Carbamodithioic acid, bis(mixed 2-ethylhexyl and 2-methylbutyl and pentyl) derivs., antimony (3+) salts

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:

17 August 2020 thru 9 October 2020

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Carbamodithioic acid, bis(mixed 2-ethylhexyl and 2-methylbutyl and pentyl) derivs., antimony (3+) salts

Molecular formula:

C33H66N3S6Sb to C51H102N3S6Sb

IUPAC Name:

Carbamodithioic acid, bis(mixed 2-ethylhexyl and 2-methylbutyl and pentyl) derivs., antimony (3+) salts

Test material form:

liquid: viscous

Specific details on test material used for the study:

PYSICAL DESCRIPTION: Highly viscous amber liquid.

PURITY: >99%

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: WT05-19
- Expiration date of the lot/batch: not applicable

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temp.
- Stability under storage conditions: stable for the duration of testing.

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

chemically-induced rat liver S9 mix

Test concentrations with justification for top dose:

Experiment I: 31.6, 100, 316, 1000, 2500, and 5000 μg/plate, with the top dose considered the limit test for this model.
Experiment II: 15.8, 50, 158, 500, 1580, and 5000 μg/plate, with the top dose considered the limit test for this model.

Vehicle / solvent:

Without metabolic activation: Tetrahydrofuran (THF)
With metabolic activation: Tetrahydrofuran (THF)

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

THF

True negative controls:

yes

Remarks:

Aqua distillata (distilled water)

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Strains TA100 and TA1535 without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

THF

True negative controls:

yes

Remarks:

Aqua distillata (distilled water)

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine (4-NOPD)

Remarks:

TA 98 and TA 1537 without metabolic activation

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

THF

True negative controls:

yes

Remarks:

Aqua distillata (distilled water)

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

WP2 uvrA without metabolic activation

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

THF

True negative controls:

no

Remarks:

Aqua distillata (distilled water)

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

All strains; with metabolic activation

Details on test system and experimental conditions:

The test followed the plate incorporation method, in which the following materials were mixed and poured over the surface of a minimal agar plate:
•50 µL of the test solution at each dose level and the solvent control (pre-experiment)
•25 µL of the test solution at each dose level and the solvent control (experiments I and II)
•100 µL of the negative or prepared positive control substance
•500 µL S9 mix or substitution buffer
•100 µL bacteria suspension (cf. Preparation of Bacteria, pre-culture of the strain)
•2000 µL overlay agar maintained at approximately 45°C
Plates were prepared in triplicate at each experimental point and uniquely identified. After pouring, plates were placed on a level surface until the agar gelled then inverted and incubated at approximately 37°C until growth was adequate for enumeration (approximately 48 hours).

Rationale for test conditions:

A pre-experiment for toxicity was performed with tester strains TA98 and TA100 at 3.16, 10, 31.6, 100, 316, 1000, 2500, and 5000 µg/plate with no significant toxicity observed.

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 E. coli WP2 uvrA (pKM101) 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 as compared to the reversion rate of the solvent control.

According to the 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.

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 the 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.

Results and discussion

Test results

Test results 1

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

True negative controls validity:

valid

Positive controls validity:

valid

Test results 2

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

True negative controls validity:

valid

Positive controls validity:

valid

Test results 3

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

True negative controls validity:

valid

Positive controls validity:

valid

Test results 4

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

True negative controls validity:

valid

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

True negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation). 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. No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II. All criteria of validity were met.

Applicant's summary and conclusion

Conclusions:

The test substance did not elicit evidence of bacterial mutagenicity in the Ames assay.

Executive summary:

In a GLP guideline bacterial reverse mutation assay using the plate incorporation method conducted according to OPPTS 870.5100, OECD 471, and EC method B.13/14, the substance did not elicit evidence of bacterial mutagenic activity with or without rat liver S9 metabolic activation with strains TA1535, TA1537, TA98 , TA100 or E. coli WP2 uvrA.