2,5,7,10,11,14-hexaoxa-1,6-distibabicyclo[4.4.4]tetradecane

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:

22 Feb 2021 - 01 Dec 2021

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

After plating, the Petri dishes were incubated with the test item but the temperature of 37 ± 1°C may not have been maintained for entire duration (48-72h). This deviation does not impact the validity of the study, as shown by the historical data.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,5,7,10,11,14-hexaoxa-1,6-distibabicyclo[4.4.4]tetradecane

EC Number:

249-820-2

EC Name:

2,5,7,10,11,14-hexaoxa-1,6-distibabicyclo[4.4.4]tetradecane

Cas Number:

29736-75-2

Molecular formula:

C6H12O6Sb2

IUPAC Name:

2,5,7,10,11,14-hexaoxa-1,6-distibabicyclo[4.4.4]tetradecane

Test material form:

not specified

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch number of test material: 29011356
- Expiration date of the lot/batch: 07/2021 (Draft report, including results presented by 03/2021)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Immediately upon receipt, the test item was registered, then stored at room temperature protected from air under inert gas, in accordance with the provider instructions.
- Stability under storage conditions: see above
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: Prior to the implementation of the study, trials for solubility were performed and demonstrated that the test item was not soluble in dimethylsulfoxide at initial concentrations ranging from 100 to 25 mg/mL or in ethanol, acetone or tetrahydrofuran in which 2 phases that sedimented in the tube were observed with the test item.
In return, ATEG could be suspended in sterile water at 50 mg/mL.


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item ATEG was suspended in sterile water (Fresenius, Batch 13 PLP 253) 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. Lower concentrations were also prepared with sterile water by serial dilutions and used at 100 µL/plate.

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

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: The S9 fraction was prepared at Institut Pasteur de Lille (IPL)

- method of preparation of S9 mix: This preparation is carried out using the method described by Ames et al. (1975) in male OFA Sprague Dawley rats induced by Aroclor 1254 (origin - Monsanto, Saint Louis, U.S.A) 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

- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): 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.
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.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Sterile Water

Controls

Positive controls:

yes

Remarks:

Solvent controls, Positive controls At the same time, solvent controls (0.1 mL solvent/plate) were processed under the same conditions but using 6 plates with and without metabolic activity

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-anthramine

Details on test system and experimental conditions:

MUTAGENICITY ASSAYS
Formulation of the test item
The test item ATEG was suspended in sterile water (Fresenius, Batch 13 QAP 171) at a maximal initial concentration of 15 mg/mL in order to obtain the top dose of 1500 µg/plate when added at 100 µL/plate.

Lower concentrations were also prepared with sterile water by serial dilutions and used at 100 µL/plate.

Treatment without metabolic activation
The following technique was performed for each strain used in the test: 0.1 mL of a bacterial suspension from a culture agitated overnight at ca. 37°C and 0.1 mL of the test item at the relevant initial concentrations were successively added to 2 mL of top agar, supplemented with 10 % of 0.5 mM biotin histidine solution, maintained in a state of superfusion at ca. 45°C. The content of each tube was agitated, then spread out on a Petri plate containing 20 mL of minimal agar. Three plates were used per dose. The plates were then incubated at ca. 37°C for approximately 48 h. At the end of the expression time, colonies of revertants were counted for each plate.

CONTROLS
• Solvent controls, Positive controls
At the same time, solvent controls (0.1 mL solvent/plate) were processed under the same conditions but using 6 plates (Gatehouse et al., 1994). Appropriate reference positive controls were also included.

• Sterility of the media
At each assay, 3 Petri plates containing 20 mL of minimal agar received 2 mL of top agar only and were incubated under the same conditions of assay for the control of media sterility.
Sterility controls are presented in Appendix No. 2, Table 4.
No colony was observed after approximately 48 hours at ca. 37°C. The results were satisfactory.

Treatment with metabolic activation
The method was the same as that described above except that, immediately before spreading onto the plates, 0.5 mL of the S9-mix metabolic activation system (see § 3.4) was added to the top agar.

CONTROLS
Solvent controls, positive controls and assay for the control of media sterility were included and processed as for the mutagenicity assay without metabolic activation.

Evaluation criteria:

After approximately 48 or 72 hours of incubation at ca. 37°C, the prototrophic revertant colonies that had developed on the plates were counted, eventually using a colony counter. The results are
expressed as the mean number of revertants per plate and, for each dose of the test item, the following ratio was established:

Mean number of revertants per plate in presence of the test item / Mean number of revertants per plate without the test item (solvent control) .

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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

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

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:

no cytotoxicity

Any other information on results incl. tables

In two independent assays performed both with and without metabolic activation (the second assay with S9-mix was performed according to the pre-incubation protocol), no biologically or statistically significant increases in the mean number of revertants were noted in the five Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 tested, in the presence of ATEG.

The test item ATEG was thus not mutagenic in these conditions.

Applicant's summary and conclusion

Conclusions:

The search for a mutagenic potential of the test item ATEG (batch 29011356) 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 tested both in presence and in absence of metabolic activation, in two independent assays according to OECD No. 471 (2020), using the highest dose compatible with the solubility of the test item, i.e. 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:

T

The search for a mutagenic potential of the test item ATEG (batch 29011356) 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 tested both in presence and in absence of metabolic activation, in two independent assays according to OECD No. 471 (2020), using the highest dose compatible with the solubility of the test item, i.e. 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.