Phosphoric acid, mono- and di-C16-18-alkyl esters

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

It was concluded that the test material was considered to be non-mutagenic in an Ames test.

It was concluded that the test material did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

It was concluded that the test material did not show evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes, in this in vitro test system under the experimental conditions described.

Link to relevant study records

Reference

Reference 1

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:

April - August 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: USA, EPA (TSCA) OCSPP harmonized guidelines 40 CFR 799.9510 TSCA bacterial reverse mutation test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable

Metabolic activation:

with and without

Metabolic activation system:

S-9

Test concentrations with justification for top dose:

Experiment 1: Concentrations of 1.5, 5, 15, 50, 150, 500, 1500 & 5000 µg/plate.
Experiment 2: Concentrations of 50, 150, 500, 1500 & 5000 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:Tetrahydrofuran
Justification: The test material was insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL and acetone at 100 mg/mL but fully soluble in tetrahydrofuran at 200 mg/mL in solubility checks performed in-house within the testing laboratory. Tetrahydrofuran was therefore selected as the vehicle.

Untreated negative controls:

yes

Remarks:

ENNG: 2µg/plate for WP2uvrA, 3µg/plate for TA100, 5µg/plate for TA1535; 9AA: 80µg/plate for TA1537; 4NQO: 0.2µg/plate for TA98.

Negative solvent / vehicle controls:

yes

Remarks:

Tetrohydrofuran

True negative controls:

not specified

Positive controls:

yes

Remarks:

2AA: 1µg/plate for TA100, 2µg/plate for TA1535 and TA1537, 10µg/plate for WP2uvrA; BP: 5µg/plate for TA98.

Positive control substance:

benzo(a)pyrene

other: 2-aminoanthracene (2AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
The test material was accurately weighed and approximate half-lg dilutions prepared in tetrahydrofuran by mixing on a vortex mixer and sonication for 30 minutes at 40°C in the day of each experiment. Tetrahydrofuran is toxic to the bacterial cells at above 50µl after employing the pre-incubation modification, therefore all of the formulation for Experiment 2 were prepared at concentrations four times greater than required on Vogel-Bonner agar plates. To compensate, each formulation was dosed using 0.025 mL (25µL) aliquots. All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneiry and stability of the test material formulation is not a requirement of the test guidelines and was, therefore not determined. This is an exeption with regards 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. 2mm sodium alumino-silicate pellets with a nominal pore diameter of 4E-4 microns.

TEST PROCEDURE
Experiment 1 - Plate Incorporation Method
- Dose: The maximum concentration was 5000µg/plate. Eight concentrations of the test material (1.5, 5, 15, 50, 150, 500, 1500 amd 5000 µg/plate) were assayed in triplicate against each tester strain.
- Without activation: 0.025mL of the concentration of test material or vehicle or 0.1 mL of appropriate positive control was added to 2mL of trace amino-acid supplemented media containing 0.1mL of one of the bacterial strain cultures and 0.5mL of phosphate buffer. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Each concentration of test material, positive control and each bacterial strain, was assayed using triplicates plates.
- With activation: The procedure was as above, except that following the addition of the test material formulation and bacterial culture, 0.5mL of S-9 mix was added to the trace amino-acid supplemented media in place of phosphate buffer.

Experiment 2 - Pre-Incubation Method
- Dose: The dose range used for Experiment 2 was determined by the results of Experiment 1, 50, 150, 500, 1500 amd 5000 µg/plate.
- Witout activation: 0.1mL of the appropriate bacterial strain culture, 0.5mL of phosphate buffer and 0.025mL of the test material formulation or vehicle or 0.1mL of appropriate positive control were incubated at 37 ± 3°C for 20 minutes (with shaking) prior to addition of 2mL of aminio-acid supplemented media and subsequent plating onto Vogel-Bonner plates.
-With activation: The procedure was as above, except that following the addition of the test material formulation and bacterial strain culture, 0.5mL of S-9 mix was added to the tube instead of phosphate buffer. All testing for this experiment was performed in triplicate.
All plates were incubated at 37 ± 3°C for 20 minutes for approximately 48 hours.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results:
negative

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

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 May 2017 to 16 February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Physical State/Appearance: White waxy solid
Purity: 100%, UVCB substance
Storage Conditions: 15-25 deg.C, in the dark

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Culture of Lymphocytes
Human blood was collected aseptically from two healthy, non-smoking, adult (between 18-35 years of age) donors, pooled in equal volumes from each donor and were stimulated to undergo cell division by the addition of phytohaemagglutinin (PHA). All cultures were then incubated at 34 to 39°C.

Media
HML Media: RPMI 1640, supplemented with 10% fetal calf serum, 0.2 IU/mL sodium heparin, 20 IU/mL penicillin / 20 µg/mL streptomycin and 2.0 mM L glutamine.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Vehicle / solvent:

Ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

colchicine

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Vehicle and positive control cultures were included in all appropriate test conditions.

S9 fraction, prepared from male Sprague-Dawley derived rats dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver, was from Envigo Shardlow and stored at -90 to -70 deg.C.

S9 mix contains: S9 fraction (10% v/v), MgCl2 (8 mM), KCl (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADP (4 mM). All the cofactors were filter sterilized before use.

Prior to commencing testing the solubility of the test material was assessed in dimethyl sulphoxide (DMSO), acetone and ethanol. The vehicle which afforded the highest solubility was ethanol in which the test material dissolved at 10 mg/mL; the test material was practically insoluble in DMSO and acetone. Ethanol was, therefore, used as the vehicle and when dosed at 1% v/v, a 10 mg/mL solution provided a maximum achievable final concentration of 100 micro g/mL. Because of the infrequent use of ethanol as a vehicle for this assay in this laboratory, untreated control cultures were also included in each test.

The osmolality of the test material in medium was tested at 100 micro g/mL; no fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed compared with the vehicle control. No fluctuations in pH of the medium were observed at 100 µg/mL of more than 1.0 unit compared with the vehicle control. The maximum final concentration tested in the preliminary toxicity test was 100 micro g/mL as this was considered to be the maximum achievable concentration in a suitable vehicle.

In this test system, human lymphocytes are cultured in vitro and are stimulated to divide by adding phytohaemagglutinin (PHA) to the culture, resulting in a high mitotic yield (Evans and O’Riordan 1975, Nowell 1960).

In this study, blood taken from healthy adult non-smoking donors was pooled and diluted with tissue culture medium. The cultures were incubated in the presence of PHA before being treated with the test material. Following treatment (3-hour exposure) or during treatment (20-hour exposure), cytokinesis was blocked using the inhibitor Cytochalasin B. Binucleate cells were then examined for the presence of micronuclei.

The study consisted of a preliminary toxicity test and a main micronucleus test. In both types of tests, the cells were exposed for either 3 hours or 20 hours in the absence of exogenous metabolic activation (S9 mix) or for 3 hours in the presence of S9 mix.

Rationale for test conditions:

Prior to commencing testing the solubility of the test material was assessed in dimethyl sulphoxide (DMSO), acetone and ethanol. The vehicle which afforded the highest solubility was ethanol in which the test material dissolved at 10 mg/mL; the test material was practically insoluble in DMSO and acetone. Ethanol was, therefore, used as the vehicle and when dosed at 1% v/v, a 10 mg/mL solution provided a maximum achievable final concentration of 100 micro g/mL.

Because of the infrequent use of ethanol as a vehicle for this assay in this laboratory, untreated control cultures were also included in each test.

Evaluation criteria:

The following criteria were applied for assessment of assay acceptability:

The concurrent vehicle control must be considered acceptable for addition to the laboratories historical vehicle control database (lie below or close to the upper control limit).

Concurrent positive controls must induce responses that are compatible with the laboratories historical positive control database and produce statistically significant increases compared with the concurrent vehicle control.

The criteria for selection of the top dose concentration are consistent with those outlined in the study plan.

Statistics:

Analysis of data

Cytostasis = 100-100{(CBPIT-1)/(CBPIC-1)}

Where CBPI = No. mononucleate cells +2 x No. binucleate cells + 3 x No. multinucleate cells/Total number of cells

T = test material treatment culture
C = solvent control culture

Thus, a CBPI of 1 (all cells are mononucleate) is equivalent to 100% cytostasis.

The numbers of micronucleated cells in each treatment group will be compared with the solvent control value using appropriate statistical tests.

The analysis of the number of micronucleated cells per 1000 cells will assume that the replicate is the experimental unit. An arcsine transformation will be used to transform the data. The treated groups will then be compared to control using Williams’ tests (Williams 1971, 1972). The positive control will be compared to negative control using a t-test. Trend tests will also been carried out using linear contrasts by group number. These will be repeated, removing the top dose group, until there are only 3 groups.

Data will be analyzed using SAS (SAS Institute 2002) and Quasar (Quasar 1.5 2016). Statistical significance will be declared at 5%.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In both the absence and presence of S9 mix, following 3-hour treatment, and in the absence of S9 mix, following 20-hour treatment, the test material did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared with the vehicle controls.

The positive control compounds (mitomycin C, colchicine and cyclophosphamide) caused statistically significant increases in the number of binucleate cells containing micronuclei under appropriate conditions, demonstrating the efficacy of the S9 mix and the sensitivity of the test system.

Conclusions:

It was concluded that the test material did not show evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes, in this in vitro test system under the experimental conditions described.

Executive summary:

This study was designed to assess the potential of the test material to cause an increase in the induction of micronuclei in cultured human peripheral blood lymphocytes in vitro.

The study consisted of a preliminary toxicity test and a main micronucleus test.

Human lymphocytes in whole blood culture, stimulated to divide by addition of phytohaemagglutinin (PHA) 48 hours prior to treatment, were exposed to the test material for 3 hours in both the absence and presence of exogenous metabolic activation (S9 mix) and for 20 hours in the absence of S9 mix. The maximum final concentration to which the cells were exposed was 100 micro g/mL, dosed at 1% v/v, in order to test up to the maximum achievable concentration in a suitable vehicle. Vehicle (ethanol) and positive control cultures were included in all appropriate test conditions.

Three test material concentrations were assessed for determination of induction of micronuclei. The highest concentration selected (100 micro g/mL) was the highest concentration tested. In all exposure conditions there were no reductions in the cytokinesisblock proliferative index (CBPI) at any concentration tested and the concentrations of the test material selected for micronucleus analysis were 25, 50 and 100 µg/mL.

In both the absence and presence of S9 mix, following 3-hour treatment, and in the absence of S9 mix, following 20-hour treatment, the test material did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared with the vehicle controls.

The positive control compounds (mitomycin C, colchicine and cyclophosphamide) caused statistically significant increases in the number of binucleate cells containing micronuclei under appropriate conditions, demonstrating the efficacy of the S9 mix and the sensitivity of the test system.

It was concluded that the test material did not show evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes, in this in vitro test system under the experimental conditions described.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 September 2017 to 19 November 2018

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

US EPA (1998) Health Effects Test Guidelines. OPPTS 870.5300 In vitro mammalian
cell gene mutation test. EPA 712-C-98-221.

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

OJ L 142/262.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Health and Welfare. Evaluation and Licensing Division, Pharmaceutical and Medical Safety Bureau, Notification No. 1604, 1 November 1999.

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

Physical State/Appearance: White waxy solid
Purity: 100%, UVCB substance
Storage Conditions: 15-25 deg.C, in the dark

Target gene:

TK +/-, locus of the L5178Y mouse lymphoma cell line

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS
L5178Y mouse lymphoma (3.7.2c) cells (Clive and Spector, 1975); these cells are heterozygous at the thymidine kinase locus, TK +/-. Spontaneous thymidine kinase deficient mutants, TK -/-, were eliminated from the cultures by a 24-hour incubation in the presence of methotrexate, thymidine, hypoxanthine and glycine two days prior to storage at -196 to -150°C, in heat-inactivated donor horse serum (HiDHS) containing 10% DMSO. Cultures were used within ten days of recovery from frozen stock. Cell stocks were periodically checked for freedom from mycoplasma contamination.

MEDIA
The following media, obtained from a suitable supplier, were used:
R0: RPMI 1640, buffered with 2 mg/mL sodium bicarbonate, supplemented with 2.0 mM L-glutamine and 50 µg/mL gentamicin.
R10p: R0, supplemented with 0.1% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 10% v/v. (R10p medium was used for cell culture unless otherwise specified). Selective medium consisted of R10p containing 4 µg/mL trifluorothymidine (TFT).

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Preliminary toxicity test:
0.2, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50 and 100 micro g/mL

Mutation tests:
-S9 mix (3 hours)
3.13, 6.25, 12.5, 25, 50 and 100 micro g/mL

+S9 mix (3 hours)
3.13, 6.25, 12.5, 25, 50 and 100 micro g/mL

-S9 mix (24 hours)
3.13, 6.25, 12.5, 25, 50 and 100 micro g/mL

The solubility of the test material was assessed in dimethyl sulphoxide (DMSO), acetone and ethanol. The vehicle which afforded the highest solubility was ethanol in which the test material dissolved at 10 mg/mL. Ethanol was, therefore, used as the vehicle for this study and when dosed at 1% v/v (the maximum volume recommended in the test guideline for this type of vehicle), a 10 mg/mL solution provided a maximum final concentration of 100 micro /mL. In all tests the maximum achievable concentration using a vehicle compatible with this test system was 100 micro g/mL.

Vehicle / solvent:

Ethanol

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

Details on test system and experimental conditions:

S9 fraction, prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver, was purchased from a commercial source and stored at -90 to -70 deg.C.

S9 mix was prepared immediately before use and contained: S9 fraction (5% v/v), glucose-6-phosphate (6.9 mM), NADP (1.4 mM) in R0. The co-factors were prepared, neutralised with 1N NaOH and filter sterilised before use.

The test material was formulated and diluted in ethanol (ACS reagent grade), shortly before dosing. The final volume added to the cultures was 1% v/v. All concentrations are expressed in terms of the test material as received.

Main test:
3-hour Treatment in the Absence and Presence of S9 Mix

The procedure for preparing the cell suspension was the same as for the preliminary toxicity test and the definitive test. Cultures contained a total of 1.2 x 107 cells in a final volume of 20 mL. The final concentration of the S9 fraction was 2% v/v, if present. Duplicate cultures were prepared throughout for each concentration of test material and positive control. Quadruplicate cultures were prepared for vehicle controls. Aliquots of 200 micro L of test material dilution (at 100 times the desired final concentration), vehicle or positive control were added to the appropriate culture. The cultures were then incubated, with continuous shaking, for 3 hours at 34 to 39 deg.C. At least four serial dilutions of the test material were tested.

Following the 3-hour treatment, the cells were washed once, re-suspended in R10p to nominally 2 x 105 cells/mL (assuming no cell loss) and incubated (at 34 to 39 deg.C, 5% (v/v) CO2) for a further 48 hours to allow for expression of mutant phenotype. The cultures were sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 105 cells/mL with R10p where necessary. After 48 hours cultures with a density of more than 1 x 105 cells/mL were assessed for cloning efficiency (viability) and mutant potential by plating in 96-well plates. Cultures that were assessed were chosen at the Study Director’s discretion. Cloning efficiency was assessed by plating 1.6 cells/well, two plates being prepared per culture. Mutant potential was assessed by plating 2 x 103 cells/well in selective medium, two plates being prepared per culture. The plates were placed in a humidified incubator at 34 to 39¿C in an atmosphere of 5% CO2 in air.

After the plates had been incubated for 10 to 12 days, the number of empty wells was assessed for each 96-well plate (P0). P0 was used to calculate the cloning efficiency (CE) and mutant frequency (MF). The colony size distribution in the vehicle and positive controls was examined to ensure that there was an adequate recovery of small colony mutants. The criteria for sizing colonies was based on morphology and generally was less than 25% of the well’s diameter for small colonies and greater than 25% of the well’s diameter for large colonies. The maximum concentration assessed for mutant frequency in the main test was 100 micro g/mL in both the absence and presence of S9 mix respectively.

24-hour Treatment in the Absence of S9 Mix

A test with a 24-hour treatment in the absence of S9 mix was carried out. Duplicate 20 mL cultures containing 6 x 106 cells were treated for 24 hours with 200 micro.L of test material, vehicle or positive control. Quadruplicate cultures were prepared for vehicle controls. At the end of the exposure period, the cells were washed once, re-suspended in 20 mL R10p and counted to ascertain treatment growth. The cultures were then diluted to 2 x 105 cells/mL with R10p as
appropriate, incubated (at 34 to 39 deg.C, 5% (v/v) CO2) and sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 105 cells/mL with R10p where necessary. Following this, the procedure was the same as in the 3-hour treatment. The maximum concentration assessed for mutant frequency in the main test was 100 micro g/mL.

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

Positive controls validity:

valid

Preliminary Toxicity Test

No precipitate was observed by eye at the end of treatment following a 3-hour exposure.

Exposure to the test material at concentrations from 0.2 to 100 micro g/mL in the absence and presence of S9 mix caused no significant reductions in relative suspension growth (RSG) values at any concentration tested. Following a continuous exposure for 24 hours, no precipitate was observed by eye at the end of treatment. Exposure to concentrations from 0.2 to 100 micro g/mL caused no significant reductions in RSG values at any concentration tested.

Concentrations used in the main test were based upon these data.

Main Mutation Test - 3-hour Treatment in the Absence of S9 Mix

Cultures were exposed to the test material at concentrations from 3.13 to 100 micro g/mL. No precipitate was observed by eye at the end of treatment. Cultures exposed to the test material at concentrations from 6.25 to 100 micro g/mL were assessed for determination of mutation frequency. There were no reductions in relative total growth (RTG) at any concentration tested. There were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. The positive control, methyl methanesulphonate, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.

Main Mutation Test - 3-hour Treatment in the Presence of S9 Mix

Cultures were exposed to the test material at concentrations from 3.13 to 100 micro g/mL. No precipitate was observed by eye at the end of treatment. Cultures exposed to the test material at concentrations from 6.25 to 100 micro g/mL were assessed for determination of mutation frequency. There were no reductions in RTG at any concentration tested. There were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF, within acceptable levels of toxicity.

The positive control, benzo[a]pyrene, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.

Main Mutation Test - 24-hour Treatment in the Absence of S9 Mix

Cultures were exposed to the test material at concentrations from 3.13 to 100 micro g/mL. No precipitate was observed by eye at the end of treatment. Cultures exposed to the test material at concentrations from 6.25 to 100 micro g/mL were assessed for determination of mutation frequency. There were no reductions in RTG at any concentration tested. There were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF, within acceptable levels of toxicity. The positive control, methyl methanesulphonate, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.

In all tests the concurrent vehicle and positive control were within acceptable ranges.

Conclusions:

It was concluded that the test material did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

Executive summary:

The test material was tested for mutagenic potential in an in vitro mammalian cell mutation assay. This test system is based on detection and quantitation of forward mutation in the subline 3.7.2c of mouse lymphoma L5178Y cells, from the heterozygous condition at the thymidine kinase locus (TK+/- ) to the thymidine kinase deficient genotype (TK-/-).

The study consisted of a preliminary toxicity test and three independent mutagenicity assays. The cells were exposed for either 3 hours or 24 hours in the absence of exogenous metabolic activation (S9 mix) or 3 hours in the presence of S9 mix.

The test material was soluble at 10 mg/mL in ethanol, which provided a maximum final concentration of 100 micro g/mL when administered at 1% v/v (the maximum dose volume recommended in the test guideline for this type of vehicle). 100 micro g/mLwas, therefore, used as the maximum concentration in the preliminary toxicity test, in order to test up to the limit of solubility.

In the preliminary toxicity test following a 3-hour exposure to the test material at concentrations from 0.2 to 100 micro g/mL, there were no significant reductions in relative suspension growth (RSG) at any concentration tested in either the absence or presence of S9 mix. Following a 24-hour exposure in the absence of S9 mix there were no significant reductions in RSG at any concentration tested. The concentrations assessed for determination of mutant frequency in the main test were based upon these data, the objective being to assess concentrations which span the complete toxicity range of approximately 10 to 100% relative total growth (RTG), or to test up the maximum achievable concentration using a suitable vehicle. Based on the results of the preliminary assay, the main mutagenicity test was conducted at concentrations of 3.13 to 100 micro g/mL.

In the main mutagenicity test following 3-hour treatment in the absence and presence of S9 mix, there were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. The maximum concentration assessed for mutant frequency in the 3-hour treatment in both the absence and presence of S9 mix was 100 micro g/mL (the highest achievable concentration). In the absence and presence of S9 mix there were no reductions in relative total growth (RTG) at any concentration tested.

In the 24-hour treatment, the maximum concentration assessed for mutant frequency was 100 micro g/mL (the highest achievable concentration). No increase in mutant frequency exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF. There were no significant reductions in RTG at any concentration tested.

In all tests the concurrent vehicle and positive control were within acceptable ranges.

It was concluded that the test material did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

It was concluded that the test material was considered to be non-mutagenic in an Ames test, it did not demonstrate mutagenic potential in this in vitro cell mutation assay and did not show evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes, in this in vitro test system.