Antimony

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:

Study initiation date (date Study Director signed Study Plan): 12/02/21
Experimental start date: 15/02/21
Experimental completion date: 26/03/21

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

The only deviation to the OECD guideline No. 471 (2020) was that, after plating, the Petri dishes were incubated with the test item for ca. 48-72h, as recommended in the guideline, knowing however that the temperature of 37 ± 1°C may not have been maintained for the entire duration of exposure (due to the elapsed time between the loading of the heat chamber and the return to equilibrium of the temperature). However, this deviation does not impact the validity of the study, as shown by the historical data for solvent controls, that are comparable to the "acceptable range of background revertant counts for the routinely used strains" reported by Gatehouse et al. (1990) and used as a guide in the laboratory. Moreover, results obtained with reference mutagens routinely demonstrate the sensitivity of the test method.

Deviations:

yes

Remarks:

Deviation outlined in version / remarks section As noted this this deviation does not impact the validity of the study

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Antimony

EC Number:

231-146-5

EC Name:

Antimony

Cas Number:

7440-36-0

Molecular formula:

Sb

IUPAC Name:

antimony

Test material form:

not specified

Details on test material:

- Test material as cited by the study: antimony
- Average particle size: 10 µm
- Lot number: 88711G
- Source: supplied by Kojyundo Chemical Lab. Co., Ltd.
- Storage: kept at room temperature in a dark place until use

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 10678/40
- Expiration date of the lot/batch: N/A


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature (+15 to +28°C), protected from light, air and humidity (in tightly closed contained)

- Stability under storage conditions: Stable mineral

- Stability under test conditions: see above

- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: Prior to the implementation of this current study, trials for solubility were performed in the frame of the study FSP-IPL 210105 (Simar, 2021) and demonstrated that the Sb metal could be suspended in sterile water at 50 mg/mL. A homogeneous suspension was obtained with sedimenting particles in few minutes.

- Reactivity of the test substance with the solvent/vehicle /test medium (if applicable): The stability of the test item in the solvent was unknown. Therefore, preparations for treatments in the dose-range finding assay or in the main assays were performed just before use.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: See above

- Preliminary purification step (if any): None

- Preparation of a nanomaterial dispersion (incl. dilution): N/A

- Final dilution of a dissolved solid, stock liquid or gel:
The test item Sb metal was prepared in sterile water (Fresenius, Batch 13PFP081) at a maximal initial concentration of 50 mg/mL in order to obtain the top dose of 5000 µg/plate when added at 100 µL/plate.
As the preparation presented sedimenting particles, it was well agitated before sampling from treatment.
Lower concentrations were also prepared with sterile water by serial dilutions and used at 100 µL/plate.

- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material): Solution - see above

OTHER SPECIFICS
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added:
- other information:

Method

Target gene:

Histidine

Species / strain

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: male OFA Sprague Dawley rats induced by Aroclor 1254 (origin - Monsanto, Saint Louis, U.S.A)

- method of preparation of S9 mix:
This preparation is carried out using the method described by Ames et al. (1975) according to the standard operating procedures of the Institut Pasteur de Lille.

- concentration or volume of S9 mix and S9 in the final culture medium
The S9-mix contained per mL:
• S9 fraction: 0.1 mL
• MgCl2, 0.4 M: 0.02 mL
• KCl, 1.65 M: 0.02 mL
• Phosphate buffer, 0.2 M pH 7.4: 0.5 mL
• NADP, 0.1 M: 0.04 mL
• Glucose-6-phosphate, 1 M: 0.005 mL
• H2O: 0.315 mL
All the solutions (except S9 fraction) were mixed the day of each assay, filtered through a sterilizing membrane and preserved in a refrigerated place pending use. The S9 fraction was added extemporaneously.

- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)
For each test, the S9-mix alone was added to 3 plates in order to check the sterility.
No colony was observed after approximately 48 hours at ca. 37°C. The results were satisfactory and demonstrated the sterility of the S9-mix.

Test concentrations with justification for top dose:

In order to choose the range of doses for the main mutagenicity assay, the toxic activity of the test item (by microscopic examination of the background growth and revertants colonies counting) and its solubility in the conditions of the main assay were determined.

The test item Sb metal was prepared in sterile water (Fresenius, Batch 13PFP081) at a maximal initial concentration of 50 mg/mL in order to obtain the top dose of 5000 µg/plate when added at 100 µL/plate. As the preparation presented sedimenting particles, it was well agitated before sampling from treatment. Lower concentrations were also prepared with sterile water by serial dilutions and used at 100 µL/plate.

The dose-range finding assay was carried out in all the strains to be tested under the same conditions as the first mutagenicity test with and without metabolic activation but using only one plate per dose instead of 3. No positive controls are included. The plates were incubated for approximately 48 hours at ca. 37°C, and the revertants were counted.

The limiting factor for maximum dose in the dose-range finding assay was the maximum dose according to OECD No. 471 (2020), i.e. 5000 µg/plate.

No precipitate was observed.

The examination of the background growth demonstrated that Sb metal induced in all strains a slight to strong toxicity at the highest dose tested of 5000 µg/plate both with and without metabolic activation, with no bacterial growth in strains TA1535 and TA1537 in absence of metabolic activation and in strains TA1535 and TA100 with metabolic activation. The lowest dose of 1500 µg/plate induced a slight toxicity in strain TA1537.
Furthermore, no colonies were present in strain TA102 at 5000 µg/plate both with and without metabolic activation.
Therefore, the maximum doses retained for the first mutagenicity assay were:
o TA1535, TA1537 and TA98 with and without metabolic activation: 3000 µg/plate
o TA100 and TA102 with and without metabolic activation: 1500 µg/plate

Vehicle / solvent:

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

- Justification for choice of solvent/vehicle:

- Justification for percentage of solvent in the final culture medium:

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:

cytotoxicity

Remarks:

at upper limit of 5000 ug per plate

Test results 2

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at upper limit of 5000 ug per plate

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at upper limit of 5000 ug per plate

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 5

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Upper limit 1500 ug per plate

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH:
- Data on osmolality:
- Possibility of evaporation from medium:
- Water solubility:
- Precipitation and time of the determination:
- Definition of acceptable cells for analysis:
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES:

In order to choose the range of doses for the main mutagenicity assay, the toxic activity of the test item (by microscopic examination of the background growth and revertants colonies counting) and its solubility in the conditions of the main assay were determined.

Formulation of the test item
The test item Sb metal was prepared in sterile water (Fresenius, Batch 13PFP081) at a maximal initial concentration of 50 mg/mL in order to obtain the top dose of 5000 µg/plate when added at 100 µL/plate. As the preparation presented sedimenting particles, it was well agitated before sampling from treatment. Lower concentrations were also prepared with sterile water by serial dilutions and used at 100 µL/plate.

Treatment
The dose-range finding assay was carried out in all the strains to be tested under the same conditions as the first mutagenicity test with and without metabolic activation but using only one plate per dose instead of 3. No positive controls are included. The plates were incubated for approximately 48 hours at ca. 37°C, and the revertants were counted.

The limiting factor for maximum dose in the dose-range finding assay was the maximum dose according to OECD No. 471 (2020), i.e. 5000 µg/plate.

Results for solubility in the conditions of the test
No precipitate was observed.

Results for toxic activity in the conditions of the test
The examination of the background growth demonstrated that Sb metal induced in all strains a slight to strong toxicity at the highest dose tested of 5000 µg/plate both with and without metabolic activation, with no bacterial growth in strains TA1535 and TA1537 in absence of metabolic activation and in strains TA1535 and TA100 with metabolic activation. The lowest dose of 1500 µg/plate induced a slight toxicity in strain TA1537.
Furthermore, no colonies were present in strain TA102 at 5000 µg/plate both with and without metabolic activation (see Table 1).
Therefore, the maximum doses retained for the first mutagenicity assay were:
o In strains TA1535, TA1537 and TA98 with and without metabolic activation: 3000 µg/plate
o In strain TA100 and TA102 with and without metabolic activation: 1500 µg/plate

Verification of the absence of microorganisms in the test item in the conditions of the assay
A volume of 0.1 mL of the three highest concentrations of the test item was added to 2 mL of top agar in a state of superfusion. The mixture was spread out on 3 Petri plates containing 20 mL of minimal agar. The plates were incubated for approximately 48 hours at ca. 37°C. The plates were examined for any bacterial growth.
No colony was visible. The results were satisfactory.


STUDY RESULTS
- Concurrent vehicle negative and positive control data

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible
- Statistical analysis; p-value if any
- Any other criteria: e.g. GEF for MLA

Ames test:
- Signs of toxicity: - see tables below and range finding results above. Sb metal induced in all strains a slight to strong toxicity at the highest dose tested of 5000 µg/plate both with and without metabolic activation, with no bacterial growth in strains TA1535 and TA1537 in absence of metabolic activation and in strains TA1535 and TA100 with metabolic activation (see Table 1). The lowest dose of 1500 µg/plate induced a slight toxicity in strain TA1537

- Individual plate counts - see tables below

- Mean number of revertant colonies per plate and standard deviation - see tables below


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:
- Negative (solvent/vehicle) historical control data:

Any other information on results incl. tables

The following results tables are attached to this RSS:

• Recapitulative results


• Individual results


• Historical data


• Control of S9

Applicant's summary and conclusion

Conclusions:

The search for a mutagenic potential of the test item Sb metal (batch 10678/40) was done by means of the Ames’ test in the five Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 tested both in presence and in absence of metabolic activation, in 2 or 3 independent assays according to OECD No. 471 (2020), using the highest dose compatible with the toxic activity, i.e. 3000 or 1500 µg/plate.

The acceptance criteria for the assay were considered as fulfilled. The current study was valid.

Under these experimental conditions, no mutagenic activity was revealed

Executive summary:

The search for a mutagenic potential of the test item Sb metal (batch 10678/40) sponsored by International Antimony Association was done by means of the Ames’ test in the five Salmonella typhimurium strains

• TA1535,


• TA1537,


• TA98,


• TA100 and


• TA102

The strains were tested both in presence and in absence of metabolic activation, in 2 or 3 independent assays according to OECD No. 471 (2020), using the highest dose compatible with the toxic activity, i.e. 3000 or 1500 µg/plate.

 

The acceptance criteria for the assay were considered as fulfilled. The current study was valid.

 

Under these experimental conditions, no mutagenic activity was revealed