparation from the liver of Phenobarbital/ beta-Naphtha flavone induced sprague-Dawley rats.
Test concentrations with justification for top dose:
Exp. 1 (plate incorporation test, with and without metabolic activation): 1.5, 5, 15, 50, 150, 500, 1500, and 5000 µg/plate
Exp. 2 (pre-incubation method, with and without metabolic activation): 15, 50, 150, 500, 1500, and 5000 µg/plate
Vehicle / solvent:
sterile distilled water
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene
Remarks:
The positive controls N-ethyl-N'-nitro-N-nitrosoguanidine, 9-Aminoacridine, and 4-Nitroquinoline-1-oxide were used without S9 mix, the positive controls 2-Aminoanthracene and benzo(a)pyrene with S9 mix.
Details on test system and experimental conditions:
METHOD OF APPLICATION: Experiment 1 = Plate Incorporation Test; as independent repeat Experiment 2 = preincubation method
Every concentration is assayed in triplicate against each tester strain, all with and without the addition of a rat liver homogenate metabolizing system.

DETERMINATION OF CYTOTOXICITY
- Method: The plates were viewed microscopically for evidence of thinning (toxicity)
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study: 1. A dose-related increase in mutant frequency over the dose range tested. 2. A reproducible increase at one or more concentrations. 3. Biological relevance against in-house historical control ranges. 4. Statistical analysis of data as determined by UKEMS. 5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100, and E. coli WPuvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method) or Experiment 2 (pre incubation method).
The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
A mauve test item colouration was initially noted at 50 µg/plate becoming an increasingly intense purple colour as the dose concentrations increased, this observation did not prevent the scoring of revertant colonies.

TEST-SPECIFIC CONFOUNDING FACTORS
None

ADDITIONAL INFORMATION ON CYTOTOXICITY:
There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) or in the second mutation test (pre-incubation method) up to and including the limit concentration of 5000 µg/plate.

Applicant's summary and conclusion

Conclusions:
The test substance was considered to be non-mutagenic under the conditions of this test.
Executive summary:

A bacterial reverse mutation assay (Ames test, OECD TG 471) was conducted with the tester strains S. thyphimurium TA 1535, TA 1537, TA 98, TA 100, and E. coli WP2 with and without metabolic activation. The test was performed as plate incubation (Exp. 1) and, as independent repeat (Exp. 2), with the preincubation method with concentrations up to the maximum recommended dose level (5000 µg/plate).

There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in Exp. 1 or 2 up to and including the limit concentration of 5000 µg/plate.  

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Exp. 1 or 2.

The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

A mauve test item colouration was initially noted at 50 µg/plate becoming an increasingly intense purple colour as the dose concentrations increased, this observation did not prevent the scoring of revertant colonies.

In conclusion, the test substance was considered to be non-mutagenic under the conditions of this test.