Boric acid (H3BO3), solid soln. with strontium oxide, europium-doped

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial Reverse Mutation Assay/Ames test): the substance boric acid (H3BO3), solid soln. with strontium oxide, europium-doped was not mutagenic in the strains S. typhimurium TA98, TA100, TA1535, TA1537 and TA102 in the presence and absence of phenobarbital and β-naphthoflavone-induced rat liver mammalian S9 metabolic activation (OECD 471/GLP).

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Zhejiang Jingneng Phosphor Material Co., Ltd; 170825
- Expiration date of the lot/batch: 24 August 2019
- Purity: 99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, protected from light

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: The S9 liver microsomal fraction was prepared at Eurofins Munich.
- method of preparation of S9 mix: Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route. The S9 mix preparation was performed according to Ames.
- concentration or volume of S9 mix and S9 in the final culture medium: 500 µL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation),
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): a) Biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene; b) Sterility Test

Test concentrations with justification for top dose:

Preliminary testl 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
Experiment 1 & 2: 31.6, 100, 316, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Purified water

- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-NOPD (4-nitro-o-phenylene-diamine): -S9; S. typhimurium: TA98, TA1537; 10 µg/plate for TA98, 40 µg/plate for TA1537; 2-AA (2-aminoanthracene): +S9; S. typhimurium: TA98, TA100, TA1535, TA1537 and TA102;2.5 µg/plate; 10 µg/plate for TA102

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2 (plate incorporation and pre-incubation)

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

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 60 min at 37 °C
- Exposure duration/duration of treatment: 48 hrs

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Cytotoxicity can be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.

Evaluation criteria:

The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).

A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.

A biologically relevant increase is described as follows:
- if in tester strains TA98, TA100 and TA102 the number of reversions is at least twice as high
- if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher
than the reversion rate of the solvent control.

A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Statistics:

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Species / strain:

other: S. typhimurium strains TA98, TA100, TA1535, TA1537 and TA102

Remarks:

Experiment 1 & 2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Precipitation of the test item was observed in all tester strains used in experiment I and II (with and without metabolic activation). In experiment I precipitation of the test item was found at a concentration of 5000 µg/plate (with and without metabolic activation) and in experiment II at concentrations of 2500 µg/plate and higher (with and without metabolic activation). The observed precipitation did not interfere with the scoring, thus it did not impact the results

RANGE-FINDING/SCREENING STUDIES (if applicable): The toxicity of the test item was determined with tester strains TA98 and TA100 in a pre-experiment. Eight concentrations were tested for toxicity and induction of mutations with three plates each. The experimental conditions in this pre-experiment were the same as described below for the main experiment I (plate incorporation test). Toxicity may be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control. The test item was tested in the pre-experiment with the following concentrations: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate. The following concentrations were chosen for the main tests: 31.6, 100, 316, 1000, 2500 and 5000 µg/plate.

STUDY RESULTS
Ames test:
- Signs of toxicity: No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated (with and without metabolic activation) in experiment I and II.
- Individual plate counts & mean number of revertant colonies per plate and standard deviation: refer to Tables 2 and 3

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: Yes, tables 5 & 7 (2015-2017)
- Negative (solvent/vehicle) historical control data: Yes, tables 4 & 6 (2015-2017)

Conclusions:

In a reverse bacteria mutation study (Ames test), boric acid (H3BO3), solid soln. with strontium oxide, europium-doped was non-mutagenic in S. typhimurium strains TA98, TA100, TA1535, TA1537 and TA102 with and without metabolic activation.

Executive summary:

In a reverse gene mutation assay in bacteria (185428), strains of S. typhimurium strains TA98, TA100, TA1535, TA1537 and TA102 were exposed to boric acid (H3BO3), solid soln. with strontium oxide, europium-doped in water at concentrations of 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (plate incorporation; 60 minute pre-incubation) in the presence and absence of phenobarbital and β-naphthoflavone-induced rat liver mammalian S9 metabolic activation.

The positive controls induced the appropriate responses in the corresponding strains. Precipitation of the test item was observed in all tester strains used in the plate incorporation assay (5000 µg/plate) and pre-incubation assay (≥2500 µg/plate) (with and without metabolic activation) but did not interfere with the scoring. No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated with and without metabolic activation in either experiment. There was no evidence of induced mutant colonies over background in the S. typhimurium TA98, TA100, TA1535, TA1537 and TA102 strains in the presence and absence of mammalian metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Gene mutation (Bacterial Reverse Mutation Assay/Ames test):

There is one gene mutation study (Bacterial Reverse Mutation Assay/Ames test) available.

In a reverse gene mutation assay in bacteria (OECD 471/GLP), strains of S. typhimurium strains TA98, TA100, TA1535, TA1537 and TA102 were exposed to boric acid (H3BO3), solid soln. with strontium oxide, europium-doped in water at concentrations of 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (plate incorporation; 60 minute pre-incubation) in the presence and absence of phenobarbital and β-naphthoflavone-induced rat liver mammalian S9 metabolic activation. The positive controls induced the appropriate responses in the corresponding strains. Precipitation of the test item was observed in all tester strains used in the plate incorporation assay (5000 µg/plate) and pre-incubation assay (≥2500 µg/plate) (with and without metabolic activation) but did not interfere with the scoring. No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated with and without metabolic activation in either experiment. There was no evidence of induced mutant colonies over background in the S. typhimurium TA98, TA100, TA1535, TA1537 and TA102 strains in the presence and absence of mammalian metabolic activation.

Justification for classification or non-classification

Based on the available information in the dossier, the substance boric acid (H3BO3), solid soln. with strontium oxide, europium-doped (CAS No. 102110-29-2) does not need to be classified for germ cell mutagenicity when the criteria outlined in Annex I of 1272/2008/EC are applied.