1,2,3,4-Butanetetracarboxylic acid, tetramethyl ester, reaction products with 1,2,2,6,6-pentamethyl-4-piperidinol and β,β,β',β'-tetramethyl-2,4,8,10-tetraoxaspiro[5.5]undecane-3,9-diethanol

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:

16 November 2016 - 09 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: • Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 112Z5
- Expiration date of the lot/batch: 30 June 2017


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark

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:

other: Type of mutations indicated: TA1537 frame shift, TA1535 base-pair substitution

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: Type of mutations indicated: base-pair substitution

Metabolic activation:

with and without

Test concentrations with justification for top dose:

The test item was tested using the following method. The maximum concentration was 5000 ug/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test item was insoluble in sterile distilled water and dimethyl sulphoxide at 50 mg/mL but was fully soluble in acetone at 100 mg/mL in solubility checks performed in house.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene, 4-Nitroquinoline-1-oxide

Details on test system and experimental conditions:

Test for Mutagenicity: Experiment 1 - Plate Incorporation Method
Dose selection
The test item was tested using the following method. The maximum concentration was 5000 μg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Without Metabolic Activation
0.1 mL of the appropriate concentration of test item, solvent vehicle or appropriate positive control was added to 2 mL of molten, trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.

With Metabolic Activation
The procedure was the same as described previously (see 3.3.2.2) except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.

Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

Test for Mutagenicity: Experiment 2 – Pre-Incubation Method
As Experiment 1 was deemed negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation.

Dose selection
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was as follows:
Salmonella strains TA100 and TA1537 (with and without S9) and TA1535 (without S9): 0.15, 0.5, 1.5, 5, 15, 50, 150 and 500 μg/plate. Salmonella strains TA98 (with and without S9) and TA1535 (with S9) and E.coli strain WP2uvrA (with and without S9): 0.5, 1.5, 5, 15, 50, 150, 500 and 1500 μg/plate.
Eight test item dose levels per bacterial strain were selected in the second mutation test in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item following the change in test methodology from plate incorporation to pre-incubation.


Without Metabolic Activation
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.05 mL of the test item formulation or solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.

With Metabolic Activation
The procedure was the same as described previously (see 3.3.3.2) except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.

Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Occasional plates were manually assessed for accuracy against the automated counts.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

not specified

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated 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:

not specified

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated 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:

not specified

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated 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:

not specified

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated 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:

not specified

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

LA-63P was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Introduction

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

Methods

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and ranged between 0.15 and 1500 μg/plate, depending on bacterial strain type and presence or absence of S9-mix. Eight test item concentrations were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the toxic limit of the test item following the change in test methodology.

Results

The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. In the first mutation test (plate incorporation method), the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains initially from 150 μg/plate in both the absence and presence of S9-mix. Consequently, the toxic limit of the test item was employed as the maximum dose in Experiment 2. In the second mutation test (pre-incubation method), the test item again induced a toxic response with weakened bacterial background lawns noted in the absence of S9-mix from 150 μg/plate (TA100, TA1535 and TA1537) and 1500 μg/plate (TA98 and WP2uvrA). In the presence S9-mix, weakened bacterial background lawns were noted from 500 μg/plate (TA100 and TA1537) and 1500 μg/plate (TA1535, TA98 and WP2uvrA). The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. In the first mutation test (plate incorporation method), a test item precipitate (light and powdery in appearance) was observed at 5000 μg/plate. In the second mutation test (pre-incubation method), a test item precipitate was noted under a low power microscope at 500 μg/plate and intermittently by eye at 1500 μg/plate. The precipitate observations did not prevent the scoring of revertant colonies.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method). A small, statistically significant increase in TA1535 revertant colony frequency was observed in the absence of S9-mix at 1.5 μg/plate in the second mutation test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at 1.5 μg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.6 times the concurrent vehicle control.

Conclusion

LA-63P was considered to be non-mutagenic under the conditions of this test.