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Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-07-22 - 2020-09-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997 as corrected in 2020

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

liquid

Details on test material:

- Physical state: liquid
- Storage condition of test material: Room temperature

Specific details on test material used for the study:

- Name of test material (as cited in study report): Jeffamine D-230
- Lot No.: DRW2000305
- Physical state: Pale yellow liquid
- Purity: ca. 100% (w/w). This product is not specified by purity/assay but by titratable total acetylatables and amine content
- Stability under test conditions: not indicated
- Storage condition of test material: Room temperature over silica gel in the dark under nitrogen
- Final preparation of a solid: The test item was accurately weighed and, on the day of each experiment, approximate half-log dilutions prepared in sterile distilled water by mixing on a vortex mixer and sonication for 5 minutes at 40 °C. No correction for purity was required. All test item preparation and dosing was performed under yellow safety lighting.

Method

Target gene:

histidine or tryptophan locus

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

Metabolic activation:

with and without

Metabolic activation system:

10% rat liver homogenate metabolizing system

Test concentrations with justification for top dose:

The maximum concentration in the first experiment (plate incorporation method) was 5000 µg/plate (the OECD TG 471 maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 15 to 5000 μg/plate. Six test item concentrations were selected in Experiment 2 in order to ensure the study achieved at least four non-toxic dose levels as required by the test guideline, and were selected based on the lack of cytotoxicity noted in Experiment 1, and the potential for a change in the cytotoxicity of the test item following the change in test methodology from plate incorporation to pre-incubation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile distilled water
- Justification for choice of solvent/vehicle: The test item was fully miscible in sterile distilled water and dimethyl sulphoxide at 50 mg/mL in solubility checks performed in-house. Sterile distilled water was therefore selected as the vehicle.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

solvent control

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

In the absence of S9-mix 2 µg/plate for WP2uvrA 3 µg/plate for TA100 5 µg/plate for TA1535

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

solvent control

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

In the absence of S9-mix 80 µg/plate for TA1537

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

solvent control

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

In the absence of S9-mix 0.2 µg/plate for TA98

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

solvent control

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

In the presence of S9-mix 1 µg/plate for TA100 2 µg/plate for TA1535 and TA1537 10 µg/plate for WP2uvrA

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

solvent control

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

In the presence of S9-mix 5 µg/plate for TA98

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments
: two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding::
All tester strain cultures should be in the range of 0.9 to 9 x 10^9 bacteria per mL.
- Test substance added: Experiment 1: in agar (plate incorporation); Experiment 2: preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: Experiment 2 - not specified
- Exposure duration/duration of treatment: between 48 and 72 hours


METHODS FOR MEASUREMENT OF CYTOTOXICITY
Method: background growth inhibition; dicrease in numbe of revertants, microcolonies

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 (De Serres and Shelby,
1979).

2. A reproducible increase at one or more concentrations.

3. Biological relevance against in-house historical control ranges.

4. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).

5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments give clear positive or negative results, in some instances the data generated prohibit making a definite judgment about test item activity. Results of this type are reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnett’s 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

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

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:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

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:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

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:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

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:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

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:

valid

True 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).

Prior to use, the relevant strains were checked for characteristics (deep rough character, ampicillin resistance, UV light sensitivity and histidine or tryptophan auxotrophy), viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments were shown to be sterile. The test item formulation was also shown to be sterile.

The vehicle (sterile distilled water) control plates gave counts of revertant colonies within or close to the normal range. A single count for TA1535 ( solvent control dosed in the presence of S9-mix after the second mutation test) was just below the minimum level of the in-house historical untreated/vehicle control minima for the tester strain. This count was considered acceptable as the other vehicle and untreated control counts were within the expected range and the tester strain responded very well to the respective positive controls in both the presence and absence of S9-mix. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Applicant's summary and conclusion

Conclusions:

In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item did not induce an increase in the frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix). Under the conditions of this test, the test substance was considered to be non-mutagenic.