Man-made vitreous (silicate) fibres with random orientation with alkaline oxide and alkali earth oxide (Na2O+K2O+CaO+MgO+BaO) content greater than 18 % by weight

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(OECD, 1997, as corrected in 2020)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Man-made vitreous (silicate) fibres with random orientation with alkaline oxide and alkali earth oxide (Na2O+K2O+CaO+MgO+BaO) content greater than 18 % by weight

EC Number:

926-771-1

Molecular formula:

Not available.

IUPAC Name:

Man-made vitreous (silicate) fibres with random orientation with alkaline oxide and alkali earth oxide (Na2O+K2O+CaO+MgO+BaO) content greater than 18 % by weight

Test material form:

solid: fibres

Method

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:

Type and composition of metabolic activation system:
- source of S9 : lyophilized phenobarbital/beta-naphthoflavone induced rat liver S9 and cofactors mix (MutazymeTM)
- method of preparation of S9 mix: reconstituted with cold, sterile water by adding 20 mL to each vial to provide a 10% phenobarbital/beta-naphthoflavone induced rat liver S9 and cofactors mix
- concentration or volume of S9 mix and S9 in the final culture medium : 0.5 mL of S9 mix added to 0.4 mL of test concentration, 0.1 mL bacterial strain culture and 2mL of media.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): the sterility of the S9-mix was repeated, in triplicate, on the day of each experiment, Quality control certificate for enzymic activity and metabolic capacity provided in report.

Test concentrations with justification for top dose:

1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
Top dose was guideline limit dose

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile distilled water
Insoluble in most common solvents used in Ames test, distilled water gave best dosable suspension.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; in agar (plate incorporation) - 1st experiment; preincubation - 2nd experiment

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 minutes
- Exposure duration/duration of treatment: 48-72 hours

Rationale for test conditions:

Standard guideline conditions

Evaluation criteria:

The reverse mutation assay may be considered valid if the following criteria are met:
1. The vehicle control counts should be consistent with the laboratory’s historical control ranges
2. The positive control chemicals should induce increases in mean revertant numbers of ≥2-fold (in strains TA98, TA100 and WP2 uvrA) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values, confirming discrimination between different strains, and an active S-9 preparation.
3. Contamination or some other confounding effect should not adversely impact the strain or assay functioning or prevent endpoint determination.

A test article is considered to have provided a mutagenic response if the assay data are valid, and:
1. Treatments with the test article provide a concentration-related increase in revertant numbers at one or more concentrations in at least one strain with or without metabolic activation system
2. An increase in mean revertant colony numbers per plate is observed which is ≥2-fold (in strains TA98, TA100 and WP2 uvrA) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values
3. Any increase in revertant numbers is reproducible, where applicable.

Statistics:

Statistical significance was not included as part of the result evaluation

Results and discussion

Test results

Test results 1

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:

valid

Positive controls validity:

valid

Test results 2

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:

valid

Positive controls validity:

valid

Test results 3

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:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

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:

valid

Positive controls validity:

valid

Additional information on results:

Experiment 1 (plate incorporation)
There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or in the absence of S9-mix.
A test item precipitate (fibrous in appearance) was noted under an inverted microscope at and above 50 μg/plate and by eye from 500 μg/plate in both the presence and absence of S9-mix. The precipitate did not prevent the scoring of revertant colonies.
There were no biologically relevant 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 S9-mix.

Experiment 2 Repeat (pre-incubation)
There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or in the absence of S9-mix.
A test item precipitate (fibrous in appearance) was noted under an inverted microscope at and above 50 μg/plate and by eye from 500 μg/plate in both the presence and absence of S9-mix. The precipitate did not prevent the scoring of revertant colonies.
There were no biologically relevant 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 S9-mix.

Applicant's summary and conclusion

Conclusions:

The substance was considered to be non-mutagenic.

Executive summary:

In a Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item B glass microfibre 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 B glass microfibre was considered to be non-mutagenic.