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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Test:

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

Chromosome Aberration:

The test item demonstrated marked toxicity and did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that induced 55±5% mitotic inhibition or greater.  The test item,LA-63P was considered to be non-clastogenic to human lymphocytesin vitro.

Gene Mutation Assay:

The test item did not induce any toxicologically significant or concentration-related increases in mutant frequency at any of the concentration levels in the main test, in either the absence or presence of metabolic activation. The test item was shown to be non-mutagenic to V79 cells at the HPRT locus under the conditions of the test.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

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

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 / 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.

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.

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

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

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

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

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 November 2016 - 26 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy Trade and Industry (METI), and Ministry of the Environmental (MOE) Guidelines of 31 March 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In vitro chromosome aberration test in human lympocytes

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
- Solubility and stability of the test substance in the solvent/vehicle: The test item was insoluble in culture medium at 25 mg/mL and insoluble in dimethyl sulphoxide (DMSO) at 250 mg/mL but was soluble in acetone at 500 mg/mL in solubility checks performed in house.

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: donors
- Sex, age and number of blood donors if applicable: aged 18-35
- Whether whole blood or separated lymphocytes were used if applicable: whole blood cultures

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % foetal bovine serum (FBS), at approximately 37 ºC with 5 % CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA)

Additional strain / cell type characteristics:

other: Culture conditions: 9.05 mL MEM, 10% (FBS), 0.1 mL Li-heparin, 0.1 mL phytohaemagglutinin, 0.75 mL heparinized whole blood

Metabolic activation:

with and without

Test concentrations with justification for top dose:

The dose range for the Preliminary Toxicity Test was 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 µg/mL. The maximum dose was the maximum achievable dose level due to formulation difficulties.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone;

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated; 4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation.

Evaluation criteria:

The following criteria were used to determine a valid assay:
• The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory historical control data range.
• All the positive control chemicals induced a positive response (p≤0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix.
• The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
• The required number of cells and concentrations were analyzed.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.
A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.

Key result

Species / strain:

lymphocytes: Human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm
- Precipitation: A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 39.06 µg/mL, in the absence of S9 and at and above 78.13 µg/mL in the presence of S9. Precipitate of the test item was also seen on the slides of all three exposure groups at and above 625 µg/mL.

Conclusions:

LA-63P did not induce any statistically significant increases in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system. The test item was, therefore, considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

Introduction

This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scott et al., 1991).

Methods

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated; 4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation.

The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by toxicity. The dose levels selected for the Main Test were as follows:

Exposure Group

Final concentration of test item LA-63P (μg/mL)

4(20)-hour without S9-1, 2, 4, 6, 8, 10, 12, 16

4(20)-hour with S9 (2%)-2, 4, 8, 10, 12, 16, 20, 24, 32

24-hour without S9-1, 2, 4, 6, 8, 10, 12, 16

Results

All vehicle (acetone) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control items induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item demonstrated marked toxicity and did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that induced 55±5% mitotic inhibition or greater.

Conclusion

The test item, LA-63P was considered to be non-clastogenic to human lymphocytes in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 February 2017 - 27 April 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell transformation assay

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
- Purity: 100%

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Test concentrations with justification for top dose:

A concentration range of 0.78 to 200 μg/mL was used in the preliminary cytotoxicity test. The maximum concentration tested was limited by test item-induced toxicity.

Mutagenicity test - Main experiment:
30, 40, 50, 60, 70, 80, 90, 100, EMS 500 and 750 ug/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone;

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: Dimethyl benzanthracene

Details on test system and experimental conditions:

DURATION
- Exposure duration: 4 hours with S9 (2%) and 4 hours with out S9

PRELIMINARY CYTOTOXICITY TEST:
Several days before starting each experiment, a fresh stock of cells was removed from the liquid nitrogen freezer and grown up to provide sufficient cells for use in the test. The preliminary cytotoxicity test was performed on cell cultures plated out at 1 x 107 cells/225 cm2 flask approximately 24 hours before dosing. This was demonstrated to provide at least 20 x 106 available for dosing in each flask using a parallel flask, counted at the time of dosing. On dosing, the growth media was removed and replaced with serum-free Minimal Essential Medium

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly positive if, in any of the experimental conditions examined:
i) At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
ii) The increase is considered to be concentration-related.
iii) The results are outside the range of the historical negative control data for the test item concentrations.
When all these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly negative if, in all of the experimental conditions examined:
i) None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
ii) There is no concentration related increase.
iii) The results for the test item concentrations are within the range of the historical negative control data.

Statistics:

When there is no indication of any increases in mutant frequency at any concentration then statistical analysis may not be necessary. In all other circumstances comparisons will be made between the appropriate vehicle control value and each individual concentration, using Student’s t-test. Other statistical analysis may be used if they are considered to be appropriate.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Preliminary Cytotoxicity Test:

A concentration range of 0.78 to 200 μg/mL was used in the preliminary cytotoxicity test. The maximum concentration tested was limited by test item-induced toxicity.

At the end of the exposure period, precipitate of the test item was observed at and above 100 μg/mL in both the absence and presence of metabolic activation.

Mutagenicity Test – Main Experiment:

At the end of the exposure period, the onset of precipitate of the test item was observed at 70 μg/ml in both the absence and presence of metabolic activation.

here was evidence of modest concentration-related reductions in the Day 0 cloning efficiency values in both the absence and presence of metabolic activation. There was no evidence of any reductions in the Day 7 cloning efficiencies in any of the concentration levels, therefore indicating that residual toxicity had not occurred.

The test item did not induce any toxicologically significant or concentration-related increases in the mutant frequency at any of the concentration levels in the main test, in either the absence or presence of metabolic activation.

The vehicle control values were all considered to be within an acceptable range, and the positive controls gave marked increases in mutant frequency, indicating the test and the metabolic activation system were operating as expected.

Conclusions:

The test item did not induce any toxicologically significant or concentration-related increases in mutant frequency per survivor in either the absence or presence of metabolic activation. The test item was therefore considered to be non-mutagenic to V79 cells at the HPRT locus under the conditions of this test.

Executive summary:

Introduction

The purpose of this study is to assess the potential mutagenicity of a test item, supplied by the Sponsor, on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line.

Methods

Chinese hamster (V79) cells were treated with the test item at eight concentrations, in duplicate, together with vehicle (acetone) and positive controls in both the absence and presence of metabolic activation.

The concentrations used in the main test were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by a combination of test item-induced toxicity and the onset of test item precipitate in both the absence and presence of metabolic activation, as recommended by the OECD 476 guidelines. The concentrations of test item plated for cloning efficiency and expression of mutant colonies were as follows:

4-hour without S9- 30, 40, 50, 60, 70

4-hour with S9 (2%)- 30, 40, 50, 60, 70

Results

The vehicle (acetone) controls gave mutant frequencies within the range expected of V79 cells at the HPRT locus.

The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.

The test item did not induce any toxicologically significant or concentration-related increases in mutant frequency at any of the concentration levels in the main test, in either the absence or presence of metabolic activation.

Conclusion

The test item was shown to be non-mutagenic to V79 cells at the HPRT locus under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

LA-63P did not show any mutagenic activity in all three in-vitro genetic toxicity studies therefore the test substance is classified as non mutagenic.