Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From September 29, 1994 to January 6, 1995
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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Department of Health. Report on Health and Social Subjects Number 35. Guidelines for the testing of chemicals for mutagenecity (1989)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes: Human lymphocytes collected from healthy male donor blood
Details on mammalian cell type (if applicable):
No details
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction obtained from male Sprague Dawley rats treated withAroclor 1254 and mixed with co-factors
Test concentrations with justification for top dose:
Concentration of the test substance in first test-
9.8, 19.5, 39.1, 78.1, 156.3, 312.5, 625, 1250, 2500 and 5000 µg/mL both in the presence and absence of metabolic activation

Concentration of the test substance in second test-
After 18 h harvest
625, 1250, 2500 and 5000 µg/ml both in the absence and presence of metabolic activation

After 32 h harvest
In absence of S9 mix: 156.3, 312, 625, 1250, 2500 and 5000 µg/ml
In presence of S9 mix: 625, 1250, 2500 and 5000 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: the test substance is fairly soluble in water (>500 mg/mL). No precipitate was formed at the highest concentration of 5000 µg/mL.
5000 µg/mL was chosen as the highest concentration due to possible artefactual effects on chromosomal aberrations due to high osmolality of higher concentrations.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
dissolved in dimethyl sulfoxide, used at a concentration of 250, 500 and 750 µg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
dissolved in sterile distilled water, used at a concentration of 2.5, 5, 10, 15 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 h
- Exposure duration: 3 h
- Expression time (cells in growth medium):
First test: In presence of S9 mix: 15 h; In absence of S9 mix: 18 h
Second test: In presence of S9 mix: 15 or 29 h; In absence of S9 mix: 18 or 32 h

- Selection time (if incubation with a selection agent): Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells):
First test: In presence of S9 mix: 17 h; In absence of S9 mix: 20 h
Second test: In presence of S9 mix: 17 or 31 h; In absence of S9 mix: 20 or 34 h

SELECTION AGENT (mutation assays): Not applicable
SPINDLE INHIBITOR (cytogenetic assays): Colchicine
STAIN (for cytogenetic assays): Giemsa with dilution factor 1 part Giemsa to 9 parts buffered distilled water

NUMBER OF REPLICATIONS: Duplicate cultures were used for each concentration for all tests

NUMBER OF CELLS EVALUATED: 1000 cells in each culture was examined for the proportion of mitotic cells (except for positive control treated cells)

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Not determined in this study
- Determination of endoreplication: Not determined in this study
- Other:
Metaphase analysis: Approximately 100 metaphase figures were examined from each culture for chromatid break, chromosome break, chromatid gap, chromatid exchange, chromosome exchange and chromosome gap.

The dose levels causing a decrease in mitotic index to 50 % of the control value were chosen as the highest dose for metaphase analysis. The intermediate and low doses were selected for the analysis.

Only cells with 44-46 chromosomes were analysed.
Evaluation criteria:
Chromosomal aberrations were scored according to the classification of ISCN1985 (International System for Human Cytogenetic Nomenclature)
Criteria for evaluating results:
Cells evaluated per dose group
1. Proportion of mitotic cells per 1000 cells in each culture recorded
2. Dose level causing approx. 50 % decrease in mitotic index (compared to the solvent control value) selected
3. 100 metaphase figures examined from each culture
4. Cells with 44-48 chromosomes analysed
5. Recording of polyploidy and endoreduplicated cells also done
6. Chromosomal aberration scoring done as per ISCN (1985)

Criteria for accepting validity of the test:
1. Negative and positive control values lie within current historical control range of laboratory

Criteria for judging response of the test:
Positive -
1. Statistically significant increases (P<0.01) in frequency of aberrant chromosomes in metaphase
2. Increases in frequency of aberrant chromosomes in metaphase exceeds 99 % of confidence limits for current historical control range
3. Reproducible increases between replicate cultures
4. Increases not associated with variations in pH, osmolality of the treatment or extreme toxicity
5. Evidence of dose-response relationship to support conclusion

Negative-
No statistically significant increases in frequency of aberrant chromosomes in metaphase observed above concurrent control frequencies
Statistics:
Fischer’s test was used to compare the number of aberrant metaphases figures in each treatment group with the solvent control (Fischer, 1973)
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:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not reported
- Effects of osmolality:
First test
An increase in the osmolality of the cultures at 5000 µg/mL was observed both in the absence and presence of S9 mix as compared to solvent control. However, this increase is not considered significant as these increases in osmolality were less than 50 mOsmol/kg

Second test (18 h harvest)
In the absence of S9 mix: No significant increase in the osmolality at 2500 µg/mL was observed.
In the presence of S9 mix: A significant increase of 52 mOsmol/kg above the mean solvent control value occurred at 5000 µg/mL.

Second test (32 h harvest)
In the absence of S9 mix: Significant increase in osmolality of 52 mOsmol/kg observed at 5000 µg/mL.
In the presence of S9 mix: No significant increase in osmolality was observed at any dose level.

- Evaporation from medium: Not reported
- Water solubility: the test substance is fairly soluble in water (>500 mg/mL).
- Precipitation: No precipitation occurred at the highest concentration of 5000 µg/mL
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES: Screening studies are not performed in this study

COMPARISON WITH HISTORICAL CONTROL DATA: Not reported

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Mitotic index

First test
In the absence of S9 mix: the test substance reduced the mitotic index to 51 % of the solvent control value at 5000 µg/mL and to 88 % at 2500 µg/mL.
In the presence of S9 mix: Reduction in mitotic index to 71 % of the solvent control value at 5000 µg/mL.

Second test (18 h harvest)
In the absence of S9 mix: Reduction in the mitotic index to 40 % of the solvent control value at 5000 µg/mL and to 85 % at 2500 µg/mL
In the presence of S9 mix: Reduction in the mitotic index to 86 % of the solvent control value at 5000 µg/mL

Second test (32 h harvest)
In the absence of S9 mix: Reduction in the mitotic index to 50 % of the solvent control value at 5000 µg/mL and to 76 % at 1250 µg/mL
In the presence of S9 mix: No toxic responses were observed.

Metaphase analysis

First test:

Both in the presence as well as in the absence of S9 mix, no statistically significant increase in the proportion of abberant cells was observed.

Second test (18 h harvest):

In the absence of S9 mix: Significant increase (5.5 %, which falls just outside the historical control range) in the proportion of metaphase figures with chromosomal aberrations at 2500 µg/mL.

In the presence of S9 mix: No statistically significant increase in the proportion of metaphase figures were observed.

Second test (32 h harvest): No significant increase in the proportion of metaphase figures were observed both in the presence and absence of metabolic activation. Both the positive controls caused large significant increase in aberrant metaphase figures.

Conclusions:
Under the study conditions evidence of cytotoxicity was observed from 1250 µg/mL in the absence of metabolic activation and only at 5000 µg/mL in the presence of metabolic activation. Based on the above results, it is concluded that the test substance shows no evidence of clastogenic activity in the in vitro human lymphocyte chromosomal aberration assay.
Executive summary:

A study was conducted to determine the clastogenic potential of the test substance according to OECD Guideline 473, in compliance with GLP. Duplicate cultures of human lymphocytes obtained from a healthy male donor were treated with the test substance (dissolved in water) both in the absence and presence of metabolic activation for 3 h. Concentrations of 9.8, 19.5, 39.1, 78.1, 156.3, 312.5, 625, 1250, 2500 and 5000 µg/mL were used in the first test where a harvest/fixation time of 17 or 20 h were kept. Concentrations of 625, 1250, 2500 and 5000 µg/mL were used for an 18 h harvest time in the second test both in the absence and presence of S9 mix. For a 32 h harvest test 156.3, 312, 625, 1250, 2500 and 5000 µg/mL was used in the absence of S9 mix and 625, 1250, 2500 and 5000 µg/mL in the presence of S9 mix. Cytotoxicity was determined by mitotic index. Metaphase analysis was performed for 100 cells per culture. Both the positive controls (cyclophosphamide and ethylmethane sulphonate) caused large, statistically significant increases in the proportion of abberant cells. The test substance caused no substantial increase in the proportion of metaphase figures containing chromosomal aberrations at any dose levels when compared with the solvent control. Under the study conditions evidence of cytotoxicity was observed from 1250 µg/mL in the absence of metabolic activation and only at 5000 µg/mL in the presence of metabolic activation. Based on the above results, it is concluded that the test substance shows no evidence of clastogenic activity in the in vitro human lymphocyte chromosomal aberration assay (Akhurst, 1995).

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 19, 2008 to July 7, 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
not specified
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
not specified
Qualifier:
equivalent or similar to guideline
Guideline:
other: EU Commission Directive 2000/32/EC
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
thymidine kinase, TK +/- lcous
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media:
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: No data
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: Before freezing the stock cells, the TK+/- heterozygote cells were cleansed of homozygous TK -/- mutant cells by culturing in THMG medium in for 24 h.
Metabolic activation:
with and without
Metabolic activation system:
2 % S9
Test concentrations with justification for top dose:
Preliminary test: 6.95 to 1780 μg/mL

Mutagenicity test:
Experiment 1: 111.25 to 1780 μg/mL
Experiment 2: 111.25 to 1780 μg/mL
Vehicle / solvent:
Not applicable
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation (S9) Migrated to IUCLID6: Sigma batch - 116K0765; 400 µg/mL and 150 µg/mL for Experiment 1 and Experiment 2 respectively
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation (S9) Migrated to IUCLID6: Acros batch - A0164185; 2 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period:
- Exposure duration:
Preliminary test - 4 h (with and without S9)
24 h (without S9)
Experiment 1 - 4 h (with and without S9)
Experiment 2 - 4 h (with S9)
24 h (without S9)
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): 2 h

SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT)
SPINDLE INHIBITOR (cytogenetic assays): Not applicable
STAIN (for cytogenetic assays): MTT was used as a vital stain to assist in scoring of mutant cells

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: 2000 cells/well

DETERMINATION OF CYTOTOXICITY
- Method: Relative total growth (RTG); Relative suspension growth (% RSG)
Evaluation criteria:
1. The normal range for mutant frequency per survivor in laboratory is 50-200 x 10-6 for the TK+/- locus in L5178Y cells. Vehicle controls results should ideally be within this range.
2. Experiments where the vehicle control values are markedly greater than 250 x 10-6 mutant frequency per survivor are not normally acceptable and will be repeated.
3. Positive control chemicals should induce at least three to five fold increases in mutant frequency greater than the corresponding vehicle control.
4. For a test material to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value.
Statistics:
The experimental data was analysed using UKEMS recommended statistical guidelines.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
10 mM
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: Osmolality did not increase by more than 50 mOsm
- Evaporation from medium: No data
- Water solubility: Not applicable
- Precipitation: No
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity test was performed to determine maximum dose levels. The dose range used in the preliminary toxicity test was 6.95 to 1780 μg/mL. Maximum dose levels were selected using the following criteria:
i) Maximum recommended dose level, 5000 μg/mL or 10 mM.
ii) The presence of excessive precipitate where no test material-induced toxicity was observed.
iii) Test material-induced toxicity, where the maximum dose level used should produce 10 to 20 % survival (the maximum level of toxicity required).

COMPARISON WITH HISTORICAL CONTROL DATA:
No

ADDITIONAL INFORMATION ON CYTOTOXICITY: None
Conclusions:
Under the study conditions the test substance was considered to be non-mutagenic to L5178Y cells.
Executive summary:

A study was conducted to determine the potential mutagenicity of the test substance according to OECD Guideline 476, EU Method B17 and Commission Directive 2000/32/EC, in compliance with GLP. Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test substance at six dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-h exposure groups both in the absence and presence of metabolic activation (2 % S9). In Experiment 2, the cells were treated with the test substance at six dose levels using a 4-h exposure group in the presence of metabolic activation (1% S9) and a 24 h exposure group in the absence of metabolic activation. The dose range of test substance was selected following the results of a preliminary toxicity test and was 111.25 to 1780 µg/mL throughout the mutagenicity test. The maximum dose level used was the 10 mM limit dose. No precipitate of test substance was observed at any of the dose levels. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. The test substance did not induce any statistically significant or dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment using a dose range that included the 10 mM limit dose. Under the study conditions the test substance was considered to be non-mutagenic to L5178Y cells (Flanders, 2008).

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From September 17, 2008 to October 07, 2008
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:
EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
Not applicable
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
10% liver S9 in standard co-factors
Test concentrations with justification for top dose:
Preliminary test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1,500 and 5,000 μg/plate

Mutation test:
Experiment 1 (Range-finding test): 50, 150, 500, 1,500 and 5,000 μg/plate
Experiment 2 (Main test): 50, 150, 500, 1,500 and 5,000 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Sterile distilled water
- Justification for choice of solvent/vehicle: Test material was fully soluble (50 mg/mL) in sterile distilled water
Untreated negative controls:
yes
Remarks:
Concurrent untreated controls
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without metabolic activation (S9) Migrated to IUCLID6: 2 μg/plate for WP2uvrA-, 3 μg/plate for TA100 and 5 μg/plate for TA1535
Untreated negative controls:
yes
Remarks:
Concurrent untreated controls
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without metabolic activation (S9) Migrated to IUCLID6: 80 μg/plate for TA1537
Untreated negative controls:
yes
Remarks:
Concurrent untreated controls
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without metabolic activation (S9) Migrated to IUCLID6: 0.2 μg/plate for TA98
Untreated negative controls:
yes
Remarks:
Concurrent untreated controls
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with metabolic activation (S9) Migrated to IUCLID6: 5 μg/plate for TA98
Untreated negative controls:
yes
Remarks:
Concurrent untreated controls
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 1 μg/plate for TA100, 2 μg/plate for TA1535 and TA1537, and 10 μg/plate for WP2uvrA-
Remarks:
with metabolic activation (S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (direct plate incorporation)


DURATION
- Preincubation period: Not applicable
- Exposure duration:
Preliminary test: 48 h (with and without S9)
Mutation test:
Experiment 1 (Range-finding test): 48 h (with and without S9)
Experiment 2 (Main test): 48 h (with and without S9)

- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): Not applicable


SELECTION AGENT (mutation assays): Not applicable
SPINDLE INHIBITOR (cytogenetic assays): Not applicable
STAIN (for cytogenetic assays): Not applicable


NUMBER OF REPLICATIONS: Triplicate


NUMBER OF CELLS EVALUATED: Not applicable
Evaluation criteria:
Criteria for determining a positive result: Dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation.

A test material was considered non-mutagenic (negative) in the test system if the above criteria were not met.

Statistics:
The experimental data was analysed using UKEMS recommended statistical guidelines.
Key result
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
Key result
Species / strain:
E. coli WP2 uvr A
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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not applicable
- Effects of osmolality: Not applicable
- Evaporation from medium: Not applicable
- Water solubility: 50 mg/mL
- Precipitation: No
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES:
The dose range for the range-finding test was determined in a preliminary toxicity assay and was 50, 150, 500, 1,500 and 5,000 μg/plate.
- The test substance caused no visible reduction in the growth of the bacteria at any dose level. The test substance was, therefore, tested up to the maximum recommended dose level of 5,000 μg/plate.
- No test substance precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
- No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA: No comparison performed.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Not applicable

Preliminary Toxicity Test

The test substance was non-toxic to the strains (TA100 and WP2uvrA-) of bacteria.

Table 1: Numbers of revertant colonies for the toxicity assay

With (+) or without (-) S9-mix

Strain

Dose (µg/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1,500

5,000

-

TA100

132

132

126

115

128

126

154

144

114

167

132

+

TA100

129

157

172

157

162

125

142

144

148

144

137

-

WP2uvrA-

23

30

31

19

18

29

20

29

31

23

21

+

WP2uvrA-

37

48

37

26

35

43

24

27

44

35

30

Mutation test:

No visible reduction in the growth of the bacterial background lawn was observed at any dose level. Therefore, the test substance was tested upto the maximum recommended dose level of 5,000 µg/plate.

Neither the test substance precipitate nor any significant increases in the frequency of revertant colonies were observed on the plates of any of the doses tested in either the presence or absence of S9-mix.

Significant increases in the frequency of revertant colonies were observed with all the positive control chemicals which confirmed the activity of the S9-mix and the sensitivity of the bacterial strains.

Table 2: Test Results: Range-Finding Test - Without Metabolic Activation

Test Period

Number of revertants (mean number of colonies per plate)

With or without S9-Mix

Test substance concentration (μg/plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

-

0

120
93
95

(103)
15.0#

17
14
18

(16)
2.1

18
21
29

(23)
5.7

17
11
21

(16)
5.0

12
12
5

(10)
4.0

-

50

91
81
83

(85)
5.3

18
18
21

(19)
1.7

26
26
31

(28)
2.9

15
16
13

(15)
1.5

11
10
9

(10)
1.0

-

150

97
86
81

(88)
8.2

18
21
18

(19)
1.7

21
32
29

(27)
5.7

17
21
18

(19)
2.1

13
10
9

(11)
2.1

-

500

99
96
92

(96)
3.5

22
18
20

(20)
2.0

32
25
32

(30)
4.0

19
18
18

(18)
0.6

16
11
5

(11)
5.5

-

1500

101
78
81

(87)
12.5

15
20
18

(18)
2.5

30
25
30

(28)
2.9

10
20
30

(20)
10.0

3
5
7

(5)
2.0

-

5000

100
102
114

(105)
7.6

18
15
22

(18)
3.5

34
21
36

(30)
8.1

22
15
20

(19)
3.6

6
12
11

(10)
3.2

Positive
controls
S9-Mix
-

Name
Concentration
(μg/plate)
No. colonies
per plate

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

386
371
448

(402)
40.8

162
146
188

(165)
21.2

676
648
715

(680)
33.7

105
114
108

(109)
4.6

537
490
443

(490)
47.0

Table 3: Test Results: Range-Finding Test - With Metabolic Activation

Test Period

Number of revertants (mean number of colonies per plate)

With or without S9-Mix

Test substance concentration (μg/plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

+

0

100
98
102

(100)
2.0#

18
12
12

(14)
3.5

38
38
33

(36)
2.9

22
33
26

(27)
5.6

10
14
14

(13)
2.3

+

50

102
99
115

(105)
8.5

10
17
19

(15)
4.7

34
36
33

(34)
1.5

17
25
22

(21)
4.0

7
13
9

(10)
3.1

+

150

129
93
121

(114)
18.9

21
17
13

(17)
4.0

37
44
32

(38)
6.0

13
22
29

(21)
8.0

7
9
10

(9)
1.5

+

500

104
99
92

(98)
6.0

12
13
16

(14)
2.1

43
33
43

(40)
5.8

25
27
25

(26)
1.2

7
7
10

(8)
1.7

+

1500

93
85
95

(91)
5.3

23
14
12

(16)
5.9

38
44
34

(39)
5.0

25
18
29

(24)
5.6

9
13
13

(12)
2.3

+

5000

113
112
84

(103)
16.5

19
16
18

(18)
1.5

36
32
37

(35)
2.6

21
17
38

(25)
11.2

7
7
9

(8)
1.2

Positive
controls
S9-Mix
+

Name
Concentration
(μg/plate)
No. colonies
per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

1048
595
547

(730)
276.4

288
296
278

(287)
9.0

429
444
472

(448)
21.8

132
157
178

(156)
23.0

283
205
190

(226)
49.9

Table 4: Test Results: Main Test - Without Metabolic Activation

Test Period

Number of revertants (mean number of colonies per plate)

With or without S9-Mix

Test substance concentration (μg/plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

-

0

107
133
119

(120)
13.0#

21
18
22

(20)
2.1

33
23
18

(25)
7.6

18
18
15

(17)
1.7

13
10
19

(14)
4.6

-

50

98
106
117

(107)
9.5

17
10
25

(17)
7.5

27
22
18

(22)
4.5

18
18
16

(17)
1.2

9
11
14

(11)
2.5

-

150

95
104
129

(109)
17.6

19
26
33

(26)
7.0

23
26
26

(25)
1.7

17
21
17

(18)
2.3

14
11
4

(10)
5.1

-

500

112
120
108

(113)
6.1

30
13
22

(22)
8.5

21
30
26

(26)
4.5

13
12
16

(14)
2.1

13
12
11

(12)
1.0

-

1500

120
122
110

(117)
6.4

36
20
23

(26)
8.5

27
26
25

(26)
1.0

14
10
15

(13)
2.6

11
12
9

(11)
1.5

-

5000

112
128
122

(121)
8.1

20
16
18

(18)
2.0

21
30
25

(25)
4.5

26
21
15

(21)
5.5

7
10
5

(7)
2.5

Positive
controls
S9-Mix
-

Name
Concentration
(μg/plate)
No. colonies
per plate

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

665
674
675

(671)
5.5

125
111
126

(121)
8.4

616
646
655

(639)
20.4

237
183
187

(202)
30.1

418
357
345

(373)
39.1

Table 5: Test Results: Main Test - With Metabolic Activation

Test Period

Number of revertants (mean number of colonies per plate)

With or without S9-Mix

Test substance concentration (μg/plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

+

0

136
119
107

(121)
14.6#

20
18
10

(16)
5.3

38
31
34

(34)
3.5

17
37
23

(26)
10.3

14
7
16

(12)
4.7

+

50

142
114
104

(120)
19.7

11
13
12

(12)
1.0

45
35
35

(38)
5.8

18
28
27

(24)
5.5

11
3
15

(10)
6.1

+

150

112
106
115

(111)
4.6

12
19
17

(16)
3.6

44
30
46

(40)
8.7

30
18
31

(26)
7.2

18
12
9

(13)
4.6

+

500

90
122
123

(112)
18.8

15
15
21

(17)
3.5

41
31
33

(35)
5.3

30
27
29

(29)
1.5

12
9
11

(11)
1.5

+

1500

121
104
91

(105)
15.0

16
12
13

(14)
2.1

38
35
42

(38)
3.5

26
23
31

(27)
4.0

14
11
21

(15)
5.1

+

5000

111
92
114

(106)
11.9

15
20
16

(17)
2.6

44
33
38

(38)
5.5

29
28
33

(30)
2.6

13
10
12

(12)
1.5

Positive
controls
S9-Mix
+

Name
Concentration
(μg/plate)
No. colonies
per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

609
936
963

(836)
197.1

167
192
189

(183)
13.7

194
202
222

(206)
14.4

141
141
145

(142)
2.3

175
194
194

(188)
11.0

Abbreviations: ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine; 4NQO: 4-Nitroquinoline-1-oxide; 9AA: 9-Aminoacridine; BP: Benzo(a)pyrene; 2AA: 2-Aminoanthracene

# Standard deviation

Conclusions:
Under the study conditions no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation, therefore the test substance was considered to be non-mutagenic.
Executive summary:

A study was conducted to determine the potential mutagenicity of the test substance according to OECD Guidelines 471, EU Method B.13/14, USA, EPA (TSCA) OPPTS harmonised guidelines, Japanese regulatory guidelines including METI, MHLW and MAFF, in compliance with GLP. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA- were treated with the test substance using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and was 50 to 5000 μg/plate. The experiment was repeated on a separate day using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test substance formulations. The vehicle (sterile distilled water) 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 test substance caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test substance was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. No test substance precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. Under the study conditions no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation, therefore the test substance was considered to be non-mutagenic (Bowles, 2009).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A study was conducted to determine the potential mutagenicity of the test substance according to OECD Guidelines 471, EU Method B.13/14, USA, EPA (TSCA) OPPTS harmonised guidelines, Japanese regulatory guidelines including METI, MHLW and MAFF, in compliance with GLP. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA- were treated with the test substance using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and was 50 to 5000 μg/plate. The experiment was repeated on a separate day using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test substance formulations. The vehicle (sterile distilled water) 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 test substance caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test substance was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. No test substance precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. Under the study conditions no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation, therefore the test substance was considered to be non-mutagenic (Bowles, 2009).

A study was conducted to determine the clastogenic potential of the test substance according to OECD Guideline 473, in compliance with GLP. Duplicate cultures of human lymphocytes obtained from a healthy male donor were treated with the test substance (dissolved in water) both in the absence and presence of metabolic activation for 3 h. Concentrations of 9.8, 19.5, 39.1, 78.1, 156.3, 312.5, 625, 1250, 2500 and 5000 µg/mL were used in the first test where a harvest/fixation time of 17 or 20 h were kept. Concentrations of 625, 1250, 2500 and 5000 µg/mL were used for an 18 h harvest time in the second test both in the absence and presence of S9 mix. For a 32 h harvest test 156.3, 312, 625, 1250, 2500 and 5000 µg/mL was used in the absence of S9 mix and 625, 1250, 2500 and 5000 µg/mL in the presence of S9 mix. Cytotoxicity was determined by mitotic index. Metaphase analysis was performed for 100 cells per culture. Both the positive controls (cyclophosphamide and ethylmethane sulphonate) caused large, statistically significant increases in the proportion of abberant cells. The test substance caused no substantial increase in the proportion of metaphase figures containing chromosomal aberrations at any dose levels when compared with the solvent control. Under the study conditions evidence of cytotoxicity was observed from 1250 µg/mL in the absence of metabolic activation and only at 5000 µg/mL in the presence of metabolic activation. Based on the above results, it is concluded that the test substance shows no evidence of clastogenic activity in the in vitro human lymphocyte chromosomal aberration assay (Akhurst, 1995).

A study was conducted to determine the potential mutagenicity of the test substance according to OECD Guideline 476, EU Method B17 and Commission Directive 2000/32/EC, in compliance with GLP. Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test substance at six dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4 -h exposure groups both in the absence and presence of metabolic activation (2 % S9). In Experiment 2, the cells were treated with the test substance at six dose levels using a 4-h exposure group in the presence of metabolic activation (1% S9) and a 24 h exposure group in the absence of metabolic activation. The dose range of test substance was selected following the results of a preliminary toxicity test and was 111.25 to 1780 µg/mL throughout the mutagenicity test. The maximum dose level used was the 10 mM limit dose. No precipitate of test substance was observed at any of the dose levels. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. The test substance did not induce any statistically significant or dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment using a dose range that included the 10 mM limit dose. Under the study conditions the test substance was considered to be non-mutagenic to L5178Y cells (Flanders, 2008).

Overall, the evidence from in vitro studies indicates that the test substance is not genotoxic. No further in vivo testing was conducted.

Justification for classification or non-classification

The available in vitro genotoxicity data suggests that the substance does not warrant classification for this endpoint according to CLP (EC 1272/2008) criteria.