(1-λ1-oxidanyl-2,2,6,6-tetramethylpiperidin-4-yl) benzoate

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:

April to September 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 2015

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1-hydroxy-2,2,6,6-tetramethylpiperidin-4-yl benzoate

EC Number:

807-702-3

Cas Number:

3225-26-1

Molecular formula:

C16H22NO3

IUPAC Name:

1-hydroxy-2,2,6,6-tetramethylpiperidin-4-yl benzoate

Test material form:

solid: flakes

Specific details on test material used for the study:

Batch No GP509, 95.7% purity.

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain 4

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with

Metabolic activation system:

Test bacteria were also exposed to the test item in the presence of an appropriate metabolic activation system, which is a cofactor-supplemented post-mitochondrial S9 fraction.

The post-mitochondrial fraction (S9 fraction) was prepared by the Microbiological Laboratory of according to Ames et al. [1] and Maron and Ames [2]. The documentation of the preparation of this post-mitochondrial fraction is stored in the reagent notebook in the Microbiological Laboratory which is archived yearly. The composition of solution refers to a final volume of 1000 mL.

Test concentrations with justification for top dose:

Based on the results of the preliminary tests, 100 mg/mL stock solution was prepared in DMF, which was diluted by serial dilutions in several steps to obtain the dosing formulations for lower doses. The maximum test concentration was 5000 μg test item/plate.

Examined concentrations in the Initial Mutation Test and Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate.

Examined concentrations in the Complementary Confirmatory Mutation Test (Salmonella typhimurium TA98, TA1535 and TA1537 strains without metabolic activation) were 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 µg/plate.

Vehicle / solvent:

Based on the available information and the results of the solubility testing, DMF was selected as vehicle (solvent) of the study (the selected vehicle was compatible with the survival of the cells and the S9 activity using the pre-incubation method). The results of the Preliminary Compatibility Test are summarized in Table 6.

Controls

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

other: 4-nitro-1,2-phenylene-diamine

Details on test system and experimental conditions:

Procedure for Exposure in the Initial Mutation Test:

A standard plate incorporation procedure was performed as an Initial Mutation Test. Bacteria (cultured in Nutrient Broth No.2 as described in Section 5.3.6.) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system.

Molten top agar was prepared and kept at 45°C. The equivalent number of minimal glucose agar plates (three plates per concentration and for each control) was properly labelled. The test item and other components were prepared freshly and added to the overlay (45°C).
Study code: 15/087-007M Final Report Page 25 of 57


The content of the tubes: top agar 2000 µL vehicle (solvent) or test item solution (or reference controls) 50 µL overnight culture of test strain 100 µL phosphate buffer (pH 7.4) or S9 mix 500 µL

This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (solvent) and positive controls. After preparation, the plates were incubated at 37°C for 48±1 hours.


Procedure for Exposure in the Confirmatory Mutation Test and Complementary Confirmatory Mutation Test:


A pre-incubation procedure [1][2][5][6][7] was performed as a Confirmatory Mutation Test since in the Initial Mutation Test no positive effect was observed (see details in Section 6.). The same method was used in the Complementary Confirmatory Mutation Test.

Bacteria (cultured in Nutrient Broth No.2. as described in 5.3.6.) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system. The equivalent number of minimal glucose agar plates was properly labelled. Molten top agar was prepared and kept at 45°C.

Before the overlaying, 50 µL of test item formulations or its vehicle (or positive reference controls or their solvent), 100 µL of the overnight culture of bacterial cells and the 0.5 mL of S9 mix (activated test conditions) or phosphate buffer pH 7.4 (nonactivated test conditions) were added into the appropriate tubes to provide direct contact between bacteria and the test item. These tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 min at 37ºC in a shaking incubator.

After the incubation period, 2 mL of molten top agar were added to the tubes, and then the content mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37°C for 48±1 hours.

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

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle 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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

The reported data of this mutagenicity assay show (see Appendix 2 to 6) that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test item 4-Hydroxy-TEMPO benzoate had no mutagenic activity in the examined bacterial strains under the test conditions of the study,