Registration Dossier

Diss Factsheets

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

Constituent 1
Chemical structure
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 / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 1535
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 resultsopen allclose all
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
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
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
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
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,