Coconut oil, reaction products with boric acid (H3BO3), diethanolamine and glycerol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental phase of this study was performed between 17 March 2015 and 21 May 2015.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Test guideline 4

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

Meets the requirements of the Japanese Regulatory Authorities including METI, MHLW and MAFF, OECD Guidelines for Testing of Chemicals No. 471 "and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Deviations:

no

Remarks:

EXAMPLE:

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Coconut oil, reaction products with boric acid (H3BO3), diethanolamine and glycerol

EC Number:

806-731-9

Cas Number:

1428353-74-5

Molecular formula:

Substance is a UVCB.

IUPAC Name:

Coconut oil, reaction products with boric acid (H3BO3), diethanolamine and glycerol

Test material form:

other: liquid

Details on test material:

Identification: TS13001Physical state/Appearance:amber colored liquidExpiry Date: 30 January 2015Storage Conditions:room temperature in the darkIntended use/Application: Lubricating oil additive

Specific details on test material used for the study:

test itemInformation as provided by the Sponsor. A copy of the test item certificate of analysis, generated by Harlan Laboratories Ltd., is included as Appendix 3.Batch:TS13001Purity:100%Physical state/Appearance:Extremely viscous amber liquidExpiry Date:12 March 2017Storage Conditions:Room temperature, in the dark, under nitrogenNo correction was made for purity.

Method

Target gene:

Histidine for Salmonella.Tryptophan for E.Coli

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/beta­naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/platemain test: Experiment one: 50, 150, 500, 1500 and 5000 µg/plateExperiment two: 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO- Justification for choice of solvent/vehicle: In solubility checks performed in-house, SP5076 was noted as immiscible in sterile distilled water at 50 mg/mL, but fully miscible in dimethyl sulphoxide at the same concentration and acetone at 100 mg/mL. Therefore, dimethyl sulphoxide was selected as the vehicle.

Controls

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

The vehicle control used was dimethyl sulphoxide.

True negative controls:

yes

Remarks:

2-Aminoanthracene (2AA) and Benzo(a)pyrene (BP)

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Details on test system and experimental conditions:

Test SystemTester StrainsThe five strains of bacteria used, and their mutations, are as follows:Salmonella typhimuriumStrains: TA1537 , TA98Genotype: his C 3076; rfa-; uvrB-: , his D 3052; rfa-; uvrB-;R-factorType of mutations indicated: frame shift mutationsStrains: TA1535 , TA100Genotype: his G 46; rfa-; uvrB-: , his G 46; rfa-; uvrB-;R-factorType of mutations indicated: base-pair substitutionsEscherichia coliStrain: WP2uvrAGenotype: trp-; uvrA-:Type of mutations indicated: base-pair substitutionAll 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 the University of California, Berkeley, on culture discs, on 04 August 1995 and from the British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987. All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34.In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 1459314 02/19) and incubated at 37 °C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.Test and Control Items PreparationTest ItemIn solubility checks performed in-house, SP5076 was noted as immiscible in sterile distilled water at 50 mg/mL, but fully miscible in dimethyl sulphoxide at the same concentration and acetone at 100 mg/mL. Therefore, dimethyl sulphoxide was selected as the vehicle.The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer and sonication for 5 minutes at room temperature on the day of each experiment. 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 has been reflected in the GLP compliance statement. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10-4 microns.Control ItemsVehicle, negative (untreated) and positive controls were used in parallel with the test item. The vehicle control used was dimethyl sulphoxide.The positive control items used in the series of plates without S9-mix were as follows:N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG): 2 μg/plate for WP2uvrAN-ethyl-N'-nitro-N-nitrosoguanidine (ENNG): 3 μg/plate for TA100N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG): 5 μg/plate for TA15359-Aminoacridine (9AA): 80 μg/plate for TA15374-Nitroquinoline-1-oxide (4NQO): 0.2 μg/plate for TA98In addition, 2-Aminoanthracene (2AA) and Benzo(a)pyrene (BP), which are non-mutagenic in the absence of metabolizing enzymes, were used in the series of plates with S9-mix at the following concentrations:2-Aminoanthracene (2AA):1 μg/plate for TA1002-Aminoanthracene (2AA):2 μg/plate for TA1535 and TA15372-Aminoanthracene (2AA):10 μg/plate for WP2uvrABenzo(a)pyrene (BP):5 μg/plate for TA98Microsomal Enzyme FractionLot No. PB/βNF S9 01 March 2015 was used in this study. The S9 Microsomal fraction was prepared in-house from male rats induced with Phenobarbitone/β-Naphthoflavone at 80/100 mg/kg/day, orally, for 3 days prior to preparation on day 4. The S9 homogenate was produced by homogenizing the liver in a 0.15M KCl solution (1g liver to 3 mL KCl) followed by centrifugation at 9000 g. The protein content of the resultant supernatant was adjusted to 20 mg/mL. Aliquots of the supernatant were frozen and stored at approximately -196 °C. Prior to use, each batch of S9 was tested for its capability to activate known mutagens in the Ames test.This procedure was designed and conducted to cause the minimum suffering or distress to the animals consistent with the scientific objectives and in accordance with the Harlan Laboratories Ltd, Shardlow, UK policy on animal welfare and the requirements of the United Kingdom’s Animals (Scientific Procedure) Act 1986 Amendment Regulations 2012. The conduct of the procedure may be reviewed, as part of the Harlan Laboratories Ltd, Shardlow, UK Ethical Review Process.S9-Mix and AgarThe S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.S95.0 mL1.65 M KCl/0.4 M MgCl2 1.0 mL0.1 M Glucose-6-phosphate2.5 mL0.1 M NADP2.0 mL0.2 M Sodium phosphate buffer (pH 7.4)25.0 mLSterile distilled water14.5 mLA 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.Top agar was prepared using 0.6% Bacto agar (lot number 4045080 01/19) 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 numbers 39090 05/15 and 39139 05/15).Test ProcedureTest for Mutagenicity (Experiment 1 - Range-Finding Test) – Plate Incorporation MethodDose selectionThe test item was tested using the following method. The maximum concentration was 5000 μg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.Without Metabolic Activation0.1 mL of the appropriate concentration of test item, vehicle or appropriate positive control was added to 2 mL of molten trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.With Metabolic ActivationThe procedure was the same as described previously (see 3.5.1.2) except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten trace amino-acid supplemented media instead of phosphate buffer.Incubation and ScoringAll of the plates were incubated at 37 °C± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Several manual counts were required, predominantly due to interference in the base agar e.g. minor precipitation of salts/dust particles or bubbles slightly distorting the counts.Test for Mutagenicity (Experiment 2 - Main Test) – Plate Incorporation MethodThe plate incorporation method was employed for Experiment 2 in the presence and absence of metabolic activation to confirm initial findings.Dose selectionThe dose ranges used for Experiment 2 (main test) were determined by the results of Experiment 1 (the range-finding test) and were as follows:All Salmonella strains: (1.5, 5, 15, 50, 150, 500, and 1500 μg/plate)Escherichia coli strain: (5, 15, 50, 150, 500, 1500, 5000 µg/plate)Additional dose levels and an expanded dose range were selected in order to achieve both four non-toxic dose levels and the toxic limit of the test item.Without Metabolic Activation The procedure was the same as described previously (see 3.5.1.2). All testing for this experiment was performed in triplicate.With Metabolic ActivationThe procedure was the same as described previously (see 3.5.1.3). All testing for this experiment was performed in triplicate.Incubation and ScoringAll of the plates were incubated at 37 °C± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Acceptance CriteriaThe reverse mutation assay may be considered valid if the following criteria are met: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) and Mortelmans and Zeiger (2000).All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. Acceptable ranges are presented in the Study Plan (Appendix 2). Combined historical negative and solvent control ranges for 2013 and 2014 are presented in Appendix 4.All tester strain cultures should be in the range of 0.9 to 9 x 109 bacteria per mL.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. The historical ranges of the positive control reference items for 2013 and 2014 are presented in Appendix 4.There should be a minimum of four non-toxic test item dose levels.There should be no evidence of excessive contamination.

Evaluation criteria:

There are several criteria for determining a positive result. For a test item to score positive in this test system it should normally have induced at least a twofold increase in revertant colony frequency for TA98, TA100 and WP2uvrA and a threefold increase for TA1535 and TA1537 (especially if accompanied by an out-of-historical range response). The effect must be reproducible in an independent assay with evidence of a dose-related response. However, if required, the study conclusion can be evaluated using one, or all of the following to determine the overall result of the study:A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).A reproducible increase at one or more concentrations.Biological relevance against in-house historical control ranges.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.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Prior to use, the master strains were checked for characteristics, 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 was shown to be sterile. The test item formulation was also shown to be sterile. These data are not given in the report. Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.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 are presented in Appendix 1.The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. SP5076 caused a visible reduction in the growth of the bacterial background lawn in all Salmonella strains (from 500 µg/plate) and in WP2uvrA (5000 µg/plate) both with and without metabolic activation. Therefore, in the main test (Experiment 2), the test item was either tested up to the maximum recommended dose level of 5000 μg/plate (WP2uvrA) or the toxic limit (Salmonella strains). In the second mutation test, SP5076 again caused a visible reduction in the growth of the bacterial background lawn in Salmonella strains (initially from 150 µg/plate) and in WP2uvrA (5000 µg/plate). No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix either by eye or under an inverted microscope.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 in two separate experiments. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Applicant's summary and conclusion

Conclusions:

The test substance was considered to be non-mutagenic under the conditions of this test.