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

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 June 2020 to 10 July 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997 as corrected in 2020

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

August 1998

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: ICH S2(R1) guideline

Version / remarks:

June 2012; Federal Register, 77: 33748–33749

Deviations:

no

Test guideline 5

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

294-785-9

EC Name:

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

Cas Number:

91770-03-5

IUPAC Name:

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

Test material form:

liquid

Details on test material:

- Physical state/appearance: dark brown liquid
- Storage conditions: room temperature in the dark over silica gel

Method

Target gene:

Histidine and tryptophan

Species / strain

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

Controls

Controls 1

Untreated negative controls:

yes

Controls 2

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

Controls 3

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)

Controls 4

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)

Controls 5

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)

Controls 6

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)

Controls 7

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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).

Applicant's summary and conclusion

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.