Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial gene mutation test

Key Study:

In this in vitro assessment of the mutagenic potential of the test item, histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Escherichia coli, strain WP2 uvrA (pKM101), were exposed to the test item diluted in dimethyl sulfoxide (DMSO). DMSO was also used as a vehicle control. It was concluded that the test item showed no evidence of mutagenic activity in this bacterial system under the test conditions employed (Envigo Research Limited, Ltd, 2018).

 

In vitro micronucleus test:

Key Study:

This study was designed to assess the potential of the test item to cause an increase in the induction of micronuclei in cultured human peripheral blood lymphocytes in vitro. Under the conditions of the study, it was concluded that the test item 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 (Envigo Research Limited, Ltd, 2018).

 

In vitro gene mutation in mammalian cells test:

Key Study:

The test item 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-/-). Under the conditions of the study, it was concluded that the test item did not demonstrate mutagenic potential in this in vitro cell mutation assay (Envigo Research Limited, Ltd, 2018).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 August 2016 to ****
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries. Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Guideline 2-1-19-1, Agricultural Production Bureau, November 24, 2000.
Deviations:
no
Qualifier:
according to
Guideline:
other: Official notice of MHLW, METI and MOE (31 March 2011) YAKUSHOKUHATSU 0331 No 7 SEIKYOKU No 5 KANPOKIHATSU No 110331009
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
CAS RN: 15337-18-5
Purity: 96.6%
Description: Yellow viscous liquid
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
S9 Rat Liver, induced with phenobarbitone/β-naphthoflavone
Test concentrations with justification for top dose:
Test 1 and Test 2: 5, 15, 50, 150, 500, 1500, 5000 µg.
5000 µg is the standard top dose recommended in the regulatory guidelines that this assay follows. Other concentrations used were a series of ca half-log10 dilutions of the highest concentration.
Vehicle / solvent:
dimethyl sulfoxide (DMSO)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
sodium azide
benzo(a)pyrene
mitomycin C
other: 2-Nitrofluorene (in the absence of S9 Mix), 2-Aminoanthracene (in the presence of S9 Mix)
Details on test system and experimental conditions:
The strains of S. typhimurium and E. coli were obtained from established commercial sources and were stored at ca -80°C as aliquots of nutrient broth cultures. Each batch of frozen strain was tested for amino acid requirement and, where applicable, for cell membrane permeability (rfa mutation), sensitivity to UV light, and the pKM101 plasmid, which confers resistance to ampicillin. The responses of the strains to a series of reference mutagens were also assessed.

For use in tests, an aliquot of frozen culture was added to 25 mL of nutrient broth and incubated, with shaking, at 37 ºC for 10 hours.

Preparation of S9 Fraction
S9 fraction was prepared from male Sprague-Dawley derived rats, dosed with phenobarbital/5,6-benzoflavone to stimulate mixed-function oxidases in the liver and stored at approximately -80°C.

Preparation of S9 Mix
The S9 mix contained: S9 fraction (10% v/v), MgCl2 (8 mM), KCl (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in water.

First Test (Test 1)
Aliquots of the test item solutions, positive control or vehicle control were placed in glass tubes. S9 mix (0.5 mL) or 0.1 M pH 7.4 sodium phosphate buffer (0.5 mL) was added, followed by 0.1 mL of a 10-hour bacterial culture and 2 mL of agar containing histidine (0.05 mM), biotin (0.05 mM) and tryptophan (0.05 mM). The mixture was thoroughly shaken and overlaid onto previously prepared Petri dishes containing 25 mL minimal agar. Each Petri dish was individually labelled with a unique code, identifying the contents of the dish. Three Petri dishes were used for each treatment. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test item, S9 mix and sodium phosphate buffer. All plates were incubated at approximately 37 ºC for between 48 and 72 hours. After this period, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter (Perceptive Instruments Sorcerer). Some plates were scored manually because of the presence of precipitate.
Any toxic effects of the test item may be detected by a substantial reduction in mean revertant colony counts (≤ 50% reduction), by a sparse or absent background bacterial lawn, or both.

Second Test (Test 2)
As a clear negative response was obtained in the first test, the pre-incubation assay was used for the second test, containing mixtures of bacteria, buffer or S9 mix and test dilution, were incubated at 37°C for 30 minutes with shaking before the addition of the agar overlay (2 mL). The maximum concentration chosen was again 5000 µg/plate..
Evaluation criteria:
If exposure to a test item produces a reproducible increase in mean revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, it is considered to exhibit mutagenic activity in this test system. If exposure to a test item does not produce a reproducible increase in mean revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis was performed.
Key result
Species / strain:
other: TA1537, TA98, TA1535, TA100, and E. coli WP2 uvrA (pKM101)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
First Test (Test 1)
No evidence of toxicity was obtained following exposure to the test item at any concentration tested in all strains used. Precipitate was observed on all plates, in the presence and absence of S9 mix (metabolic activation), at 5000 µg/plate. A maximum exposure concentration of 5000 µg/plate was, therefore, selected for use in the second test.
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test item at any concentration up to and including 5000 µg/plate in either the presence or absence of S9 mix.

Second Test (Test 2)
Toxicity (observed as a reduction in revertant colony numbers) was seen following exposure to the test item in strain TA1537 at 50 µg/plate in the presence of S9 mix. This reduction in colony number was not dose dependent, appears to be anomalous when considered with the remaining cultures in each exposure condition and therefore, was considered an effect of experimental variation.
No evidence of toxicity was observed in any other strains at any concentration tested in either the absence or presence of S9 mix.
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test item at any concentration up to and including 5000 µg/plate in either the presence or absence of S9 mix.
Conclusions:
It was concluded that the test item showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.
Executive summary:

Histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Escherichia coli, strain WP2 uvrA (pKM101), were exposed to the test item diluted in dimethyl sulfoxide (DMSO). DMSO was also used as a vehicle control.

 

Two independent mutation tests were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. The first test was a standard plate incorporation assay; the second test included a pre-incubation stage.

 

Concentrations of the test item up to 5000 µg/plate were tested. Other concentrations used were a series of approximately half-log10 dilutions of the highest concentration.

 

In the first test no evidence of toxicity following exposure to the test item was observed at any concentration tested in any strains used. Precipitate was observed on all plates, in the presence and absence of S9 mix, at 5000 µg/plate. 

 

In the second test a pre-incubation method was used. Toxicity (observed as a reduction in revertant colony numbers) was seen following exposure to the test item in strain TA1537 at 50 µg/plate in the presence of S9 mix. This reduction in colony number was not dose dependent, appears to be anomalous when considered with the remaining cultures in each exposure condition, and therefore, was considered an effect of experimental variation.

 

No evidence of mutagenic activity was seen at any concentration of the test item in either mutation test.

 

The concurrent positive controls verified the sensitivity of the assay and the metabolizing activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the current historical control range for the laboratory.

 

It was concluded that the test item showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 May 2016 to 06 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
CAS RN: 15337-18-5
Appearance: Yellow viscous liquid
Purity: 96.6%
Lot number: 2016119622
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 diluted with HML
media. 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
Test concentrations with justification for top dose:
The maximum final concentration to which the cells were exposed in the preliminary test was 2000 μg/mL, dosed at 0.5% v/v. Cells in the main test were exposed to the highest concentration showing a visible precipitate. Concentrations for micronucleus evaluation were based upon cytotoxicity, assessed as a reduction in CBPI equivalent to 55 ± 5% (approximately).
Vehicle / solvent:
Acetone
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Colchicine
Details on test system and experimental conditions:
Positive Controls
In the absence of S9 mix
- Identity: Mitomycin C
- Exposure concentrations: 0.3 μg/mL (3-hour treatment), and 0.1 μg/mL (20-hour treatment)

- Identity: Colchicine
- Exposure concentrations: 0.07 μg/mL (3-hour treatment), and 0.02 μg/mL (20-hour treatment)

In the presence of S9 mix
- Identity: Cyclophosphamide
- Exposure concentrations: 10 μg/mL (3-hour treatment)

S9 Metabolizing System
- S9 Fraction: Prepared from male Sprague-Dawley derived rats dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver, and stored at -90 to -70°C.

- Preparation of S9 Mix: 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).

Test Concentrations
- Preliminary toxicity test:
-S9 and +S9 mix 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μg/mL

- Main tests:
-S9 mix (3 hours) 0.25, 2.5, 5, 10, 15 and 20 μg/mL
+S9 mix (3 hours) 0.25, 2.5, 5, 10, 15 and 20 μg/mL
-S9 mix (20 hours) 0.0025, 0.025, 0.25, 2.5, 5, 10, 15
and 20 μg/mL

- Additional Main tests:
-S9 mix (3 hours) 0.1, 10, 15, 20, 22.5, 25, 27.5, 30 and 35 μg/mL
+S9 mix (3 hours) 0.1, 10, 15, 20, 22.5, 25, 27.5, 30
and 35 μg/mL

Main Test Procedures
3-Hour Treatment in the Absence and Presence of S9 Mix and 20-Hour Treatment in the Absence of S9 Mix:
- positive control cultures included
- duplicate cultures prepared at each treatment level and positive control
- quadruplicate cultures were prepared for vehicle controls
- two slides were prepared from each culture

Microscopic Examination
- examined by fluorescence microscopy
- incidences of mononucleate, binucleate and polynucleate cells were assessed per 500 cells per culture
- presence of an unusual number of, for example, cells undergoing mitosis, polyploid cells, necrotic cells and debris, if any, was also noted
- incidence of micronucleated cells per 1000 binucleate cells per culture were scored where possible.
Evaluation criteria:
Acceptance 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.
Tests that did not fulfill the required criteria were rejected and therefore are not reported.
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

Preliminary Toxicity Test

In all exposure conditions the highest concentration tested was 2000 μg/mL and precipitate was observed by eye at 15.63 μg/mL and above after 3-hour treatment and at 62.5 μg/mL and above after 20-hour treatment. These data were used to select concentrations for the main test.

 

Main Test

3-Hour Treatment in the Absence of S9 Mix

Concentrations of the test item used for the main micronucleus test were 0.25, 2.5, 5, 10, 15 and 20 μg/mL. No precipitate was observed by eye at the end of treatment at any concentration tested. As neither the required 55±5% cytotoxicity nor a precipitating concentration was achieved, the test was discontinued and an additional test performed using 0.1, 10, 15, 20, 22.5, 25, 27.5, 30 and 35 μg/mL. Precipitate was observed by eye at 30 μg/mL. A reduction in CBPI compared with vehicle control values equivalent to 30.7% cytotoxicity, was obtained. Concentrations of the test item selected for micronucleus analysis were 0.1, 20 and 30 μg/mL.

 

The test item did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared with the vehicle controls. Mean micronucleus induction in the vehicle control was below the upper control limit of the laboratory historical control database. The positive control compounds (mitomycin C and colchicine) caused statistically significant increases in the number of binucleate cells containing micronuclei, demonstrating the sensitivity of the test system.

 

3-Hour Treatment in the Presence of S9 Mix

Concentrations of the test item used for the main micronucleus test were 0.25, 2.5, 5, 10, 15 and 20 μg/mL. No precipitate was observed by eye at the end of treatment at any concentration tested. As neither the required 55±5% cytotoxicity nor a precipitating concentration was achieved, the test was discontinued and an additional test performed using 0.1, 10, 15, 20, 22.5, 25, 27.5, 30 and 35 μg/mL. Precipitate was observed by eye at 30 μg/mL. A reduction in CBPI compared with vehicle control values, equivalent to 36.1% cytotoxicity, was obtained. Concentrations of the test item selected for micronucleus analysis were 0.1, 20 and 30 μg/mL.

 

The test item did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared with the vehicle controls. Mean micronucleus induction in the vehicle control was below the upper control limit of the laboratory historical control database. The positive control compound (cyclophosphamide) caused a statistically significant increase in the number of binucleate cells containing micronuclei, demonstrating the efficacy of the S9 mix and the sensitivity of the test system.

 

20-Hour Treatment in the Absence of S9 Mix

Concentrations of the test item used for the main micronucleus test were 0.0025, 0.025, 0.25, 2.5, 5, 10, 15 and 20 μg/mL. No precipitate was observed by eye at the end of treatment at any concentration tested. A reduction in CBPI compared with vehicle control values, equivalent to 53.2% cytotoxicity, was obtained at 15 μg/mL. Concentrations of the test item selected for micronucleus analysis were 0.25, 5 and 15 μg/mL.

 

The test item did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared with the vehicle controls. Mean micronucleus induction in the vehicle control was below the upper control limit of the laboratory historical control database. The positive control compounds (mitomycin C and colchicine) caused statistically significant increases in the number of binucleate cells containing micronuclei, demonstrating the sensitivity of the test system.

Conclusions:
The test item did not show any 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 item 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 five main micronucleus tests. Human lymphocytes in whole blood culture, stimulated to divide by addition of phytohaemagglutinin (PHA), were exposed to the test item 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. Untreated, vehicle (acetone) and positive control cultures were included in all appropriate test conditions.

 

Three test item concentrations were assessed for induction of micronuclei. The highest concentration selected was that which caused a reduction in cytokinesis-block proliferative index (CBPI) equivalent to 55±5% cytotoxicity. Following 3-hour treatment in the absence of S9 mix, a reduction in CBPI equivalent to 30.7% cytotoxicity was obtained at 30 μg/mL. Concentrations of the test item selected for micronucleus analysis were 0.1, 20 and 30 μg/mL. Following 3-hour treatment in the presence of S9 mix, a reduction in CBPI equivalent to 36.1% cytotoxicity was obtained at 30 μg/mL. Concentrations of the test item selected for micronucleus analysis were 0.1, 20 and 30 μg/mL. In the absence of S9 mix following 20-hour treatment, a reduction in CBPI equivalent to 53.2% cytotoxicity was obtained at 15 μg/mL. Concentrations of the test item selected for micronucleus analysis were 0.25, 5 and 15 μg/mL.

Concentrations selected for evaluation were based upon the assessment of 500 cells per culture for the incidences of mononucleate, binucleate and polynucleate cells by fluorescent microscopy. One thousand binucleate cells per culture were evaluated for micronuclei.

 

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 item 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 item did not show any 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.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 September 2017 to ****
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Specific details on test material used for the study:
CAS RN: 15337-18-5
Purity: 96.6%
Appearance: Yellow viscous liquid
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% dimethyl sulphoxide (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 g/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:
3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL

Mutation tests:
-S9 mix (3 hours): 0.5, 5, 15, 30, 40, 50 and 60 µg/mL
+S9 mix (3 hours): 0.5, 5, 15, 30, 40, 50 and 60 µg/mL
-S9 mix (24 hours): 0.05, 0.5, 5, 10, 15, 20, 25 and 30 µg/mL

The concentrations assessed for determination of mutant frequency in the main test were based upon the preliminary toxicity test data, the objective being to test up to the maximum solubility using a suitable vehicle.

The solubility of the test item was assessed in DMSO and acetone as part of a separate study. The vehicle which afforded the highest solubility was acetone in which the test item dissolved at 400 mg/mL. Acetone was, therefore, used as the vehicle for this study and when dosed at 1% v/v, a 200 mg/mL solution provided a maximum final concentration of 2000 µg/mL. The maximum final concentration tested in the preliminary toxicity test was 2000 µg/mL.

The osmolality of the test item in medium was tested at 2000 µg/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 2000 µg/mL of more than 1.0 unit compared with the vehicle control.
Vehicle / solvent:
Acteone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION
In medium

S9 METABOLIZING SYSTEM
- S9 Fraction: Prepared from male Sprague-Dawley derived rats dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver (stored at -90 to -70°C).

- Preparation of S9 Mix: S9 mix contains: 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.

FORMULATION OF TEST ITEM
The test item was formulated and diluted in acetone (analytical reagent grade), shortly before dosing. The final volume added to the cultures was 1% v/v for a maximum final concentration of 2000 µg/mL tested in the preliminary toxicity test.

PRELIMINARY TEST
Cultures contained a total of 6 x 10^6 cells for 3-hour exposures and 3 x 10^6 cells for 24-hour exposures. The final concentration of the S9 fraction was 2% v/v, if present. One culture was prepared for each concentration of the test item for each test condition. Vehicle controls were tested in duplicate for each test condition.
Aliquots of test item dilution or vehicle were added to each culture prior to incubation for 3 hours (continuous shaking at 34 to 39 ºC) or 24 hours (static incubator, at 34 to 39 ºC, 5% (v/v) CO2). At the end of the exposure period, the cells were washed, re-suspended in R10p, incubated (at 34 to 39 ºC, 5% (v/v) CO2) and sampled after 24 and 48 hours to assess growth in suspension. Relative suspension growth (RSG) was used to determine the concentrations of test item used in the main test. There was evidence of toxicity in the preliminary toxicity test, so the maximum concentrations tested in the main mutation test in all the 3-hour exposure conditions was 60 µg/mL and in the 24-hour exposure condition was 30 µg/mL.

MAIN TEST
3-hour Treatment in the Absence and Presence of S9 Mix:
Cultures contained a total of 1.2 x 10^7 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 item and positive control. Quadruplicate cultures were prepared for vehicle controls. Aliquots of test item dilution, vehicle or positive control were added, and then all cultures were incubated, (with continuous shaking, for 3 hours at 34 to 39 ºC). At least four serial dilutions of the test item were tested.

Following the 3-hour treatment, the cells were washed once, re-suspended in R10p to nominally 2 x 10^5 cells/mL and incubated for a further 48 hours. Cultures were then sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 10^5 cells/mL with R10p where necessary and after 48 hours cultures with a density of more than 1 x 10^5 cells/mL were assessed for cloning efficiency (viability) and mutant potential by plating in 96-well plates. Cloning efficiency was assessed by plating 1.6 cells/well, two plates being prepared per culture. Mutant potential was assessed by plating 2 x 10^3 cells/well in selective medium, two plates being prepared per culture. The plates were placed in a humidified incubator at 34 to 39 ºC 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 maximum concentration assessed for mutant frequency in the main test was 30 and 40 µg/mL in the absence and presence of S9 mix respectively.

24-hour Treatment in the Absence of S9 Mix:
Duplicate 20 mL cultures containing 6 x 10^6 cells were treated for 24 hours with an aliquot of test item 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 10^5 cells/mL with R10p as appropriate, incubated 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 10^5 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 10 µg/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
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Conclusions:
The test item did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described. 
Executive summary:

The test item was tested for mutagenic potential in an in vitro mammalian cell mutation assay, based on detection and quantitation of forward mutation in the 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 item was soluble at 200 mg/mL in acetone. A final concentration of 2000 µg/mL, dosed at 1% v/v, was used as the maximum concentration in the preliminary toxicity test.

 

Following a 3-hour exposure to the test item at concentrations from 3.91 to 2000 µg/mL, relative suspension growth (RSG) was reduced from 113 to 3% and from 62 to 7% in the absence and presence of S9 mix respectively. Following a 24-hour exposure in the absence of S9 mix RSG was reduced from 47 to 0%. 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).

 

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 concentrations assessed for mutant frequency in the 3-hour treatment in both the absence and presence of S9 mix were 30 and 40 µg/mL respectively. In the absence and presence of S9 mix the mean RTG was reduced to 16 and 19% respectively.

 

In the 24-hour treatment, the maximum concentration assessed for mutant frequency was 10 µg/mL. No increase in mutant frequency exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF. The mean RTG was reduced to 19%. In all tests the concurrent vehicle and positive control values were within acceptable ranges.

 

The test item 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)

Additional information

Justification for classification or non-classification