Fatty acids, tall-oil, reaction products with boric acid (H3BO3) and diethanolamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: The test item was considered to be non-mutagenic (OECD 471, EU Method B13/B14, ICH S2(R1) and relevant Japanese guidelines)

 

Gene mutation in mammalian cells: The test material was considered to be non-mutagenic to CHO cells under the conditions of two hypoxanthine-guanine phosphoribosyl transferase (HGPRT) tests.

 

Mouse lymphoma assay: The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF), consequently it is considered to be non-mutagenic in this assay (OECD 490, EU Method B.17 and OPPTS 870.5300).

 

Chromosome aberration test: The test item was considered to be non-clastogenic to human lymphocytes in vitro (OECD 473)

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:

10 June 2020 to 10 July 2020

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:

21 July 1997 as corrected in 2020

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

August 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH S2(R1) guideline

Version / remarks:

June 2012; Federal Register, 77: 33748–33749

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF.

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine and tryptophan

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital / β-naphthaflavone induced S9 mix

Test concentrations with justification for top dose:

- Experiment 1 (plate incorporation method): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
- Experiment 2 (pre-incubation method): 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate (eight test item concentrations selected depending on bacterial strain type and presence or absence of S9-mix)

Vehicle / solvent:

Dimethyl formamide

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl formamide (Acros Organics)

Remarks:

Experiment 1: batch 1871962, purity ≥ 99.75 %, expiry date June 2021 based on individual bottle open date; Experiment 2: batch 1871962, purity ≥ 99.75 %, expiry date July 2021 based on individual bottle open date

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

ENNG (CAS 4245-77-6; batch 67F-3700; purity treated as 100 %; expiry date 18 Sep 2021; solvent DMSO)

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

9-aminoacridine

Remarks:

9AA (CAS 90-45-9; batch S32398-438; purity 99.9 %; expiry date 01 October 2021; solvent DMSO)

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

4NQO (CAS 56-57-5; batch WXBC1554V; purity 98 %; expiry date 02 December 2021; solvent DMSO)

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

other: 2-aminoanthracene

Remarks:

2AA (CAS 613-13-8; batch STBB1901M9; purity 97.5 %; expiry date 08 October 2021; solvent DMSO)

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

benzo(a)pyrene

Remarks:

BP (CAS 50-32-8; batch 090M1400V; purity 96 %; expiry date 12 October 2021; solvent DMSO)

Details on test system and experimental conditions:

STUDY CONTROLS
- The vehicle control used was dimethyl formamide. The negative (untreated) controls were performed to assess the spontaneous revertant colony rate. The solvent and negative controls were performed in triplicate.
- The positive control items used demonstrated a direct and indirect acting mutagenic effect depending on the presence or absence of metabolic activation. The positive controls were performed in triplicate.

STERILITY CONTROLS
- Top agar and histidine/biotin or tryptophan in the absence of S9-mix (in triplicate).
- Top agar and histidine/biotin or tryptophan in the presence of S9-mix (in triplicate).
- The maximum dosing solution of the test item in the absence of S9-mix only (test in singular prior to Experiment 1).

MICROSOMAL ENZYME FRACTION
- The Phenobarbitone / β-Naphthoflavone induced S9 microsomal fractions (Sprague-Dawley) were purchased from Moltox.
- Lot No: 4146 was used in this study with the protein level adjusted to 20 mg/mL.
- A copy of the S9 Certificate of Efficacy was provided in Appendix 3 of the full study report.

S9 MIX AND AGAR
- The S9-mix was prepared before use using sterilised co-factors and maintained on ice for the duration of the test.
- The S9 mix contained S9 (5.0 mL); 1.65 M KCl/0.4 M MgCl2 (1.0 mL); 0.1 M glucose-6-phosphate (2.5 mL); 0.1 M NADP (2.0 mL); 0.2 M sodium phosphate buffer pH 7.4 (25.0 mL); sterile distilled water (14.5 mL).
- A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.

MEDIA
- Top agar was prepared using 0.6 % Bacto agar (lot number 9105946; expiry date 01/2024) and 0.5 % sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar.
- Vogel-Bonner Minimal agar plates were purchased from SGL Ltd (lot number 54834; expiry date 07/2020 and lot number 54898; expiry date 07/2020).

BACTERIA
- The five strains of bacteria used are shown in the table below together with their mutations.
- All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through the deletion of the uvrB- bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin. In the strains TA98 and TA100, the R-factor plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error-prone repair pathway. The plasmid also confers ampicillin resistance which acts as a convenient marker (Mortelmans and Zeiger, 2000). In addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would normally act to remove and repair the damaged section of the DNA molecule (Green and Muriel, 1976 and Mortelmans and Riccio, 2000).
- The bacteria used in the test were obtained from British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987 or Trinova Biochem GmbH on 27 June 2017.
- All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34. Batches of culture are prepared from master stocks on a regular basis (approximately monthly) and coded, these are then routinely tested for appropriate characteristics, viability and mutation frequency to ensure acceptability criteria is met.
- In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 2547490; expiry date 07/2024) and incubated at 37 ± 3 °C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

TEST ITEM PREPARATION AND ANALYSIS
- The test item was immiscible or only partially miscible in sterile distilled water and dimethyl sulphoxide at 50 mg/mL and immiscible in acetone at 100 mg/mL but was fully miscible in dimethyl formamide at 50 mg/mL. Dimethyl formamide was therefore selected as the vehicle.
- The test item was accurately weighed and, on the day of each experiment, approximate half-log dilutions prepared in high purity dimethyl formamide by mixing on a vortex mixer. No correction for purity was required. Dimethyl formamide is considered an acceptable vehicle for use in this test system (Maron et al., 1981). All test item preparation and dosing was performed under yellow safety lighting.
- All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and was reflected in the GLP compliance statement.

DOSE SELECTION FOR EXPERIMENT 1 – PLATE INCORPORATION METHOD
- 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.
- The maximum concentration was 5000 μg/plate because this is the maximum recommended dose level given by OECD test guideline 471.

EXPERIMENT 1 - WITHOUT METABOLIC ACTIVATION
- An aliquot (0.1 mL) of the appropriate concentration of test item, solvent vehicle or 0.1 mL of the appropriate positive control was added together with 0.1 mL of the bacterial strain culture, 0.5 mL of phosphate buffer and 2 mL of molten, trace amino-acid supplemented media. 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.

EXPERIMENT 1 - WITH METABOLIC ACTIVATION
- The procedure was the same as described previously except that following addition of the test item formulation and bacterial culture, S9-mix (0.5 mL) was added to the molten trace amino-acid supplemented media instead of phosphate buffer.

EXPERIMENT 1 - INCUBATION AND SCORING
- All of the plates were incubated at 37 ± 3 °C for between 48 and 72 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).
- Sporadic manual counts were performed due to differences in colony size which prevented an accurate automated count.

DOSE SELECTION FOR EXPERIMENT 2 – PRE-INCUBATION METHOD
- Since the result of Experiment 1 was considered negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation (S9-mix).
- The dose range used for Experiment 2 was determined by the results of Experiment 1 and was as follows:
(i) TA100 (absence of S9) and TA1537 (presence of S9): 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 μg/plate.
(ii) TA1535, TA1537 (absence of S9) and TA100 (presence of S9): 0.15, 0.5, 1.5, 5, 15, 50, 150 500 μg/plate.
(iii) TA98 (absence of S9) and TA1535 (presence of S9): 0.5, 1.5, 5, 15, 50, 150 500, 1500 μg/plate.
(iv) WP2uvrA (absence and presence of S9) and TA98 (presence of S9): 1.5, 5, 15, 50, 150 500, 1500, 5000 μg/plate.
- Eight test item concentrations per bacterial strain were selected in Experiment 2 in order to ensure the study achieved at least four non-toxic dose levels as required by the test guideline, and were selected based on the cytotoxicity noted in Experiment 1, and the potential for a change in the cytotoxicity of the test item following the change in test methodology from plate incorporation to pre-incubation.

EXPERIMENT 2 - WITHOUT METABOLIC ACTIVATION
- An aliquot (0.1 mL ) of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the appropriate concentration of test item formulation, 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.

EXPERIMENT 2 - WITH METABOLIC ACTIVATION
- The procedure was the same as described previously 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.

EXPERIMENT 2 - INCUBATION AND SCORING
- All of the plates were incubated at 37 ± 3 °C for between 48 and 72 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).

ACCEPTANCE CRITERIA
- The reverse mutation assay may be considered valid if the following criteria are met:
(i) All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al., (1975), Maron and Ames (1983), Mortelmans and Zeiger (2000), Green and Muriel (1976) and Mortelmans and Riccio (2000).
(ii) All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls). Acceptable ranges are TA 1535 (7 to 40); TA100 (60 to 200); TA1537 (2 to 30); TA98 (8 to 60); WP2uvrA (10 to 60). Combined historical negative and solvent control ranges for 2018 and 2019 were presented in Appendix 2 of the full study report.
(iii) All tester strain cultures should be in the range of 0.9 to 9 x 10E+09 bacteria per mL.
(iv) Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation (S9-mix). The historical ranges of the positive control reference items for 2018 and 2019 were presented in Appendix 2 of the full study report.
(v) There should be a minimum of four non-toxic test item dose levels.
(vi) There should be no evidence of excessive contamination.

MAJOR COMPUTERISED SYSTEMS
- Perceptive Instruments – Ames Study Manager v1.24 and Ames Sorcerer v3.

Rationale for test conditions:

- The purpose of the study was to evaluate the test item for the ability to induce reverse mutations, either directly or after metabolic activation, at the histidine or tryptophan locus in the genome of five strains of bacteria.
- The study was based on the in vitro technique described by Ames et al., (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000), in which mutagenic effects are determined by exposing mutant strains of Salmonella typhimurium to various concentrations of the test item. The Salmonella typhimurium strains have a deleted excision repair mechanism which makes them more sensitive to various mutagens and they will not grow on media which does not contain histidine. When large numbers of these organisms are exposed to a mutagen, reverse mutation to the original histidine independent form takes place. These are readily detectable due to their ability to grow on a histidine deficient medium. Using these strains of Salmonella typhimurium revertants may be produced after exposure to a chemical mutagen, which have arisen as a result of a base-pair substitution in the genetic material (miscoding) or as a frameshift mutation in which genetic material is either added or deleted. Additionally, a mutant strain of Escherichia coli (WP2uvrA) which requires tryptophan and can be reverse mutated by base-pair substitution to tryptophan independence (Green and Muriel, 1976 and Mortelmans and Riccio, 2000) is used to complement the Salmonella strains.
- Since many compounds do not exert a mutagenic effect until they have been metabolized by enzyme systems not available in the bacterial cell, the test item and the bacteria are also incubated in the presence of a liver microsomal preparation (S9-mix) prepared from rats pre-treated with a mixture known to induce an elevated level of these enzymes.

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:
(i) A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
(ii) A reproducible increase at one or more concentrations.
(iii) Biological relevance against in-house historical control ranges.
(iv) A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
(v) Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
- A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
- Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

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.

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS
- Prior to use, the relevant strains were checked for characteristics (deep rough character, ampicillin resistance, UV light sensitivity and histidine or tryptophan auxotrophy), viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments were shown to be sterile. The test item formulation was also shown to be sterile. These data were not given in the report.
- Results for the negative controls (spontaneous mutation rates) and viability are presented in Table 1 of the full study report and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
- The vehicle (dimethyl formamide) 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 and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
- The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive and vehicle controls, both with and without metabolic activation (S9-mix), were presented in the full study report (Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2). The results were also expressed graphically in Figure 1 to Figure 4 of the full study report.
- Information regarding the equipment and methods used in these experiments as required by the Japanese Ministry of Economy, Trade and Industry and Japanese Ministry of Health, Labour and Welfare were presented in Appendix 1 of the full study report.
- A history profile of vehicle, untreated and positive control values (reference items) was presented in Appendix 2 of the full study report.

EXPERIMENT 1 – PLATE INCORPORATION
- The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 μg/plate.
- The test item induced a visible reduction in the growth of the bacterial background lawns and/or substantial reductions in the revertant colony frequency of all of the Salmonella tester strains in the first mutation test (plate incorporation method) in the absence of metabolic activation (S9-mix), initially from 150 μg/plate for TA100, TA1535 and TA1537 and 500 μg/plate for TA98.
- In the presence of metabolic activation (S9-mix), weakened bacterial background lawns and/or substantial reductions in the revertant colony frequency were initially noted from 150 μg/plate for TA100 and TA1537, 500 μg/plate for TA1535 and 1500 μg/plate for TA98.
- No toxicity was noted for Escherichia coli strain WP2uvrA at any test item dose level in either the absence and presence of metabolic activation (S9-mix).
- No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix).
- 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).

EXPERIMENT 2 – PRE-INCUBATION
- The maximum dose level of the test item in the second experiment was 5000 μg/plate or the toxic limit, depending on bacterial strain type and presence/absence of metabolic activation (S9-mix).
- The test item induced a stronger toxic response after employing pre-incubation methodology with visible reductions in the growth of the bacterial background lawns noted for all of the Salmonella tester strains dosed in the absence of metabolic activation (S9-mix), initially from 50 μg/plate for TA100 and TA1537, 150 μg/plate for TA1535 and 500 μg/plate for TA98.
- In the presence of metabolic activation (S9-mix), weakened bacterial background lawns were initially noted from 50 μg/plate for TA1537, 150 μg/plate for TA100, 500 μg/plate for TA1535 and 1500 μg/plate for TA98.
- As noted in the first mutation test, no toxicity was observed for Escherichia coli strain WP2uvrA at any test item dose level in either the absence and presence of metabolic activation (S9-mix).
- No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix).
- 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).

Conclusions:

The test item did not induce an increase in the frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix). Under the conditions of this investigation, the test item was considered to be non-mutagenic.

Executive summary:

GUIDELINE

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, the ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749) 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 (plate incorporation) was based on OECD TG 471 and was 1.5 to 5000 μg/plate. The experiment was performed 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 0.05 and 5000 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 ensure the study achieved at least four non-toxic dose levels as required by the test guideline. The dose levels were selected based on the cytotoxicity noted in Experiment 1 plus the potential for a change in the cytotoxicity of the test item following the change in test methodology from plate incorporation to pre-incubation.

 

RESULTS

The vehicle (dimethyl formamide) 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 and 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 OECD TG 471 recommended dose level of 5000 μg/plate. The test item induced a visible reduction in the growth of the bacterial background lawns and/or substantial reductions in the revertant colony frequency of all of the Salmonella tester strains in the first mutation test (plate incorporation method), initially from 150 μg/plate in both the absence and presence of metabolic activation (S9-mix). No toxicity was noted for Escherichia coli strain WP2uvrA at any test item dose level in either the absence and presence of metabolic activation (S9-mix).

 

Based on the results of Experiment 1, the same maximum dose level (5000 μg/plate) or the toxic limit was employed in the second mutation test (pre-incubation method), depending on bacterial strain type and presence or absence of S9-mix. The test item induced a stronger toxic response employing pre-incubation methodology with visible reductions in the growth of the bacterial background lawns noted for all of theSalmonellatester strains, initially from 50 μg/plate in both the absence and presence of metabolic activation (S9-mix). Once again, no toxicity was noted for Escherichia coli strain WP2uvrA at any test item dose level in either the absence and presence of metabolic activation (S9-mix).

 

No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix) in Experiments 1 and 2.

 

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 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).

 

CONCLUSION

The test item did not induce an increase in the frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix). Under the conditions of this investigation, the test item was considered to be non-mutagenic.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

05 April 1983 to 03 June 1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Done according a method similar to OECD 471 current at the time of the study but does not include strain TA102 or a repeat experiment. The former is not considered to be significant and the latter was corrected by the performance of an additional study, included as supporting data as another endpoint study record. Only duplicate plates per dose level were used but this is acceptable in the OECD 471 guideline.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

no repeat experiment

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine locus

Species / strain / cell type:

S. typhimurium, other: TA1535, TA100, TA1537, TA98 and TA1538

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver metabolic activation system

Test concentrations with justification for top dose:

10, 30, 100, 300, 1000 ug/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

2.5 ug/plate TA100 and TA1535 -S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

10 ug/plate TA98 and TA1538

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

100 ug/plate TA1537

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-anthramine

Remarks:

2.5 ug/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- in agar (plate incorporation)

DURATION
- Exposure duration: 2 days

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: other: Evaluation of the growth of the background lawn

OTHER EXAMINATIONS:
- Other: Some of the colonies of TA1535 were picked off and grown in histidine deficient media to determine if they were true revertants or not. Only one colony was a revertant indicating that there wa no mutagenicity.

Evaluation criteria:

A test substance is generally considered mutagenic if it induces a concentration-dependent response in which the number of revertants per plate exceeds two times the spontaneous mtation rate (negative control)

Statistics:

Not done

Species / strain:

S. typhimurium, other: TA1535, TA100, TA1537, TA98 and TA1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Reductions in revertant counts and reduced background lawn

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

At the maximum dose level tested (1000 ug/ml) there were reductions in colony counts and a thining of the growth of the backgraound lawn of non-revertant bacteria. In the with-S9 group for TA98, TA100 and TA1535 there was also observed a high number of small colonies. There were considered to be survivors of toxicity rathe than true revertants and this was tested by picking a number of colonies and attempting to grow them on minimal media. Only one colony was shown to be a true revertant and it was concluded that this was not evidence of mutagenicity.

Remarks on result:

other: all strains/cell types tested

Concentration ug/plate	S9	TA98	TA100	TA1535	TA1537	TA1538
DMSO	-	21	215	35	7	14
10	-	18	209	28	12	15
30	-	16	184	36	5	16
100	-	15	82	30	9	21
300	-	24	79	15	2	8
1000	-	6	5	36	1	1
Positive control	-	425	401	739	901	1395

Concentration ug/plate	S9	TA98	TA100	TA1535	TA1537	TA1538
DMSO	+	44	201	50	9	40
10	+	41	246	41		34
30	+	45	241	27	8	31
100	+	32	251	29	10	43
300	+	40	84	35	11	48
1000	+	31^	452^	581^	0	4
Positive control	+	1754	1 655	229	183	377

^ = colonies on these plates were very small and did not appear to be true revertants. Several colonies of strain TA1535 were streaked on minmal agar plates and observed after 3 days to determine if they were true revertants. Only 1 colony appeared to be a true revertant.

Conclusions:

The test item did not induce significant mutations in any of the Salmonella strains in the quantitative mutagenesis assay, either in the presence or absence of metabolic activation. Thus, under the conditions of the assay employed, the test item is determined to be non-mutagenic in the salmonella/microsome mutagenesis assay.

Executive summary:

Test Guidance

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Method and Material

The test item was tested in the Ames revertant assay both with and without metabolic activation. The study used five strains of Salmonella that were in common use at the time of the study. The mutagenicity test used only duplicate plates per dose level, but this is acceptable according to the current version of OECD 471. Only a single experiment was performed but this again is acceptable according to the current version of OECD 471. Furthermore, an additional study was performed by the same laboratory on the same substance at a later date, and this confirmed the negative result.

Results

The conclusion of the study was that the test item did not induce significant mutations in any of the Salmonella strains in the quantitative mutagenesis assay, either in the presence or absence of metabolic activation.

Conclusion

Thus, under the conditions of the assay employed, the test item was determined to be non-mutagenic in the salmonella/microsome mutagenesis assay.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

12 June 1984 to 14 June 1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Done according a method similar to OECD 471 current at the time of the study but does not include strain TA102 or a repeat experiment. This study is included as supporting data to the key study endpoint record. Only duplicate plates per dose level were used but this is acceptable in the OECD 471 guideline.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

no repeat experiment

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine locus

Species / strain / cell type:

S. typhimurium, other: TA1535, TA100, TA1537, TA98 and TA1538

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver metabolic activation system

Test concentrations with justification for top dose:

50, 100, 200, 300, 400 ug/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

2.5 ug/plate TA100 and TA1535 -S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

10 ug/plate TA98 and TA1538

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

100 ug/plate TA1537

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-anthramine

Remarks:

2.5 ug/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- in agar (plate incorporation)

DURATION
- Exposure duration: 2 days

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: other: Evaluation of the growth of the background lawn

OTHER EXAMINATIONS:
- Other: Ten of the colonies of TA1535 were picked off and grown in histidine deficient media to determine if they were true revertants or not. Only two colonies were true revertants indicating that there was no mutagenicity.

Evaluation criteria:

A test substance is generally considered mutagenic if it induces a concentration-dependent response in which the number of revertants per plate exceeds two times the spontaneous mtation rate (negative control)

Statistics:

Not done

Species / strain:

S. typhimurium, other: TA1535, TA100, TA1537, TA98 and TA1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Reductions in revertant counts and reduced background lawn

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

At the maximum dose level tested (400 ug/ml) there were reductions in colony counts and a thining of the growth of the backgraound lawn of non-revertant bacteria. In TA1535 there was also observed a number of small colonies. There were considered to be survivors of toxicity rathe than true revertants and this was tested by picking ten colonies and attempting to grow them on minimal media. Only two colonies was shown to be a true revertant and it was concluded that this was not evidence of mutagenicity.

Remarks on result:

other: all strains/cell types tested

Concentration ug/plate	S9	TA98	TA100	TA1535	TA1537	TA1538
DMSO	-	24	93	16	5	9
50	-	17	78	16	4	2
100	-	19	61	12	6	15
200	-	15	42	6	4	15
300	-	7a	9a	7a	0a	15
400	-	2a	7a	35ab	0a	8a
Positive control	-	722	867	690	1666	1364

a = toxic effect sobserved

Concentration ug/plate	S9	TA98	TA100	TA1535	TA1537	TA1538
DMSO	+	30	92	19	7	29
50	+	34	93	14	8	16
100	+	39	81	18	10	36
200	+	32	15a	12	5	8a
300	+	22a	6a	9	3	25
400	+	8a	6a	30ab	1a	31
Positive control	+	902	1294	237	62	924

b = colonies on these plates were very small and did not appear to be true revertants. Ten colonies of were streaked on minmal agar plates and observed after 3 days to determine if they were true revertants. Only 2 colonies appeared to be true revertants.

Conclusions:

The test item did not induce significant mutations in any of the Salmonella strains in the quantitative mutagenesis assay, either in the presence or absence of metabolic activation. Thus, under the conditions of the assay employed, the test item is determined to be non-mutagenic in the salmonella/microsome mutagenesis assay.

Executive summary:

Test Guidance

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Method and Material

The test item was tested in the Ames revertant assay both with and without metabolic activation. The study used five strains of Salmonella that were in common use at the time of the study. The mutagenicity test used only duplicate plates per dose level, but this is acceptable according to the current version of OECD 471. Only a single experiment was performed but this again is acceptable according to the current version of OECD 471. Furthermore, this study was performed as an additional study by the same laboratory on the same substance as one done at an earlier date, and this confirmed the negative result.

Results

The conclusion of the study was that the test item did not induce significant mutations in any of the Salmonella strains in the quantitative mutagenesis assay, either in the presence or absence of metabolic activation.

Conclusion

Thus, under the conditions of the assay employed, the test item was determined to be non-mutagenic in the salmonella/microsome mutagenesis assay.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1984-05-30 to 1984-06-21

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

restriction becuase experimental procedures were slightly different from the current OECD guidance.

Principles of method if other than guideline:

Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) is a purine salvage enzyme which provides a scavenger pathway for the synthesis of purine nucleotides. Mutants deficient in HGPRT activity are selected by growing mammalian cells in medium containing 6-thioguanine (6-TG). These purine analogues are HGPRT substrates and are converted to toxic nucleotides, thus killing the wild type cells. Mutant cells, those lacking HGPRT activity, cannot metabolize the purine analogues and thus survive the selection step. The best established HGPRT system has been developed at the Oak Ridge laboratories under Dr. Hsie, and it quantifies the frequency of mutation at the HGPRT locus in Chinese hamster cells (either lung cells, V79, or in ovary cells, CHO line) (O'Neill et al,1977a, O;Neill, et at, 1977b, Shaw and Hsie, 1978, O'Neill-and Hsie, 1979).

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) gene.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: no data available
- Properly maintained: no data available
- Periodically checked for Mycoplasma contamination: no data available
- Periodically checked for karyotype stability: no data available
- Periodically "cleansed" against high spontaneous background: no data available

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 rat liver microsome fraction from Aroclor-induced Sprague-Dawley rats (Litton Bionetics lot No. RDL 127).

Test concentrations with justification for top dose:

Without S9 (mg/ml) : 0.29,0.19, 0.15,0.12,0.09,0.07,0.05,0.03,0.015
With S9 (mg/ml): 0.34,0.24,0.18,0.14,0.12,0.1,0.07,0.05,0.03,0.015

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test material was a complex mixture and DMSO was selected as the solvent because the test material was readily soluble in it at the required concentration.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Benzo(a)pyrene, Ethylmethanesulphonate

Remarks:

Ethylmethanesulphonate (EMS) at 240 ug/ml for non-activated assay; B(a)P at 10 ug/ml in the presence of metabolic activation.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;

DURATION
- Exposure duration: 4 hours +S9, 16 hours -S9
- Expression time (cells in growth medium): at least 7 days
- Selection time (if incubation with a selection agent):at least 7 days

SELECTION AGENT (mutation assays): 6-TG

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: plating efficiency

Evaluation criteria:

A substance was regarded as mutagenic if observing statistically significant differences between the mutation frequencies of the test concentrations and those of the negative controls.

Statistics:

The data were transformed and analysed using analysis of variance methods.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Range-finding Experiments (data from BSC project # 11586, section 7.6.1 Key HPRT Gorodecki 1983 LZ)

The effects of test substance on the survival of CHO cells in the absence/presence of the activation system were evaluated in previous project (BSC 11586). The test sample exhibited similar toxicity under activated and non-activated conditions. Based on the results of the range-finding experiments, a concentration range of 0.03-0.47 mg/ml was tested in both initial activated and non-activated mutagenesis assays.

 

Non-activated Mutagenesis Experiments

In the previous experiment (data from BSC project #11586), nine concentrations ranging from 0.03-0.47 mg/ml were tested, however, only five survived the expression period. A test sample concentration of 0.12 mg/ml resulted in approximately 13% survivors. The background mutant frequencies of the DMSO controls were 17.7 and 14.6 x 10^-6. The mutant frequencies of the F10 medium control were 9.9 and 15.0 X10^-6. These values are within the acceptable background range of 0-20 outlined by  the EPA Gene-Tox Program.  None of the test sample concentrations induced mutant frequencies significantly different from the negative controls.  The  positive control, EMS  (234 ug/ml)  induced significant increases in mutation frequency (811 and 968 x 10^-6).

In experiment no.  2  without activation, nine concentrations ranging from 0.015-0.29 mg/ml were tested; six survived the expression period. A test sample concentration of 0.12 mg/ml resulted in approximately 8% survivors.  Toxicity similar to experiment no. 1 was observed. The background mutant frequencies of the DMSO controls were 46 .1 and 26.1 x 10^-6 The mutant frequencies of the medium controls were 16.9 and 17.8 x 10^-6. The average of these values are higher than those reported in the first experiment, but does not affect the interpretation of the data. One test sample concentration, 0.07 mg/ml, had a single point with a mutant frequency higher than the negative controls (89.8 x 10^-6), however the duplicate flask had a much lower mutant frequency  (15  x  10^-6) . None  of  the other test sample concentrations had mutant frequencies different from the negative controls.  The  positive control, EMS  (234 ug/m } induced significant increases in mutant frequency (558 and 478 x 10^-6).

 

Activated Mutagenesis Assays

The test substance was also tested in the presence of a metabolic activation system. In experiment no. l (data from BSC project # 11586) nine concentrations ranging from 0.03-0.47 mg/ml were tested; seven survived the expression period. A test sample concentration  of 0.19 mg/ml resulted in approximately  37% survivors. The background mutant  frequencies of the DMSO + S9 controls were 14.2 and 39.8 x 10^-6. The mutant frequencies of the medium controls were 34.1 and 14.2 X 10^-6 in the presence of S9 and 25.9 X 10^-6 in its absence. Several test sample concentrations had single points with mutant frequencies higher than the negative controls.  In each case, however the mutant frequency of the duplicate flask was much lower. A concentration - related response was not observed. The positive control B (a) P (10 ug/ml) plus S9 induced significant increases in mutant frequency (247 1 and 345 X 10^-6)

Ten concentrations ranging from 0.015-0.34 mg/ml were: tested in experiment no. 2 with activation eight survived the expression period.  Toxicity similar to experiment No. 1 was observed. The background mutant frequencies of the DMSO + S9 controls were 57.6 and 18.0 X 10^-6. The mutant frequencies of the medium controls were 18.4 and 34.7 X 10^-6 in the presence of S9 and 22.2 and 9.7 X 10^-6 in its absence. The elevated value was regarded as spontaneous fluctuation. Two test sample concentrations (0 .14 and 0.18 mg/ml) had points with mutant frequencies higher than the negative controls (64.1 and 84.4 X 10^-6); a concentration-related response was not observed. None of the other test sample concentrations had mutant frequencies different from those of the negative controls. The positive control, DMBA (15 ug/ml) plus S9, induced significant increases in mutant frequency (262 and 244 X 10^-6 ).

Conclusions:

The ability of the test substance to induce mutations at the HGPRT locus in CHO cells was evaluated in duplicate independent experiments in the presence and absence of a metabolic activation system. Under the conditions of the assay employed, the compound did not exhibit mutagenic activity.

Executive summary:

Principle:

Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) is a purine salvage enzyme which provides a scavenger pathway for the synthesis of purine nucleotides. Mutants deficient in HGPRT activity are selected by growing mammalian cells in medium containing 6-thioguanine (6-TG). These purine analogues are HGPRT substrates and are converted to toxic nucleotides, thus killing the wild type cells. Mutant cells, those lacking HGPRT activity, cannot metabolize the purine analogues and thus survive the selection step. The best established HGPRT system has been developed at the Oak Ridge laboratories under Dr. Hsie, and it quantifies the frequency of mutation at the HGPRT locus in Chinese hamster cells (either lung cells, V79, or in ovary cells, CHO line) (O'Neill et al,1977a, O;Neill, et at, 1977b, Shaw and Hsie, 1978, O'Neill-and Hsie, 1979).

 

Conclusion:

The test material was considered to be non-mutagenic to CHO cells under the conditions of the test.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1983-02-28 to 1983-04-19

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

restriction because the experimental procedures were slightly different from the current OECD guidance.

Principles of method if other than guideline:

Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) is a purine salvage enzyme which provides a scavenger pathway for the synthesis of purine nucleotides. Mutants deficient in HGPRT activity are selected by growing mammalian cells in medium containing 6-thioguanine (6-TG). These purine analogues are HGPRT substrates and are converted to toxic nucleotides, thus killing the wild type cells. Mutant cells, those lacking HGPRT activity, cannot metabolize the purine analogues and thus survive the selection step. The best established HGPRT system has been developed at the Oak Ridge laboratories under Dr. Hsie, and it quantifies the frequency of mutation at the HGPRT locus in Chinese hamster cells (either lung cells, V79, or in ovary cells, CHO line) (O'Neill et al,1977a, O;Neill, et at, 1977b, Shaw and Hsie, 1978, O'Neill-and Hsie, 1979).

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) gene.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: no data available
- Properly maintained: no data available
- Periodically checked for Mycoplasma contamination: no data available
- Periodically checked for karyotype stability: no data available
- Periodically "cleansed" against high spontaneous background: no data available

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 rat liver microsome fraction from Aroclor-induced Sprague-Dawley rats (Litton Bionetics lot No. RDL 127).

Test concentrations with justification for top dose:

Without S9 (mg/ml) : 7.5, 1.75 (no survival), 1.5, 1.25, 1.0, 0.7, 0.5, 0.2, 0.1, 0.05, 0.02, 0.01,
With S9 (mg/ml) : 1.0, 0.7, 0.5, 0.2, 0.1, 0.05, 0.02, 0.01

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test material was a complex mixture and DMSO was selected as the solvent because the test material was readily soluble in it at the required concentration.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Benzo(a)pyrene, Ethylmethanesulphonate

Remarks:

Ethylmethanesulphonate (EMS) at 240 ug/ml for non-activated assay; B(a)P at 10 ug/ml in the presence of metabolic activation.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;

DURATION
- Preincubation period: Not applicable.
- Exposure duration: 4 h (activated), or 16 h (non-activated).
- Expression time (cells in growth medium): 8 days


DURATION
- Exposure duration: 4 hours +S9, 16 hours -S9
- Expression time (cells in growth medium): 9 to 10 days
- Selection time (if incubation with a selection agent): 8 to 10 days

SELECTION AGENT (mutation assays): 6-TG

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: plating efficiency

Evaluation criteria:

A substance was regarded as mutagenic if observing statistically significant differences between the mutation frequencies of the test concentrations and those of the negative controls.

Statistics:

The data were transformed and analysed using analysis of variance methods.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

All the experiments indicated evidence of greater toxicity under non-activated rather than under activated conditions. This presumably is due to the shorter exposure time used in the activated assays. Based on range-finding experiments, a series of concentrations were chosen for the mutagenicity experiments. Test sample concentration ranges of 0-0.19mg/ml for the activated assay and 0-0.12mg/ml for the non-activated assay produced cells which were evaluated for mutagenesis; higher concentrations were toxic in both assays.

 

Five concentrations were tested the induction of HGPRT- mutants in CHO cells in a non-activated assay. The background mutation frequencies for the negative control, 1% DMSO, were 17.7 and 14.6 x 10^-6. These values are within the acceptable range of 0-20 x 10^-6 outlined by the Gene Tox Program (Mutation Research, 86 (1981) 192-214). None of the test concentrations had mutation frequencies significantly different than the negative controls. The positive control, EMS (240 ug/ml) induced significant increases in mutation frequency.

Seven concentrations of test material were also tested in the presence of a metabolic activation system. The background mutation frequencies for 1% DMSO plus S9 were 14.2 and 39.8 x10^-6. The average of these background mutation frequencies (27 x 10 ^-6) exceeds the acceptable range of the Gene Tox Program. The cells used for this assay, however, were from the same source (all vials thawed 3/18/83) and were seeded only one day later than those used in the non-activated assay in which background mutation frequencies were well within the accepted range. Thus, no explanation is available for the elevated background. It is suspected, however, that the high background is a consequence of a spontaneous fluctuation. Several test sample points were higher than the background, but the replicate flask in these cases had a very low mutation frequency.  This lack of reproducibility between replicate flasks makes it unlikely that the high mutation frequencies were induced by the compound. Statistical analysis was performed (see Statistical Analysis) and showed no significant differences between the mutation frequencies of the test concentrations and those of the negative controls. The positive control, B {a) P (10 ug/ml) induced significantly greater mutation frequencies of 247 and 345 x 10^-6.  

Conclusions:

The ability of the test substance to induce mutations at the HGPRT gene locus in Chinese hamster ovary cells was evaluated in the presence and absence of a metabolic activation system. Under the conditions of the assay employed, the compound did not exhibit any mutagenic activity.

Executive summary:

Principle:

Hypoxanthine-guanine phosphoribosyl transferase (HGPRT)  is a purine salvage enzyme which provides a scavenger pathway for the synthesis of  purine nucleotides. Mutants deficient in HGPRT activity are selected by growing mammalian cells in medium containing 6-thioguanine (6-TG). These purine analogues are HGPRT substrates and are converted to toxic nucleotides, thus killing the wild type cells. Mutant cells, those lacking HGPT activity, cannot metabolize the purine analogues and thus survive the selection step. The best established HGPRT system has been developed at the Oak Ridge laboratories under Dr. Hsie, and it quantifies the frequency of mutation at the HGPRT locus in Chinese hamster cells (either lung cells, V79, or in ovary cells, CHO line) (O'Neill et al,1977a, O;Neill, et at, 1977b, Shaw and Hsie, 1978, O'Neill-and Hsie, 1979).

 

Conclusion:

The test material was considered to be non-mutagenic to CHO cells under the conditions of the test.

Reference 6

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 July 2020 to 03 August 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

29 Jully 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Method B67 of Commission Regulation (EC) No. 440/2008

Version / remarks:

26 September 2019

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes

Type of assay:

other: mouse lymphoma assay

Target gene:

Thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus)

Metabolic activation:

with and without

Metabolic activation system:

S9 Microsomal Enzyme Fraction supplied by Moltox

Test concentrations with justification for top dose:

- Test item dose levels of 2.5, 5, 10, 20, 40, 60, 80 and 100μg/mL for 4-hour exposure to the test item without S9-mix in the main experiment.
- Test item dose levels of 20, 40, 60, 80, 85, 90, 95 and 100μg/L for 4-hour exposure to the test item with S9-mix in the main experiment.

Vehicle / solvent:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Remarks:

Fisher; batch number 196455; purity 99.9 %; expiry date 01 September 2024

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

ethylmethanesulphonate

Remarks:

EMS (Sigma Aldrich; batch BCBZ8402; purity 100 % assumed; expiry date 21 October 2024; solvent DMSO)

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

cyclophosphamide

Remarks:

CP (Acros Organics; batch A0389646; purity 99.9 %; expiry date 01 October 2022; solvent DMSO)

Details on test system and experimental conditions:

CELL LINE
- The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK.
- The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.

CELL CULTURE
- The stocks of cells are stored in liquid nitrogen at approximately -196 °C.
- Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 μg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 μg/mL) and 10% donor horse serum (giving R10 media) at 37 °C with 5% CO2 in air.
- The cells have a generation time of approximately 12 hours and were sub-cultured accordingly. RPMI 1640 with 20 % donor horse serum (R20), 10 % donor horse serum (R10), and without serum (R0), are used during the course of the study. All donor horse serum was purchased heat inactivated from the supplier. Master stocks of cells were tested and found to be free of mycoplasma.

MICROSOMAL ENZYME FRACTION
- The S9 Microsomal fraction used during the course of the study was purchased from Moltox (Lot 4222; expiry date 12 March 2022). The S9 was pre-tested for acceptability by the supplier prior to purchase and was supplied with a relevant “Quality Control & Production Certificate” which was presented in Annex 2 of the full study report. The protein content was adjusted to 20 mg/mL prior to use.
- The S9 mix was prepared by mixing S9 with a phosphate buffer containing NADP (5 mM), G6 P (5 mM), KCl (33 mM) and MgCl2 (8 mM) to give a 20 % S9 concentration. The final concentration of S9 when dosed at a 10 % volume of S9-mix was 2 % for the Preliminary Toxicity Test and the Mutagenicity Test.

CELL CLEANSING
- The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate.
- Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 μg/mL), Hypoxanthine (15 μg/mL), Methotrexate (0.3 μg/mL) and Glycine (22.5 μg/mL). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium.

TEST ITEM PREPARATION
- The test item was a UVCB, therefore the maximum proposed dose level in the solubility test was initially set at 5000 μg/mL.
- The test item was insoluble in culture media at 50 mg/mL but was soluble in DMSO at 500 mg/mL during solubility checks performed in-house. Prior to each experiment, the test item was accurately weighed, dissolved in DMSO and serial dilutions prepared.
- 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 (Scott et al., 1991). The pH and osmolality readings are presented in the table below.
- The test item was formulated within two hours of it being applied to the test system; it is assumed that the test item formulation was stable for this duration. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation because it is not a requirement of the guidelines. This is an exception with regard to GLP and was reflected in the GLP compliance statement.

PRELIMINARY TOXICITY TEST
- A preliminary toxicity test was performed on cell cultures at 5 x 10E+05 cells/mL, using a 4-hour exposure period both with and without metabolic activation (S9). The dose range used in the preliminary toxicity test was 2.5 to 640 μg/mL to avoid excessive precipitating dose levels.
- Following the exposure periods the cells were washed twice with R10, resuspended in R20 medium, counted and then serially diluted to 2 x 10E+05 cells/mL.
- The cultures were incubated at 37 °C with 5 % CO2 in air and sub-cultured after 24 hours by counting and diluting to 2 x 10E+05 cells/mL. After a further 24 hours, the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post exposure toxicity, and a comparison of each exposure SG value to the concurrent solvent control performed to give a percentage Relative Suspension Growth (%RSG) value.
- Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments. Maximum dose levels were selected using the following criteria:
(i) For non-toxic test items the upper test item concentrations will be 10 mM, 2 mg/mL or 2 μL/mL whichever is the lowest. When the test item is a substance of unknown or variable composition (UVCB) the upper dose level may need to be higher and the maximum concentration will be 5 mg/mL.
(ii) Precipitating dose levels will not be tested beyond the onset of precipitation regardless of the presence of toxicity beyond this point.
(iii) In the absence of precipitate and if toxicity occurs, the highest concentration should lower the Relative Total Growth (RTG) to approximately 10 to 20 %. This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al., 2002).

MUTAGENICITY TEST
- Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 10E+06 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals for the 4-hour exposure groups in both the absence and presence of metabolic activation.
- The exposures were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration) at eight dose levels of the test item and solvent and positive controls. To each universal was added 2 mL of S9-mix if required, 0.2 mL of the exposure dilutions, (0.2 mL or 0.15 mL for the positive controls), and sufficient R0 medium to bring the total volume to 20 mL.
- Two exposure groups were used for Main Experiment:
(i) 4-hour exposure to the test item without S9-mix. The dose levels of test item used were 2.5, 5, 10, 20, 40, 60, 80 and 100μg/mL.
(ii) 4-hour exposure to the test item with S9-mix (2 %). The dose levels of test item used were 20, 40, 60, 80, 85, 90, 95 and 100μg/mL.
- The exposure vessels were incubated at 37 °C for 4 or 24 hours with continuous shaking using an orbital shaker within an incubated hood.

MEASUREMENT OF SURVIVAL, CLONING EFFICIENCY AND MUTANT FREQUENCY
- At the end of the exposure periods, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 10E+05 cells/mL. The cultures were incubated at 37 °C with 5 % CO2 in air and sub-cultured every 24 hours for the expression period of two days, by counting and dilution to 2 x 10E+05 cells/mL.
- On Day 2 of the experiment, the cells were counted, diluted to 10E+04 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 μg/mL 5-trifluorothymidine (TFT) in 96-well plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for cloning efficiency (%V) in non-selective medium.
- The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post exposure toxicity during the expression period as a comparison to the solvent control, and when combined with the cloning efficiency (%V) data, a Relative Total Growth (RTG) value.

PLATE SCORING
- Microtitre plates were scored using a magnifying mirror box after ten to twelve days incubation at 37 °C with 5 % CO2 in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutant plates were also recorded as the additional information may contribute to an understanding of the mechanism of action of the test item (Cole et al, 1990). Colonies are scored manually by eye using qualitative judgment.
- Large colonies are defined as those that cover approximately 1/4 to ¾ of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25 % of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 mL of thiazolyl blue tetrazolium bromide (MTT) solution, 2.5 mg/mL in phosphate buffered saline (PBS), was added to each well of the mutant plates. The plates were incubated for two hours. MTT is a vital stain that is taken up by viable cells and metabolised to give a brown/black color, thus aiding the visualization of the mutant colonies, particularly the small colonies.

CALCULATION OF PERCENTAGE RELATIVE SUSPENSION GROWTH (%RSG)
- The cell counts obtained immediately post exposure and over the 2-day expression period were used to calculate the Percentage Relative Suspension Growth:
(i) 4-Hour Suspension Growth (SG) = (24-hour cell count/2) * (48-hour cell count/2)
(ii) Day 0 Factor = dose 0-hour cell count/vehicle control 0-hour cell count
(iii) %RSG = [(dose SG x dose Day 0 Factor)/vehicle control SG] x 100

CALCULATION OF DAY 2 VIABILITY (%V)
- Since the distribution of colony-forming units over the wells is described by the Poisson distribution, the day 2 viability (%V) was calculated using the zero term of the Poisson distribution [P(0)] method:
(i) P(0) = number of negative wells / total wells plated
(ii) %V = -ln P(0) x 100 / number of cells/well

CALCULATION OF RELATIVE TOTAL GROWTH (RTG)
- For each culture, the relative cloning efficiency, RCE, was calculated using the equation RCE = %V / mean solvent control %V
- The RTG was then calculated for each culture using the equation RTG = (RCE * RSG) / 100

CALCULATION OF MUTATION FREQUENCY (MF)
- The mutation frequency (MF) per survivor = [(-ln P(0) selective medium)/cells per well in selective medium)]/surviving fraction in non-selective medium.

DATA EVALUATION
- The current Historical Solvent and Positive Control Mutant Frequencies was presented in Appendix 1 of the full study report. The Historical Solvent and Positive Control data is generated by the Mutant 240C program on a rolling system of the last twenty sets of archived data.
- Dose selection for the mutagenicity experiments was made using data from the preliminary toxicity test in an attempt to obtain the desired levels of toxicity. This optimum toxicity is approximately 20 % survival (80% toxicity), but no less than 10 % survival (90 % toxicity). Relative Total Growth (RTG) values are the primary factor used to designate the level of toxicity achieved by the test item for any individual dose level. However, under certain circumstances, %RSG values may also be taken into account when designating the level of toxicity achieved. Dose levels that have RTG survival values less than 10 % are excluded from the mutagenicity data analysis, as any response they give would be considered to have no biological or toxicological relevance.
- An approach for defining positive and negative responses is recommended to assure that the increased MF is biologically relevant. In place of statistical analysis generally used for other tests, it relies on the use of a predefined induced mutant frequency (i.e. increase in MF above the concurrent control), designated the Global Evaluation Factor (GEF) of 126 x 10E-06 i.e. the mutant frequency of the concurrent solvent control plus 126, which is based on the analysis of the distribution of the solvent control MF data from participating laboratories.
- Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.
- Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system.

ACCEPTABILITY OF ASSAY
- A mutation assay is considered acceptable if it meets the following criteria (the current recommendations of the IWGT are considered; (Moore et al., 2002; Moore et al., 2003; Moore et al., 2006; Moore et al., 2007):
(1) For non-toxic test items the upper test item concentrations will be 10mM, 2 mg/mL or 2μL/mL whichever is the lowest. When the test item is a substance of unknown or variable composition (UVCBs) the upper dose level may need to be higher and the maximum concentration will be 5 mg/mL. Precipitating dose levels will not be tested beyond the onset of precipitation regardless of the presence of toxicity beyond this point. In the absence of precipitate and if toxicity occurs, the highest concentration should lower the Relative Total Growth (RTG) and/or percentage Relative Suspension Growth (%RSG) to approximately 10 to 20 % of survival.
(2) The absolute Cloning Efficiency (%V) at the time of mutant selection of the solvent controls is 65 to 120 %.
(3) The total suspension growth of the solvent control following 4 hour exposure, calculated by the day 1 fold-increase in cell number multiplied by the day 2 fold increase in cell number, should be in the range of 8 to 32. Following 24-hour exposure the total suspension growth should be in the range 32 to 180.
(4) The in-house historical solvent control mutant frequency is in the range of approximately 50 to 170 x 10E-06 cells. Solvent control results should ideally be within this range, although minor errors in cell counting and dilution, or exposure to the metabolic activation system, may cause this to be slightly elevated.
(5) Every test should also be evaluated as to whether the positive controls (EMS and CP) meet at least one of the following two acceptance criteria developed by the IWGT workgroup:
(i) The positive control should demonstrate an absolute increase in total MF, that is, an increase above the spontaneous background MF [an induced MF (IMF)] of at least 300 x 10E-06. At least 40 % of the IMF should be reflected in the small colony MF.
(ii) The positive control has an increase in the small colony MF of at least 150 x 10E-06 above that seen in the concurrent untreated/solvent control (a small colony IMF of 150 x 10E-06).
(6) The upper limit of cytotoxicity observed in the positive control culture should be the same as for the experimental cultures i.e. the Relative Total Growth (RTG) and percentage Relative Suspension Growth (%RSG) should be greater than approximately 10 % of the concurrent selective control group.
(7) A minimum of four analysed duplicate dose levels is considered necessary in order to accept a single assay for evaluation of the test item.

MAJOR COMPUTERISED SYSTEMS
- None

Rationale for test conditions:

- The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.
- The use of cultured mammalian cells for mutation studies may give a measure of the intrinsic response of the mammalian genome and its maintenance process to mutagens. Such techniques have been used for many years with widely different cell types and loci. The thymidine kinase heterozygote system, TK +/- to TK -/-, was described by Clive et al., (1972) and is based upon the L5178Y mouse lymphoma cell line established by Fischer (1958). This system has been extensively validated (Clive et al., 1979; Amacher et al., 1980; Jotz and Mitchell, 1981).
- The technique used was a fluctuation assay using microtitre plates and trifluorothymidine as the selective agent and is based on that described by Cole and Arlett (1984). Two distinct types of mutant colonies can be recognised, i.e. large and small. Large colonies grow at a normal rate and represent events within the gene (base-pair substitutions or deletions) whilst small colonies represent large genetic changes involving chromosome 11b (indicative of clastogenic activity).

Evaluation criteria:

See above

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

PRELIMINARY CYTOTOXICITY TEST
- The dose range of the test item used in the preliminary toxicity test was 2.5 to 640 μg/mL.
- The results for the Relative Suspension Growth (%RSG) were as shown in the table below.
- In the 4-hour exposures, both in the absence and presence of metabolic activation (S9), there was evidence of marked reductions in the relative suspension growth (%RSG) of cells treated with the test item when compared to the concurrent solvent controls.
- A precipitate of the test item was observed at and above 160 μg/mL in both exposure groups. In the subsequent mutagenicity experiments the maximum dose was limited by a combination of test item induced toxicity and precipitate of the test item.

MUTAGENICITY TEST
- A summary of the results from the test is presented in Table 1 (below).
- The results of the microtitre plate counts and their analysis are presented in Tables 2 to 4 of the full study report.
- There was evidence of toxicity following exposure to the test item in both of the exposure groups, as indicated by the %RSG and RTG values. There was evidence of modest reductions in cloning efficiency (%V) in the absence of metabolic activation only, therefore indicating that residual toxicity occurred in this exposure group.
- Optimum levels of toxicity were achieved in both the absence and presence of metabolic activation. The excessive toxicity observed at and above 60 μg/mL in the absence of metabolic activation, and at and above 90 μg/mL in the presence of metabolic activation, resulted in these dose levels not being plated for viability or 5-TFT resistance. The toxicity observed at 85 μg/mL in the presence of metabolic activation exceeded the upper acceptable limit of 90%, therefore, this dose was excluded from the analysis. Acceptable levels of toxicity were seen with both positive control substances.
- The solvent controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional.
- The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10E-06 per viable cell in either the presence or absence of metabolic activation.. Precipitate of test item was observed at and above 60 μg/mL in the absence of metabolic activation, and at and above 90 μg/mL in the presence of metabolic activation, at the end of the exposure period.
- The numbers of small and large colonies and their analysis were presented in Table 3 and Table 5 of the full study report.

RESULTS OF PRELIMINARY CYTOTOXICITY TEST

Dose (μg/mL)	% RSG (-S9) 4-hour exposure	% RSG (+S9) 4-hour exposure
0	100	100
2.5	100	113
5	84	109
10	96	114
20	76	108
40	41	89
80	0	52
160 P	0	1
320 P	0	0
640 P	0	0

P = precipitate observed at the end of the exposure period

 

TABLE 1 – SUMMARY OF MUTAGENICITY TEST RESULTS

Test item concentration (μg/mL)	4-hour (-S9) %RSG	4-hour (-S9) RTG	4-hour (-S9) MF§	Test item concentration (μg/mL)	4-hour (+S9) %RSG	4-hour (+S9) RTG	4-hour (+S9) MF§
0	100	1	78.33	0	100	1	114.05
2.5	103	1.29	69.95	20	89	0.84	109.18
5	95	1.03	85.78	40	74	0.99	69.22
10	96	1.17	79.42	60	63	0.65	97.82
20	88	0.89	86.80	80	17	0.18	88.90
40	28	0.20	126.60	85 X	8	0.07	91.79
60 P	0	0.00	NP	90 P	2	0.00	NP
80 P	–	–	NP	95 P	13	0.00	NP
100 P	–	–	NP	100 P	0	0.00	NP
Positive control concentration (μg/mL)	4-hour (-S9) %RSG	4-hour (-S9) RTG	4-hour (-S9) MF§	Positive control concentration (μg/mL)	4-hour (+S9) %RSG	4-hour (+S9) RTG	4-hour (+S9) MF§
400 (EMS)	84	0.73	628.22	1.5 (CP)	66	0.43	1007.12

MF threshold for a positive response (-S9) = 204.33

MF threshold for a positive response (+S9) = 240.05

%RSG = Relative Suspension Growth

RTG = Relative Total Growth

MF§ = 5-TFT resistant mutants/10E+05 viable cells 2 days after exposure

X = Treatment excluded from test statistics due to toxicity

P = precipitate observed at the end of the exposure period

NP = Not plated due to toxicity / precipitate, or surplus to requirements

Conclusions:

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded theGlobal Evaluation Factor (GEF), consequently it is considered to be non-mutagenic in this assay.

Executive summary:

GUIDELINE

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guideline for Testing of Chemicals No 490 "In VitroMammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 29 July 2016, Method B67 of Commission Regulation (EC) No. 440/2008 of 26 September 2019, and the US EPA OPPTS 870.5300 Guideline.

 

METHODS

One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels in duplicate, together with solvent (DMSO), and positive controls using 4 hour exposure groups both in the absence and presence of metabolic activation (2 % S9).

 

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies were 2.5, 5, 10, 20 and 40 μg/mL for 4-hour without S9 and 20, 40, 60, 80 and 85 μg/mL for 4-hour with S9 (2 %).

 

RESULTS

The maximum dose level used was limited by a combination of test item-induced toxicity and test item precipitate. Precipitate of the test item was observed at and above 60 μg/mL in the absence of metabolic activation , and at and above 90 μg/mL in the presence of metabolic activation, at the end of the exposure period.

 

The solvent control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- 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 increases in the mutant frequency at any of the dose levels in the main test that exceeded the Global Evaluation Factor (GEF), using a dose range that achieved optimum levels of toxicity in both of the exposure groups, and at least four analysable dose levels in each exposure group, as recommended by the OECD 490 guideline.

 

CONCLUSION

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded theGlobal Evaluation Factor (GEF), consequently it is considered to be non-mutagenic in this assay.

Reference 7

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 29 June 1998 and 12 January 1999.

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Chromosome damage.

Species / strain / cell type:

other: human whole blood

Details on mammalian cell type (if applicable):

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability. The volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. The average generation time (AGT) for the regular donors used in this laboratory has been determined to be approximately 14 hours under optimal growth conditions.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 was prepared, in-house, from the livers of male Sprague-Dawley rats weighing 200g. These had received a single ip injection of Aroclor 1254 at 500 mg/kg, five days before S9 preparation. The metabolic activation fraction (S9) was stored at -196 C.

Test concentrations with justification for top dose:

Experiment 1

with S9:
0, 20, 40, 80, 120, 160 and 240 ug/ml. 1 ml of 10% S9 (ie 1% final concentration of S9) in standard co-factors was added and the cultures
Positive control (Cyclophosphamide, CP), 25ug/ml

without S9:
0, 20, 40, 80, 120, 160 and 240 ug/ml
Positive control (Ethyl methanesulphonate, EMS), 750ug/ml

Experiment 2
with S9:
0, 5, 10, 20*, 40*, 80*, 120*, 160, 240 and 320 ug/ml. 1 ml of 20% S9 (ie 2% final concentration) in standard co-factors was added and the cultures
Positive control (CP), 25ug/ml

without S9:
0, 5, 10, 20*, 40*, 80*, 120*, 160, 240 and 320 ug/ml
Positive control (EMS), 500ug/ml


*: samples were analyzed for chromosome aberrations.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test material was a complex mixture, insoluble in water, and acetone was selected as the solvent because the test material was readily soluble in it at the required concentration.

Untreated negative controls:

no

Remarks:

Cyclophosphamide, Ethylmethanesulphonate

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other:

Details on test system and experimental conditions:

METHOD OF APPLICATION: Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
9.05 ml MEM, 15% (FCS)
0.1 ml Li-heparin
0.1 ml phytohaemagglutinin-M
0.75 ml heparinised whole blood.

DURATION
- Exposure duration:
Cells cultured for 48 hours before exposure, then:
Experiment 1: 4 (16)h with and without S9
Experiment 2: 4(16)h with S9/ 20 h without S9

- Fixation time (start of exposure up to fixation or harvest of cells): . The cells were fixed by using fresh methano/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at 4 C for at least four hours to ensure complete fixation.
SPINDLE INHIBITOR (cytogenetic assays): Demecolcine (Colcemid 0.1 ug/ml) two hours before the required harvest time

STAIN: for metaphase analysis, the slides were stained in 5% Gurrs Giemsa R66 for 5 minutes, rinsed, dried and coverslipped using mounting medium
NUMBER OF REPLICATIONS: All treatments were performed in duplicate.

NUMBER OF CELLS EVALUATED: 200 per dose level

DETERMINATION OF CYTOTOXICITY
Method: 2000 nuclei counted to determine the mitotic index

Evaluation criteria:

The test substance will be regarded as clastogenic when it induced a statistically significant increase in the frequency of cells with aberrations either in the presence or absence of metabolic activation.

Statistics:

The frequency of cells with aberrations (both including and excluding gaps) and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher’s Exact test or Chi-squared test.

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no observable change in pH when test material was dosed into media; no evidence of effect from pH.
- Effects of osmolality: no observable change in osmolality when test material was dosed into media; no evidence of effect from osmolality.
- Evaporation from medium: no data available
- Water solubility: no data available
- Precipitation: yes.
- Other confounding effects: none.

Remarks on result:

other: all strains/cell types tested

Chromosome Aberration Test - Experiment 1

The dose levels of the controls and the test material are given in the table below:

	Final concentration(ug/ml)
20h-S9	0*, 20,40,80*,120*, 160*, 240, EMS 750*
20h+S9	0*, 20,40,80,120*,160*, 240*,CP25*

* dose levels selected for metaphase analysis

 

- A cloudy precipitate of the test material was observed at 80 ug/ml and above at the washing stage of the exposure regimen. Haemolysis of the red cells was recorded at 240 ug/ml.

-  The qualitative assessment of the slides determined that there were scorable metaphases at all dose levels in both treatment groups.

- The mitotic index data showed that approximately 50%mitotic inhibition was achieved at 160 ug/ml in the absence of S9 and at 240 ug/ml in the presence of S9.

- The chromosome aberration data show that both of the vehicle control cultures had frequencies of cells with chromosome aberrations that were considered acceptable. The positive control treatments gave statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore satisfactory and the test method itself was operating as expected. The test material did not induce any statistically significant increases in the frequency of cells with aberrations either in the presence or absence of metabolic activation.

- The frequency of polyploid cells data show that the test material induced a significant increase in the numbers of polyploid cells at 120 ug/ml in the absence of metabolic activation, the middle dose assessed for aberrations.

 

Chromosome Aberration Test - Experiment 2

The dose levels of the controls and the test material are given in the table below:

	Final concentration(ug/ml)
20h-S9	0*, 5,10,20*,40*,80*,120*, 160, 240, EMS 500*
20h+S9	0*, 10, 20*,40*,80*,120*,160, 240, 320,CP25*

* dose levels selected for metaphase analysis

- A cloudy precipitate was observed at and above 40 ug/ml without metabolic activation and 120 ug/ml with metabolic activation at the end of the exposure period for each treatment condition. Haemolysis was observed at and above 160 ug/ml.

- The qualitative assessment of the slides determined that there were no scorable metaphases at and above 240 ug/ml and that there were very few metaphase cells at 160 ug/ml.

- A dose related inhibition of mitotic index was observed and approximately 50% mitotic inhibition was achieved at 80 ug/ml in the absence and presence of S9.

-  The chromosome aberration data showed that all of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control treatments gave statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore satisfactory and the test method itself was operating as expected. The test material did not induce any statistically significant increases in the frequency of cells with chromosome aberrations either in the presence or absence of metabolic activation.

- The frequency of polyploid cell data showed that the test material did not induce a significant increase in the numbers of polyploid cells. However, a small increase over the control was observed in the continuous exposure group at 80 ug/ml, and in the presence of S9 at 40 ug/ml. There was no repeat of the response observed in the absence of metabolic activation in Experiment 1 and this may be due to the test material proving to be more toxic. The toxicological significance of polyploid responses is not clear, however, it is not a clastogenic effect.

Conclusions:

The test material did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either the presence or absence of a liver enzyme metabolising system in either of two separate experiments. The test substance was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

Test Guidance

The method used followed that described in the OECD Guidelines for Testing of Chemicals (1 997) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission Directive 92/69/EEC.

Method and Material

Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, ie. 4 hours exposure with the addition of an induced rat liver homogenate metabolising system (S9) with cell harvest after a 16-hour expression period and a 4-hour exposure in the absence of activation with a 16-hour expression period, this was Experiment 1. In Experiment 2 the 4-hour exposure with addition of S9 was repeated, whilst in the absence of activation the exposure time was increased to 20 hours.

Results

All vehicle (solvent) controls gave frequencies of cells with aberrations that were considered acceptable for human lymphocytes. All the positive control treatments gave statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

Conclusion

The test material did not induce any statistically significant increases in the frequency of cells with aberrations. Adequate levels of toxicity were achieved in all exposure groups. The test material was shown to be non-clastogenic to human lymphocytes in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Negative results were obtained during investigation of in vitro gene mutation in bacteria (Ames test), in vitro cytogenicity in mammalian cells (chromosome aberration study) and in vitro gene mutation in mammalian cells (HPRT assay). As a result, and in accordance with ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R.7a: Endpoint specific guidance (Version 6.0; July 2017), the substance is not considered to be genotoxic, and no further testing is required.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro

Ames test

Key study

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, the ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012;77:33748-33749) and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

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 (plate incorporation) was based on OECD TG 471 and was 1.5 to 5000 μg/plate. The experiment was performed 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 0.05 and 5000 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 ensure the study achieved at least four non-toxic dose levels as required by the test guideline, and were selected based on the cytotoxicity noted in Experiment 1, and the potential for a change in the cytotoxicity of the test item following the change in test methodology from plate incorporation to pre-incubation.

The vehicle (dimethyl formamide) 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 and 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 OECD TG 471 recommended dose level of 5000 μg/plate. The test item induced a visible reduction in the growth of the bacterial background lawns and/or substantial reductions in the revertant colony frequency of all of the Salmonella tester strains in the first mutation test (plate incorporation method), initially from 150 μg/plate in both the absence and presence of metabolic activation (S9-mix). No toxicity was noted for Escherichia coli strain WP2uvrA at any test item dose level in either the absence and presence of metabolic activation (S9-mix).

Based on the results of Experiment 1, the same maximum dose level (5000 μg/plate) or the toxic limit was employed in the second mutation test (pre-incubation method), depending on bacterial strain type and presence or absence of S9-mix. The test item induced a stronger toxic response employing pre-incubation methodology with visible reductions in the growth of the bacterial background lawns noted for all of the Salmonella tester strains, initially from 50 μg/plate in both the absence and presence of metabolic activation (S9-mix). Once again, no toxicity was noted for Escherichia coli strain WP2uvrA at any test item dose level in either the absence and presence of metabolic activation (S9-mix).

No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix) in Experiments 1 and 2.

 

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 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).

 

In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item did not induce an increase in the frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix). Under the conditions of this investigation, the test item was considered to be non-mutagenic.

Supporting studies

Two Bacterial reverse mutation assays (Ames tests) were performed according to a test protocol similar to the OECD 471 test guideline. The first study (1983) was tested at higher concentrations. The second study (1984) was a repeat study done in the same laboratory as the original study, but at lower test concentrations to reduce cytotoxicity. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains in both tests, with any dose of the test material, either with or without metabolic activation. Both tests indicate that the substance does not induce gene mutations in bacteria whereas all positive control chemicals (with and without metabolic activation) induced a significant increase in the number of colonies. The substance is therefore considered as non-mutagenic according in the Ames test.

Gene mutation in mammalian cells

The inability to induce gene mutations was confirmed in mammalian cells using an in vitro forward mutation assay in CHO cells (HGPRT test). The first study (1983) was tested at higher concentrations; the second study (1984) was a repeat study done in the same laboratory as the original study, but at lower test concentrations to reduce cytotoxicity.

None of the test substance dose levels up to the cytotoxicity limit, either with or without metabolic activation, induced a significant increase in the mutant frequency. The substance does not induce forward mutations at the HGPRT locus in CHO cells under activation and nonactivation conditions whereas both positive control chemicals (with and without metabolic activation) induced significant mutant frequency increases. The substance is therefore considered as negative for inducing forward mutations at the HGPRT locus in CHO cells under activation and non-activation conditions used in these assays. This result confirms the results of both Ames tests and extends the non-mutagenic effect of the test substance to mammalian cells.

Mouse lymphoma assay

The key study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guideline for Testing of Chemicals No 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 29 July 2016, Method B.17 of Commission Regulation (EC) No. 440/2008 of 26 September 2019, and the US EPA OPPTS 870.5300 Guideline. 

 

One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels in duplicate, together with solvent (DMSO), and positive controls using 4 hour exposure groups both in the absence and presence of metabolic activation (2% S9).

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The test item dose levels plated for viability and expression of mutant colonies were 2.5, 5, 10, 20 and 40 μg/mL (4-hour without S9) and 20, 40, 60, 80 and 85 μg/mL (4-hour with 2 % S9).

 

The maximum dose level used limited by a combination of test item-induced toxicity and test item precipitate. Precipitate of the test item was observed at and above 60 μg/mL in the absence of metabolic activation , and at and above 90 μg/mL in the presence of metabolic activation, at the end of the exposure period.

 

The solvent control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- 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 metabolising system.

The test item did not induce any increases in the mutant frequency at any of the dose levels in the main test that exceeded the Global Evaluation Factor (GEF), using a dose range that achieved optimum levels of toxicity in both of the exposure groups, and at least four analysable dose levels in each exposure group, as recommended by the OECD 490 guideline.

Chromosome aberration test

The method used in the key study followed that described in the OECD Guidelines for Testing of Chemicals (1 997) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission Directive 92/69/EEC.

 

Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, ie. 4 hours exposure with the addition of an induced rat liver homogenate metabolising system (S9) with cell harvest after a 16-hour expression period and a 4-hour exposure in the absence of activation with a 16-hour expression period, this was Experiment 1. In Experiment 2 the 4-hour exposure with addition of S9 was repeated, whilst in the absence of activation the exposure time was increased to 20 hours.

 

All vehicle (solvent) controls gave frequencies of cells with aberrations that were considered acceptable for human lymphocytes.

All the positive control treatments gave statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

 

The test material did not induce any statistically significant increases in the frequency of cells with aberrations. Adequate levels of toxicity were achieved in all exposure groups. The test material was shown to be non-clastogenic to human lymphocytes in vitro.

Justification for classification or non-classification

Negative results were obtained during investigation of in vitro gene mutation in bacteria (Ames test), in vitro cytogenicity in mammalian cells (chromosome aberration study) and in vitro gene mutation in mammalian cells (mouse lymphoma assay). As a result, and in accordance with ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R.7a: Endpoint specific guidance (Version 6.0; July 2017), the substance is not considered to be genotoxic, no further testing is required, and classification under the terms of Regulation (EC)No. 1272/2008 does not apply.