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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item was considered to be non-mutagenic under the conditions of the following tests: OECD 471, OECD 473 and OECD 490.

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:
Experimental start date: 6 September 2016 Experimental completion date: 03 October 2016
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:
other: USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Physical state/Appearance: Amber coloured gelatinous solid
Batch: E00268-014-212
Purity: 100%
Expiry Date: 10 June 2018
Storage Conditions: Room temperature in the dark
Target gene:
The purpose of the study was to evaluate EXP1505486 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.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
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:
• University of California, Berkeley, on culture discs, on 04 August 1995.
• 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 1758279 10/20) 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.
Metabolic activation:
with and without
Metabolic activation system:
liver microsomal preparation (S9-mix) prepared from rats pre treated with a mixture known to induce an elevated level of these enzymes.
Test concentrations with justification for top dose:
Experiment 1
The 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.
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was as follows:
TA1537 (without S9-mix): 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate.
TA100 and TA1535 (without S9-mix) and TA1537 (with S9-mix): 0.15, 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate.
TA100 and TA1535 (with S9-mix) and TA98 (with and without S9-mix): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 µg/plate.
WP2uvrA (with and without S9-mix): 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate.
Eight test item dose levels per bacterial strain were selected in the second mutation test in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item following the change in test methodology from plate incorporation to pre-incubation.
Vehicle / solvent:
In solubility checks performed in house, the test item was noted to be insoluble in sterile distilled water and dimethyl sulphoxide at 50 mg/mL but fully soluble in acetone at 100 mg/mL and dimethyl formamide at 50 mg/mL. Acetone was selected as the vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Direct acting compounds in the absence of S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-Aminoanthracene (2AA)
Remarks:
Indirect acting compounds in the presence of S9-mix
Details on test system and experimental conditions:
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).
Rationale for test conditions:
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 and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.
Evaluation criteria:
All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks
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 as follows
TA1535 7 to 40
TA100 60 to 200
TA1537 2 to 30
TA98 8 to 60
WP2uvrA 10 to 60

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.
There should be a minimum of four non-toxic test item dose levels.
There should be no evidence of excessive contamination.

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:
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.
Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.


Statistics:
Following Sponsor confirmation, statistical analysis was excluded from the data set.
Key result
Species / strain:
E. coli WP2 uvr A
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:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
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:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
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:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
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:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
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:
valid
Positive controls validity:
valid
Remarks on result:
other: EXP1505486 was considered to be non-mutagenic under the conditions of this test.

Please see attached tables:

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) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

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, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2. 

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. In the first mutation test (plate incorporation method) and in the absence of S9-mix, the test item caused a visible reduction in the growth of the bacterial background lawns of all of the Salmonella strains initially from 150 µg/plate (TA100, TA1535 and TA1537) and 500 µg/plate (TA98). In the presence of S9-mix reduced growth of the bacterial background lawns were noted at and above 500 µg/plate to all of the Salmonella strains. No toxicity was noted to Escherichia coli strain WP2uvrA in either the absence or presence of S9-mix. Consequently, the toxic limit or the maximum recommended dose level (5000 µg/plate) of the test item was employed in the second mutation test, depending on bacterial tester strain and absence or presence of S9-mix. In the second mutation test (pre-incubation method), the test item induced an identical toxic response with reduced growth of the bacterial background lawns noted from 150 µg/plate (absence of

S9-mix) and 500 µg/plate (presence S9-mix). No toxicity was noted to Escherichia coli strain WP2uvrA in either the absence or presence of S9-mix. A test item precipitate (particulate and greasy in appearance) was noted at and above 500 µg /plate, this observation did not prevent the scoring of revertant colonies.

There were no toxicologically 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). 

The vehicle (acetone) 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 or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Conclusions:
The test item was considered to be non-mutagenic under the conditions of this test.
Executive summary:

Introduction

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 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 was predetermined and was 1.5 to 5000 ¿g/plate. The experiment was repeated 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 dose levels per bacterial strain were selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item following the change in test methodology.

Results

The vehicle (acetone) 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 or 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 maximum recommended dose level of 5000 µg/plate. In the first mutation test (plate incorporation method) and in the absence of S9-mix, the test item caused a visible reduction in the growth of the bacterial background lawns of all of the Salmonella strains initially from 150 µg/plate (TA100, TA1535 and TA1537) and 500 µg/plate (TA98). In the presence of S9-mix reduced growth of the bacterial background lawns were noted at and above 500 µg/plate to all of the Salmonella strains. No toxicity was noted to Escherichia coli strain WP2uvrA in either the absence or presence of S9-mix. Consequently, the toxic limit or the maximum recommended dose level (5000 µg/plate) of the test item was employed in the second mutation test, depending on bacterial tester strain and absence or presence of S9-mix. In the second mutation test (pre-incubation method), the test item induced an identical toxic response with reduced growth of the bacterial background lawns noted from 150 µg/plate (absence of

S9-mix) and 500 µg/plate (presence S9-mix). No toxicity was noted to Escherichia coli strain WP2uvrA in either the absence or presence of S9-mix. A test item precipitate (particulate and greasy in appearance) was noted at and above 500 ¿g/plate, this observation did not prevent the scoring of revertant colonies.

There were no toxicologically 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 was considered to be non-mutagenic under the conditions of this test.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 April 2018 - 01 May 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)
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: in vitro mammalian cell gene mutation tests using the thymidine kinase gene (migrated information)
Specific details on test material used for the study:
Amber colored gelatinous solid
Batch - E00268-015-141
Target gene:
Thymidine kinase, 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 USED
- Source of cells: MRC Cell Mutation Unit at the University of Sussex, Brighton, UK.
- Cell cycle length, doubling time or proliferation index: approximately 12 hours
- Methods for maintenance in cell culture if applicable: Routinely cultured in RPMI 1640 medium

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/mL) and 10% donor horse serum (giving R10 media) at 37 °C with 5% CO2 in air.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
2% S9 fraction
Test concentrations with justification for top dose:
Preliminary toxicity test: 2.5 to 200 µg/mL for all three of the exposure groups. These dose levels were selected to avoid the excessive precipitate that was observed in the solubility test.
Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments. (Table 1)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable):
A preliminary toxicity test was performed on cell cultures at 5 x 10e5 cells/mL, using a 4 hour exposure period both with and without metabolic activation (S9), and at 1.5 x 10e5 cells/mL using a 24-hour exposure period without S9.
Mutagenicity test: 1 x 10e6 cells/mL in 10 mL aliquots in R10 medium for the 4-hour exposure groups in both the absence and presence of metabolic activation, and 0.3 x 10e6 cells/mL in 10 mL cultures for the 24-hour exposure group in the absence of metabolic activation.

SELECTION AGENT (mutation assays): Trifluorothymidine

NUMBER OF REPLICATIONS: The exposures were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration) at eight dose levels.
Rationale for test conditions:
The thymidine kinase heterozygote system, TK +/- to TK -/-, was described by Clive et al., (1972) and is based upon the L5178Y mouse lymphoma cell line established by Fischer (1958). This system has been extensively validated (Clive et al., 1979; Amacher et al., 1980; Jotz and Mitchell, 1981).
Evaluation criteria:
An approach for defining positive and negative responses is recommended to assure that the increased mutation frquency (MF) is biologically relevant. In place of statistical analysis generally used for other tests, it relies on the use of a predefined induced mutant frequency (i.e. increase in MF above the concurrent control), designated the Global Evaluation Factor (GEF) of 126 x 10-6, which is based on the analysis of the distribution of the vehicle control MF data from participating laboratories.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system.
Statistics:
not specified
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: The dose range of the test item used in the preliminary toxicity test was 2.5 to 200 µg/mL. The results for the Relative Suspension Growth (%RSG) are shown in table 2.

Main mutagenicity experiment: results are summarised in Table 3-5.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Relative Suspension Growth (%RSG)
Remarks on result:
no mutagenic potential (based on QSAR/QSPR prediction)

Table 2.

Dose

(µg/mL)

% RSG (-S9)

4-Hour Exposure

% RSG (+S9)

4-Hour Exposure

% RSG (-S9)

24-Hour Exposure

0

100

100

100

2.5

22

92

0

5

64

83

1

10

0

77

0

20

0

0

0

40 p

0

0

0

80 p

0

0

0

120 p

0

0

0

160 p

0

0

0

200 p

0

0

0

P = Precipitate

Table 3.

 

Treatment

(µg/mL)

 

4 hours (-S9)

 

%RSG

RTG

MF§

0

100

1.00

150.32

0.25 Ø

 

105

 

-

-

0.5

87

0.80

141.86

1

103

1.10

130.80

2

94

0.89

128.30

4

84

0.71

155.57

6

64

0.64

145.06

8

10

0.12

155.71

10 Ø

-

-

-

MF threshold for a positive response: 276.32

EMS

400

93

0.80

1080.85

Table 4.

 

Treatment

(µg/mL)

 

4 hours (+S9)

 

%RSG

RTG

MF§

0

100

1.00

139.73

1 Ø

 

108

 

-

-

2

89

0.78

168.66

4

86

0.90

161.34

6

50

0.93

134.49

10

50

0.46

173.63

12

2

0.52

138.77

16 X

0

0.02

209.38

20 Ø

-

-

-

MF threshold for a positive response: 265.73

CP 1.5

82

0.70

897.99

Table 5.

 

Treatment

(µg/mL)

 

4 hours (+S9)

 

%RSG

RTG

MF§

0

100

1.00

153.42

0.03 Ø

88

-

-

0.06 Ø

81

-

-

0.13

82

0.85

136.45

0.25

84

1.07

120.77

0.5

44

0.71

140.78

1

48

0.96

88.75

2

42

0.78

109.35

4

7

0.17

136.33

MF threshold for a positive response: 279.42

EMS 150

54

0.49

1124.04

%RSG = Relative Suspension Growth

RTG = Relative Total Growth

MF§ = 5-TFT resistant mutants/106 viable cells 2 days after exposure

EMS = Ethylmethanesulphonate

Ø = Not plated surplus to requirements / Excessive toxicity

X = Excluded due to excessive toxicity

CP = Cyclophosphamide

Conclusions:
The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of 126 x 10e-6, consequently it is considered to be non-mutagenic in this assay.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 February 2018 - 02 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy Trade and Industry (METI), and Ministry of the Environment (MOE) Guidelines of 31 March 2011
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: in vitro mammalian chromosome aberration test (migrated information)
Specific details on test material used for the study:
Physical state/Appearance: Amber colored gelatinous solid
Batch: E00268-015-141
Purity: UVCB
Target gene:
not specified
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: whole blood drawn from the peripheral circulation of a non-smoking volunteer (aged 18-35) who had been previously screened for suitability
- Cell cycle length, doubling time or proliferation index: Based on over 20 years in-house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it was considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT was 24 hours.

- Sex, age and number of blood donors if applicable:
Preliminary Toxicity Test: male, aged 28 years
Main Experiment: female, aged 22 years
Main Experiment Repeat: female, aged 31 years

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % foetal bovine serum (FBS), at approximately 37 ºC with 5 % CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).
- Properly maintained: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Microsomal Enzyme Fraction and S9-Mix
Test concentrations with justification for top dose:
Preliminary Toxicity Test: 0, 0.156, 0.312, 0.625, 1.25, 2.5, 5, 10, 20 and 40 µg/mL.
Due to the excessive toxicity of the test item, the maximum concentration was limited to 40 µg/mL.

Main Experiment:
> 4-hour exposure to the test item without S9-mix, followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 0, 1, 2, 4, 8, 12, 16 and 24 µg/mL.
> 4-hour exposure to the test item with S9-mix (2%), followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 0, 2, 4, 8, 12, 16, 24 and 32 µg/mL.
> 24-hour continuous exposure to the test item without S9-mix prior to cell harvest. The dose range of test item used was 0, 1, 2, 4, 8, 12, 16 and 24 µg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:DMSO
- Justification for choice of solvent/vehicle: solubility
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium [MEM (including serum)]

DURATION
- Preincubation period: 48 hours incubation at approximately 37 ºC, 5% CO2 in humidified air
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 20 hours

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Mitosis was arrested by addition of demecolcine (Colcemid 0.1 µg/mL) 2 (Preliminary Toxicity Test and Main Experiment) and 2.5 (Main Experiment Repeat) hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC to ensure complete fixation prior to slide preparation.
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.
The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for mitotic index evaluation.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): Where possible, 300 consecutive well-spread metaphases from each concentration were counted (150 per duplicate), where there were at least 15 cells with aberrations (excluding gaps), slide evaluation was terminated. In the positive control for the 4(20)-hour exposure group in the absence of S9, an additional slide was scored from the B culture due to the poor positive response in the A culture. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing and the ISCN (1985).

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index - A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

OTHER EXAMINATIONS:
- Determination of polyploidy: cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) (including the incidence of cells with endoreduplicated chromosomes) was also reported.
Rationale for test conditions:
Guidelines
Evaluation criteria:
The following criteria were used to determine a valid assay:
• The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory historical control data range
• All the positive control chemicals induced a positive response (p=0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix
• The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline
• The required number of cells and concentrations were analyzed where possible

Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly negative if, in any of the experimental conditions examined:
1) The number of cells with structural aberrations in all evaluated dose groups should be within the range of the laboratory historical control data.
2) No toxicologically or statistically significant increase of the number of cells with structural chromosome aberrations is observed following statistical analysis.
3) There is no concentration-related increase at any dose level.

A test item can be classified as genotoxic if:
1) The number of cells with structural chromosome aberrations is outside the range of the laboratory historical control data.
2) At least one concentration exhibits a statistically significant increase in the number of cells with structural chromosome aberrations compared to the concurrent negative control.
3) The observed increase in the frequency of cells with structural aberrations is considered to be dose-related

When all of the above criteria are met, the test item can be considered able to induce chromosomal aberrations in human lymphocytes.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.
Key result
Species / strain:
lymphocytes:
Remarks:
Human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The dose range for the Preliminary Toxicity Test was initially set up to 5000 µg/mL. However, the test item was very toxic so the maximum dose was limited to 40 µg/mL.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 20 µg/mL in the all of the exposure groups. The test item induced evidence of toxicity in the dose range used in the Preliminary Toxicity Test to all of the exposure groups. The selection of the maximum dose level for the Main Experiment was, therefore, based on the lowest precipitating dose level which coincided with the onset of toxicity in all three exposure groups.

In the 4(20)-hour exposure group in the absence of S9, 21%, 44% and 70% mitotic inhibition was achieved at 8, 12 and 16 µg/mL, respectively. Above this dose level, there were no scorable metaphases present. The maximum dose level selected for metaphase analysis was, therefore, 12 µg/mL because this dose level approached the minimum range for toxicity as specified by the OECD 473 test guideline (55±5%).

In the exposure group in the presence of S9, 36% and 83% mitotic inhibition was achieved at 16 and 24 µg/mL, respectively. Above this dose level, there were no scorable metaphases present. The maximum dose level selected for metaphase analysis was, therefore, 24 µg/mL because, although this dose level exceeded the maximum range for toxicity as specified by the OECD 473 test guideline, it was the lowest precipitating dose level.

In the 24-hour continuous exposure group in the absence of S9, 21% and 74% mitotic inhibition was achieved at 8 and 12 µg/mL, respectively. Above this dose level, there were either too few or no scorable metaphases present. The maximum dose level selected for metaphase analysis was, therefore, 12 µg/mL because, although this dose level exceeded the maximum range for toxicity as specified by the OECD 473 test guideline, it was the lowest precipitating dose level.

The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.

Polyploid cell frequency:
The test item induced a significant increase in the numbers of polyploid cells at 24 µg/mL in the 4(20)-hour exposure group in the presence of S9-mix and at 12 µg/mL in the 24-hour continuous exposure group. However, these increases were accompanied by inhibitions of Mitotic Index which were in excess of optimum toxicity and may be a consequence of toxicity rather than any genotoxic activity. No increases were observed in the 4(20)-hour exposure group in the absence of S9-mix.
Remarks on result:
no mutagenic potential (based on QSAR/QSPR prediction)
Conclusions:
The test item did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system. The test item was, therefore, considered to be non-clastogenic to human lymphocytes in vitro.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
18 May 1993 to 22 September 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
Five special tester strains of Salmonella typhimurium, which were sensitive to framshift or base pair mutagens, were employed. The purpose of this assay was to determine if the test material was capable of causing reverse mutations in these special tester strains of bacteria, using one vehicle and one type of metabolic activation. The test material was tested twice.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch: March 31, 1998

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

FORM AS APPLIED IN THE TEST: Amber solid
Target gene:
Histidine locus (from nutritional histidine dependence to histidine independence).
Species / strain / cell type:
S. typhimurium TA 98
Remarks:
frameshift sensitive
Species / strain / cell type:
S. typhimurium TA 100
Remarks:
base pair substitution and frameshift sensitive
Species / strain / cell type:
S. typhimurium TA 1535
Remarks:
base pair substitution sensitive
Species / strain / cell type:
S. typhimurium TA 1537
Remarks:
frameshift sensitive
Species / strain / cell type:
S. typhimurium TA 1538
Remarks:
frameshift sensitive
Metabolic activation:
with and without
Metabolic activation system:
Rat S9 mix; Liver homogenate from the livers of Aroclor 1252 pretreated Sprague Dawley rats
Test concentrations with justification for top dose:
- Toxicity Pretest: To determine the highest dose of test material to be used in the assay, a dose range from 1 to 10,000 ug per plate with and without metabolic activation was tested. Only strain TA98 was used. A single plate per dose point was used and the conditions were identical to those for the regular assay. Colocy counts were made after 2 d incubation and plates were evaluated for toxic response. Toxicity, seen as either a reduction in the number of revertant colonies or a reduction in the background lawn, was observed at 100 ug/plate for the saline (-S9) treated plates and 500 ug/plate for the treated (+S9) plates. Therefore, 100 ug/plate for the saline (-S9) plates and 500 ug/plate for the treated (+S9) plates were selected as the high doses to be used on the mutagenesis assay.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone; DMSO
- Justification for choice of solvent/vehicle: The determine the vehicle to be used in the assay, solubility tests using DMSO and acetone were performed. The dose tested was 10,000 ug/0.1 mL (100 mg/mL). The test material was found to be insoluble in DMSO but soluble in acetone. Therefore, the acetone was selected as the vehicle to be used on the mutagenesis asay. The positive controls, 2AA, 2NF, 9AA and MNNG, were diluted in DMSO.
Untreated negative controls:
yes
Remarks:
vehicle, DMSO and non-treated
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthacene (2AA)
Untreated negative controls:
yes
Remarks:
vehicle, DMSO and non-treated
Positive controls:
yes
Positive control substance:
9-aminoacridine
Untreated negative controls:
yes
Remarks:
vehicle, DMSO and non-treated
Positive controls:
yes
Positive control substance:
other: N-methyl-N-nitro-N-nitrosoguanidine (MNNG)
Untreated negative controls:
yes
Remarks:
vehicle, DMSO and non-treated
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Details on test system and experimental conditions:
METHOD OF APPLICATION: Stock concentrations of the test material were prepared and diluted in acetone. The test material was incorporated into the molten top agar following the addition of bacteria. S9 mix or saline was then added, the mixture vortexed and immediately poured on plates.

DURATION
- Preincubation period: 2 d at 37 °C ± 1 °C

NUMBER OF REPLICATIONS: 3 plates per dose point

MUTAGENESIS ASSAY: On the day of the assy, the test material was diluted in acetone just prior to use. The positive controls were diluted in DMSO. The nontreated plates were handled in the same manner as the treated plates except that no test material or vehicle was added. Frozen tester organisms were inoculated into broth cultures a day prior to dosing. One the day of dosing, the S9 for the metabolic activation mix was thawed and the activation mix was prepared an stored on ice. Molten top agar, tester strain, test or control material and S9 mix or saline were combined, and the total was poured into a plate containing a layer or minimal agar. After cooling, the plates were inverted and incubated,. Afer incubation, all the plates were evaluated for toxic effects and the number of revertant colonies was counted either manually or with an automatic colony counter. During the operation of the assay the individual groups were uniquely identified by the use of a separate label for each test plate.
Statistics:
- The mean plate count and standard deviation for each dose point were determined.
- An indiviual dose is considered positive if mean colony count on the test plates is equal to or greater than 3 times the mean number of spontaneous revertants on the vehicle control plates.
- A positive result for the assay is defined as a reproducible dose-related increase in the number of revertant colonies over at least 3 concentrations of test material uncliuding at lease 1 positive dose.
- A lack of response in the positive controls or spontaneous revertant frequencies which are not in keeping with historical laboratory values will render the entire test invalid.
Key result
Species / strain:
S. typhimurium TA 98
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
Key result
Species / strain:
S. typhimurium 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
Key result
Species / strain:
S. typhimurium TA 1535
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
Key result
Species / strain:
S. typhimurium TA 1537
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
Key result
Species / strain:
S. typhimurium TA 1538
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
Additional information on results:
- In the initial mutagenesis assay, neither a positive response nor a dose related increase in revertants was observed for any of the tester strains. Toxicity, either a reduction in the number of revertant colonies or a reuction in the background lawn, was noted for all 5 strains tested.
- A repeat assay was performed in order to verify the data produced in the initial assay. In the repeat mutagenesis assay, neither a positive response nor a dose related increase in revertants was observed for any of the tester strains. Toxicity either a reduction in the number of revertant colonies or a reduction in the background lawn, was noted for all 5 strains tested.

CONTROL DATA
- Positive control data: All positive controls (MNNG, 2NF, 9AA and 2AA) responded in a manner consistent with data from previous assays.
- Negative (solvent/vehicle) control data: Negative controls (vehicle, DMSO and nontreated) responded in a manner consistent with data from previous assays. The sterility control plates were without growth.
Conclusions:
The study was conducted to determine the potential of the test material to cause specific gene mutations in bacterial cells. The test material was tested twice in the Salmonella microbial mutagenesis assay using strains TA98, TA100, TA1535, TA1537 and TA1538, with acetone as a vehicle. Was tested with and without metabolic activation. The assay contained positive (2AA, 9AA, 2NF and MNNG) and negative (nontreated, DMSO and vehicle) controls which responded in a manner consistent with data from previous assays. The material did not induce a dose related increase in the the mutation frequencies in any of the tester strains. Under the conditions of the assay, the test material was not mutagenic for the Salmonella tester strains at doses up to and including 500 ug/plate.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

According to the available data and criteria of Regulation (EC) No 1272 /2008 no classification is warranted for the test substance as a mutagen.