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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames test: Under the conditions of this study the test material was considered to be non-mutagenic.

- Chromosome aberration test: Under the conditions of this study, the test material is considered to be negative for inducing chromosomal aberrations in cultured human whole blood lymphocyte cells with and without metabolic activation.

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:
27 July 2016 to 16 August 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)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
- Histidine requirement in the Salmonella typhimurium strains (Histidine operon)
- Tryptophan requirement in the Escherichia coli strain (Tryptophan operon)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
MEDIA USED
- Type and identity of media: Overnight subcultures of stock cultures were prepared in nutrient broth (Oxoid Limited) and incubated at 37 °C for approximately 10 hours.
- Properly maintained: Yes, each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates. Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). All of the strains were stored at approximately -196 °C in a iquid nitrogen freezer.
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
MEDIA USED
- Type and identity of media: Overnight subcultures of stock cultures were prepared in nutrient broth (Oxoid Limited) and incubated at 37 °C for approximately 10 hours.
- Properly maintained: Yes, each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates. Prior to use, the master strain was checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). All of the strains were stored at approximately -196 °C in a iquid nitrogen freezer.
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
- Experiment 1 (with and without metabolic activation): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate)
- Experiment 2 (with and without metabolic activation): 15, 50, 150, 500, 1500 and 5000 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (dimethyl sulphoxide)
- Justification for choice of solvent/vehicle: The test material was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in DMSO at the same concentration in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.
Untreated negative controls:
yes
Remarks:
Untreated
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) for Experiment 1; pre-incubation for Experiment 2

EXPERIMENT 1: PLATE INCORPORATION METHOD
0.1 mL of the appropriate concentration of test material, solvent vehicle or appropriate positive control was added to 2 mL of molten, trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and either 0.5 mL of S9-mix or phosphate buffer (for assays with and without metabolic activation, respectively). These were then mixed and overlayed onto a Vogel-Bonner minimal agar plate. Negative (untreated) controls were also performed on the same day as the mutation test.
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. Manual counts were performed at 5000 μg/plate because of test material precipitation. Several further manual counts were also required due to revertant colonies spreading slightly, thus distorting the actual plate count.

EXPERIMENT 2: PRE-INCUBATION METHOD
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of S9-mix or phosphate buffer (for assays with and without metabolic activation, respectively) and 0.1 mL of the test material formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner minimal agar plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method.
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. Manual counts were performed at 5000 μg/plate because of test material precipitation.

NUMBER OF REPLICATIONS: Triplicate assays were performed

DETERMINATION OF CYTOTOXICITY
- Method: Reduction in the growth of the bacterial background lawn and increases in the frequency of revertant colonies were recorded to determine cytotoxicity. The plates were viewed microscopically for evidence of thinning (toxicity).
Evaluation criteria:
ACCEPTABILITY CRITERIA
The reverse mutation assay may be considered valid if the following criteria are met:
- All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks.
- 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 presented 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 10^9 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.

EVALUATION CRITERIA
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:
1. A dose-related increase in mutant frequency over the dose range tested.
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.
Key result
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The mean number of revertant colonies and the standard deviations for the test material, positive and vehicle controls, both with and without metabolic activation, are presented in Table 1 and Table 2 for Experiment 1 and Table 3 and Table 4 for Experiment 2.
The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test material formulation was also shown to be sterile. Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. Similarly there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). A test material precipitate (cream-coloured and particulate in appearance) was noted at and above 1500 μg/plate; this observation did not prevent the scoring of revertant colonies.
There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method). A small, statistically significant increase in TA100 revertant colony frequency was observed in the absence of S9-mix at 150 μg/plate in the second mutation test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at 150 μg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.2 times the concurrent vehicle control.
The vehicle (dimethyl sulphoxide) 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.

Table 1: Number of revertants (mean) ± SD in Experiment 1- without metabolic activation

Dose Level per plate (µg)

TA100

TA1535

WP2uvrA

TA98

TA1537

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Solvent control

65

1.2

11

4.6

21

4.5

19

1.7

13

3.6

1.5

63

2.3

10

2.3

21

5.5

20

1.5

18

6.8

5

64

0.6

9

1.7

26

9.7

14

6.0

16

4.6

15

67

0.6

15

4.6

24

8.2

18

4.7

15

4.5

50

65

1.5

10

0.6

19

6.8

19

1.5

11

3.2

150

68

4.9

9

2.0

23

8.1

21

4.0

11

2.9

500

68

5.3

11

4.6

19

10.7

18

6.4

8

1.5

1500

62P

1.0

10P

1.5

18P

2.3

20P

0.6

13P

0.0

5000

62P

1.0

11P

2.6

13P

5.5

17P

5.0

9P

3.2

Positive Controls (µg)

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

536

28.3

591

69.7

856

52.1

267

1.7

313

25.1

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO: 4-Nitroquinoline-1-oxide

9AA: 9-Aminoacridine

P = Precipitate

Table 2: Number of revertants (mean) ± SD in Experiment 1- with metabolic activation

Dose Level per plate (µg)

TA100

TA1535

WP2uvrA

TA98

TA1537

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Solvent control

75

11.5

12

2.3

18

4.7

30

8.7

18

5.5

1.5

69

13.3

13

4.4

24

5.8

28

3.1

18

4.5

5

79

6.1

9

0.0

19

3.2

28

4.4

13

5.9

15

71

9.6

11

1.0

23

0.6

34

11.2

10

1.7

50

70

12.4

11

0.6

23

3.5

27

8.7

12

2.1

150

76

4.4

15

5.0

18

2.1

33

6.7

9

1.0

500

81

8.0

10

1.0

20

3.8

35

1.7

11

7.8

1500

70P

3.2

10P

1.2

21P

2.3

26P

0.6

13P

1.2

5000

66P

3.8

8P

0.6

19P

2.5

24P

3.8

11P

2.3

Positive Controls (µg)

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

1119

64.1

257

9.0

460

70.7

128

5.5

261

12.1

BP: Benzo(a)pyrene

2AA: 2-Aminoanthracene

P = Precipitate

 

Table 3: Number of revertants (mean) ± SD in Experiment 2- without metabolic activation

Dose Level per plate (µg)

TA100

TA1535

WP2uvrA

TA98

TA1537

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Solvent control

91

6.1

17

7.6

33

7.2

26

6.2

15

6.9

15

76

5.8

18

6.1

27

1.0

25

5.0

11

1.2

50

95

4.0

18

8.7

30

12.0

27

6.1

14

1.5

150

108*

12.1

21

10.7

28

12.0

19

4.0

16

3.1

500

99

1.5

13

1.7

27

6.1

24

5.2

20

2.5

1500

89P

3.1

14P

4.0

29P

0.6

18P

4.0

21P

1.2

5000

77P

4.2

11P

3.0

20P

3.2

19P

0.6

9P

1.7

Positive Controls (µg)

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

969

346.8

1862

173.4

1025

65.0

283

7.1

301

126.7

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO: 4-Nitroquinoline-1-oxide

9AA: 9-Aminoacridine

*p≤0.05

P = Precipitate

Table 4: Number of revertants (mean) ± SD in Experiment 2- with metabolic activation

Dose Level per plate (µg)

TA100

TA1535

WP2uvrA

TA98

TA1537

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Solvent control

91

1.2

17

4.2

31

6.0

24

2.1

17

8.5

15

87

13.5

18

5.1

28

5.0

26

3.8

22

4.6

50

108

13.5

13

2.9

31

6.4

32

4.7

19

1.0

150

101

12.1

13

2.6

33

2.9

26

7.9

20

4.2

500

96

9.9

10

2.6

21

10.7

30

5.1

17

4.0

1500

103P

6.1

12P

2.1

26P

8.1

29P

9.5

22P

4.0

5000

73P

5.9

10P

1.5

22P

7.0

26P

4.6

11P

2.0

Positive Controls (µg)

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

1173

25.2

181

12.9

195

16.7

108

14.4

129

3.5

BP: Benzo(a)pyrene

2AA: 2-Aminoanthracene

P = Precipitate

Conclusions:
Under the conditions of this study the test material was considered to be non-mutagenic.
Executive summary:

A bacterial reverse mutation assay was carried out in accordance with the standardised guidelines OECD 471, EU Method B13/14, US EPA OCSPP 870.5100 and the testing published by the Japanese METI, MHLW and JMAFF under GLP conditions.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test material in DMSO using both the Ames plate incorporation and pre-incubation methods at up to eight 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 Experiment 1 was predetermined and was 1.5 to 5000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test material formulations. The dose range was amended following the results of Experiment 1 and was 15 to 5000 μg/plate. Bacteria were also exposed to vehicle controls and appropriate positive controls.

The vehicle 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.

In Experiment 1 there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method). Similarly there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). A test material precipitate (cream-coloured and particulate in appearance) was noted at and above 1500 μg/plate; this observation did not prevent the scoring of revertant colonies.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation in Experiment 1. Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation in Experiment 2. A small, statistically significant increase in TA100 revertant colony frequency was observed in the absence of S9-mix at 150 μg/plate in the second mutation test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at 150 μg/plate were within the in-house historical untreated/vehicle control range for the tester strain.

Under the conditions of this study the test material was considered to be non-mutagenic.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 March 1991 to 30 April 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: chromosome aberration study in mammalian cells
Species / strain / cell type:
lymphocytes: human whole blood
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: A single human donor. Venous blood was drawn into sterile, heparinised "Vacutainers".
- Whether whole blood or separated lymphocytes were used if applicable: Whole blood
- Cell culture: Cultures were initiated by adding 0.3 mL of blood to 4.7 mL of culture medium in the range-finding assays and 0.6 mL of whole blood to 9.4 mL of culture medium in the aberrations assays in 15 mL centrifuge tubes. The cells were incubated at about 37 °C on a slope, with loose caps, in an atmosphere of about 5 % CO2 in air.

MEDIA USED
- Type and identity of media: RPMI 1640, supplemented with 15 % foetal calf serum (FCS), 1 % phytohaemagglutinin (PHA-M), 1 % penicillin and streptomycin and 1 % L-glutamine.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
- Range-finding test (without metabolic activation): 167, 501, 1670 and 5010 µg/mL
- Range-finding test (with metabolic activation): 50.1, 167, 501, 1670 and 5010 µg/mL
- Trial 1 (with and without metabolic activation): 315, 630, 1260, 2510 and 5010 µg/mL
- Trial 2 (with and without metabolic activation): 313, 625, 1250, 2500 and 5000 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent: According to the sponsor the test material was insoluble in water. Solubility was evaluated in DMSO and clear colourless solutions were obtained at concentrations of 201, 301, 401 and 501 mg/mL. These were sham dosed using 1 % in RPMI-1640 medium, but the resulting solutions had visible precipitation and oily globules. Solubility was then evaluated in ethanol, using the same dilutions; at a final concentration of 5020 µg/mL, a clear solution with very fine oily droplets and no precipitate that adhered to the sides of the dilution tube (pH 7.0) was obtained. Ethanol was therefore chosen as the solvent. The test material was dissolved at a stock concentration of 501 mg/mL in ethanol before diluting.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
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

DURATION
- Pre-incubation period: Cultures were initiated approximately 48 hours prior to treatment by adding 0.6 mL of whole blood to 9.4 mL of culture medium in 15 mL centrifuge tubes
- Exposure duration: 21 or 45 hours (non-activation assay) and 2 hours (activation assay)
- Expression time (cells in growth medium): 2 hours (non-activation assay) and 21 or 45 hours with Colcemid only present for the last 2 hours (activation assay)

SPINDLE INHIBITOR (cytogenetic assays): Colcemid 0.1 µg/mL

PROCEDURE
- Non-activation assays: Following treatment with the test material, the cultures were then washed with buffered saline and refed with complete RPMI 1640 medium containing Colcemid. Approximately two hours later the cells were harvested and slides were made.
- Activation assays: The cultures were incubated at 37 °C for two hours in the presence of the test material and the S9 reaction mixture in RPMI culture medium without FCS. After the two hour exposure period, the cells were washed twice with buffered saline and the cells were refed with complete RPMI 1640 medium. The cells were incubated with Colcemid then the cells were harvested and prepared for cytogenetic analysis.

STAIN (for cytogenetic assays): 5 % Giemsa solution

REPLICATES: 2

HARVESTING
The cell suspension was centrifuged, supernatant discarded and the cells treated with hypotonic KCl (0.075 M) for 6 to 10 minutes. This treatment helps to swell the cells and thus disperse the chromosomes. After centrifugation and removal of the KCl, the cells were fixed three times with freshly prepared fixative (absolute methanol: glacial acetic acid 3:1 v:v).

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Slides were prepared by dropping the harvested cultures on clean slides. The slides were stained and all slides were then air-dried and coverslipped using Depex mounting medium.

NUMBER OF CELLS EVALUATED: One hundred cells, if possible, from each replicate culture at four dose levels of the test material and from each of the negative and solvent control cultures were analysed for the different types of chromosomal aberrations. At least 25 cells were analysed for chromosomal aberrations from one of the positive control cultures.

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index was first analysed from one of the replicate cultures from the first set of assays to select doses for analysis. In the second set of experiments, the top four dose levels were analysed. Cells were selected for good morphology and only cells with the number of centromeres equal to the modal number 46 ± 2 (range 44 to 48) were analysed.
Rationale for test conditions:
The dosing concentrations were determined as a result of a range finding test performed with and without metabolic activation in which no reduction in mitotic index was observed for any of the tested concentrations in the range 167 to 5010 µg/mL without activation and 50.1 to 5010 µg/mL with activation.
Evaluation criteria:
The following factors were taken into account in the evaluation of the chromosomal aberrations data:
1. The overall chromosomal aberration frequency
2. The percentage of cells with any aberrations
3. The percentage of cells with more than one aberration
4. Any evidence for increasing amounts of damage with increasing dose, i.e. a positive dose response.

Chromatid and isochromatid gaps, if observed, were noted in the raw data and were tabulated. They were not, however, considered in the evaluation of the ability of the test material to induce chromosomal aberrations since they may not represent true chromosomal breaks and may possibly be induced by toxicity.
Statistics:
Statistical analysis employed the Fisher’s Exact Test with an adjustment for multiple comparisons (Sokal and Rohlf, 1981) to compare the percentage of cells with aberrations in each treatment group with the results from the pooled solvent and negative controls (the solvent and negative controls were statistically evaluated for similarity prior to the pooled evaluation). Test material significance was established where p<0.01. All factors as stated previously were taken into account and the final evaluation of the test material was based upon scientific judgement.
Key result
Species / strain:
lymphocytes: human whole blood
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
with metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING STUDY:
Mitotic index was evaluated by analysing the number of metaphases in the first 1000 cells. The analysis was performed on the highest dose levels with metaphases (four from the non-activation assay and five from the activation assay). No reduction in the mitotic index was evident in the cultures analysed either with or without activation.

CHROMOSOMAL ABERRATIONS ASSAY WITHOUT METABOLIC ACTIVATION
In the first 24 hour trial, oily droplets were observed after dosing and an oily precipitate was observed on the sides of the culture tubes prior to wash in the cultures dosed with 5010 µg/mL. A slight amount of precipitate was observed on the sides of the culture tubes prior to wash in the cultures dosed with 2510 µg/mL. No reduction in mitotic index was observed in the test cultures. Results were analysed from the cultures dosed with 630, 1260, 2510 and 5010 µg/mL. No significant increase in cells with chromosomal aberrations was observed at the concentrations analysed.
In the 24 hour repeat trial, oily droplets were observed after dosing and a precipitate was observed on the sides of the culture tubes prior to washing in the cultures dosed with 5000 µg/mL. A slight amount of precipitate was observed on the sides of the culture tubes in the cultures dosed with 2510 µg/mL. Results were analysed from the cultures dosed with 625, 1250, 2500 and 5000 µg/mL. No significant increase in cells with chromosomal aberrations was observed at the concentrations analysed.
In the 48 hour repeat trial, oily droplets were observed after dosing the cultures with 5000 µg/mL. Slight lysis of red blood cells was observed prior to washing in all test cultures. Results were analysed from the cultures dosed with 625, 1250, 2500 and 5000 µg/mL. No significant increase in cells with chromosomal aberrations was observed at the concentrations analysed.
The sensitivity of the cell culture for induction of chromosomal aberrations is shown by the increased frequency of aberrations in the cells exposed to mitomycin C, the positive control agent. The test material was considered negative for inducing chromosomal aberrations under non-activation conditions.

CHROMOSOMAL ABERRATIONS ASSAY WITH METABOLIC ACTIVATION
In the first 24 hour trial, oily droplets were observed after dosing the cultures with 5010 µg/mL. Lysis of red blood cells was observed prior to washing in all test cultures. No reduction in mitotic index was observed in the test cultures. Results were analysed from the cultures dosed with 630, 1260, 2510 and 5010 µg/mL. Due to toxicity sparse numbers of metaphases were available on the slides prepared from the cultures dosed with 1260 µg/mL and only 95 metaphases were available for analysis from one of the replicate cultures dosed with 5010 µg/mL. No significant increase in cells with chromosomal aberrations was observed at the concentrations analysed.
In the 24 hour repeat trial, oily droplets were observed after dosing the cultures with 5000 µg/mL. Lysis of red blood cells was observed prior to washing in all test cultures. Results were analysed from the cultures dosed with 625, 1250, 2500 and 5000 µg/mL. Due to toxicity, probably arising from the lysis of erythrocytes, only 73 and 76 cells were available for analysis from one of the replicate cultures dosed with 625 and 1250 µg/mL, respectively. The remainder of metaphases for a total number of 200 cells for those dose levels was analysed from the other culture. No significant increase in cells with chromosomal aberrations was observed at the concentrations analysed.
In the 48 hour repeat trial, oily droplets were observed after dosing the cultures with 5000 µg/mL. Lysis of red blood cells was observed prior to washing in all test cultures. Results were analysed from the cultures dosed with 625, 1250, 2500 and 5000 µg/mL. . Due to toxicity, probably arising from the lysis of erythrocytes, 100 metaphases were not available for analysis from one of the replicate cultures dosed with 625, 1250 and 2500 µg/mL. No significant increase in cells with chromosomal aberrations was observed at the concentrations analysed.
The successful activation by the metabolic system is illustrated by the increased incidence of cells with chromosomal aberrations in the cultures induced with cyclophosphamide, the positive control agent. The test material was considered negative for inducing chromosomal aberrations under conditions of metabolic activation.

Table 1: Mitotic index analysis

Treatment

µg/mL

Mitotic index

-S9

+S9

Negative control (RPMI-1640)

-

3.7

2.2

Solvent control (Ethanol)

(10)

4.0

3.1

Positive control wthout activation (MMC)

0.15

0.9

-

Positive control with activation (CP)

50.0

-

0.2

Test material

50.1

-

2.1

167

3.2

2.7

501

0.7

1.7

1670

3.1

2.2

5010

2.5

4.1

( ) Indicates µL/mL

Conclusions:
Under the conditions of this study, the test material is considered to be negative for inducing chromosomal aberrations in cultured human whole blood lymphocyte cells with and without metabolic activation.
Executive summary:

A chromosomal aberration assay using human lymphocytes was carried out using methodology equivalent to the standardised guideline OECD 473 under GLP conditions.

Whole blood lymphocytes from a single human donor were used for the study. The test material was dissolved in ethanol which was then diluted in the medium for exposure to the cells.

A range finding test was performed to determine the concentrations to be used for testing, both with and without metabolic activation. Without metabolic activation, mitotic index was evaluated in cultures dosed with 167, 501, 1670, and 5010 µg/mL. No reduction in mitotic index was evident; therefore a 24 hour harvest was selected for testing concentrations of 315, 630, 1260, 2510, and 5010 µg/mL in the non-activation aberrations assay. The results from this assay were confirmed in independently conducted assays, testing concentrations of 313, 625, 1250, 2500 and 5000 µg/mL with 24 and 48 hour harvests.

With metabolic activation, mitotic index was evaluated in cultures dosed with 50.1, 167, 501, 1670 and 5010 µg/mL. No reduction in mitotic index was evident; therefore a 24 hour harvest was selected for testing concentrations of 315, 630, 1260, 2510, and 5010 µg/mL in the aberrations assay with metabolic activation. The results from this assay were confirmed in independently conducted assays, testing concentrations of 313, 625, 1250, 2500, and 5000 µg/mL with 24 and 48 hour harvests.

Each concentration was tested in duplicate both with and without metabolic activation, in the form of S-9 mix. Negative (medium), solvent (ethanol) and positive controls (mitomycin C or cyclophosphamide) were also tested concurrently.

Following exposure the cells were harvested, fixed and stained with 5 % Giemsa solution. 100 cells, if possible, from each replicate culture at four test material dose levels and controls were analysed for chromosome aberrations. 25 cells were evaluated for the positive control.

No significant increase in the number of cells with chromosomal aberrations was observed at the concentrations analysed either with or without metabolic activation. Positive and negative controls were deemed to be valid.

Under the conditions of this study, the test material is considered to be negative for inducing chromosomal aberrations in cultured human whole blood lymphocyte cells with and without metabolic activation.

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

Genetic toxicity in vivo

Description of key information

- Mammalian erythrocyte micronucleus test: Under the conditions of the study, the test material is not considered to be a clastogenic agent.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 December 1986 to 09 February 1987
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: mouse micronucleus
Species:
mouse
Strain:
CD-1
Details on species / strain selection:
Mice have historically been used in the Micronucleus test and have been shown to exhibit micronuclei indicative of chromosome breakage or lagging chromosomes. Intraperitoneal administration was chosen to maximise bioavailability of the test material.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 11 weeks old
- Weight at study initiation: Males: 35 to 44 g; Females: 30 to 35 g.
- Assigned to test groups randomly: Yes using a random number table
- Housing: 5 per cage according to sex and dose group in stainless steel wire mesh cages
- Water: Tap water ad libitum
- Acclimation period: 33 days
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: Corn oil
- Lot No: 55F-0601 (Sigma Chemical Company)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS
-The test material was weighed and diluted in corn oil. A good solution was obtained and maintained at all dose levels. Dosing solutions were prepared fresh and dosed within two hours of preparation. There was no apparent change in the physical state of the test material during the assay.
- Dose volume: 10 mL/kg
Duration of treatment / exposure:
The dosing solutions were administered in a single intraperitoneal dose.
Frequency of treatment:
Once
Post exposure period:
Groups were sacrificed 30, 48 and 72 hours after exposure.
Dose / conc.:
5 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 animals per sex per group
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive control: triethylenemelamine (TEM)
- Preparation: Dissolved in 0.9 % saline
- Route of administration: Intraperitoneal
- Doses / concentrations: 0.5 mg/kg/bw (dose volume 10 mL/kg)
Tissues and cell types examined:
Slides were prepared from the bone marrow of the femora and stained. The numbers of micronucleated polychromatic erythrocytes and normochromatic erythrocytes were recorded.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The dose selection was determined as the result of the range finding test conducted at doses of 500, 1500, 3000, 4000 and 5000 mg/kg of body weight.

TREATMENT AND SAMPLING TIMES: A single dose was administered with sacrifice after 30, 48 or 72 hours.

DETAILS OF SLIDE PREPARATION: The femora were opened carefully at the proximal end with scissors until a small opening to the marrow canal became visible. A 1 mL tuberculin syringe filled with approximately 0.2 mL foetal bovine serum was inserted into the bone and the bone marrow was gently flushed (to assure maximum dispersion) into 1.0 mL of foetal bovine serum in a 3 mL conical centrifuge tube. The femora were flushed with foetal bovine serum until all the marrow was out and the bone appeared almost transparent. If necessary the distal ends were opened and flushed. The suspension was centrifuged at 1000 rpm for 5 minutes. The supernatant was removed leaving a small amount of foetal bovine serum with the remaining cell button. The button was mixed with a Pasteur pipette to assure a homogenous mixture. A small drop of the mixture was immediately placed near the frosted end of a glass microscope slide previously cleaned in absolute ethanol and pulled behind a clean slide at a 45 ° angle. The slides were quick dried on a slide warmer at approximately 56 °C. Following preparation of the smears, they were dipped in absolute methanol and allowed to air dry.

STAINING: The slides were stained according to the following procedure: Fix in absolute methanol for 5 minutes and air dry. Remove metallic film from surface of Giemsa working solution (5 % Giemsa in pH 6.8 phosphate buffer solution) using a paper towel. Stain for 20 minutes in Giemsa working solution. Rinse twice. The first rinse is in deionised water adjusted to pH 4.0 to 4.5 and the second rinse in deionised water adjusted to pH 7.0. Clean back of slide with absolute methanol. Dry on 56 °C slide warmer. Clear in xylene. Mount in Permount with cover glass.

METHOD OF ANALYSIS: The slides were screened for good preparation, i.e. well spread, undamaged, perfectly stained. One thousand (1000) PCE per animal were counted for the presence of micronuclei. The data were expressed as the number of micronucleated PCE versus total normal PCE in 1000 total PCE per animal. A total of 1000 polychromatic and normochromatic erythrocytes (NCE) was also counted per animal. These data were expressed as the ratio of polychromatic erythrocytes to normochromatic erythrocytes.
Evaluation criteria:
Micronuclei are uniform, darkly stained, typically round bodies in the cytoplasm of PCE. Occasionally, micronuclei appear almond, or tear drop shaped. Inclusions in PCEs which are reflective, improperly shaped or stained, or which are not in the focal plane of the cell are judged to be artefacts and are not scored as micronuclei. Cells containing more than one micronucleus are only scored as micronucleated PCE.
Assessment of a test material as positive is based upon its ability to produce a statistically significant increase in the number of micronucleated polychromatic erythrocytes (PCE) as compared to the vehicle control.

CRITERIA FOR A VALID TEST
If the spontaneous rate of micronuclei in the polychromatic erythrocytes is less than 0.5 % and the positive control is statistically greater (p≤0.05) than the spontaneous and at least seven animals per group survived the treatment, the results are deemed acceptable.
Statistics:
A one-tailed t-test was used to make pairwise comparisons between each treatment group with the vehicle control for statistically significant increases in the number of micronucleated PCE. The PCE/NCE ratio was also calculated based on 1000 erythrocytes for each animal. The proportion of PCE per 1000 erythrocytes per animal was evaluated by pairwise t-tests after an arcsin transformation was performed. Statistical significance was judged at the P≤0.05 and P≤0.01 levels.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Some animals exhibited decreased body tone and/or abdominal gait and/or body drop
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 500, 1500, 3000, 4000 and 5000 mg/kg/bw
- Clinical signs of toxicity in test animals: No signs were observed at any dose level immediately after administration. At 500 mg/kg/bw no signs were observed at 4 hours post-dose and only one male had a decrease in body tone at 24, 48 and 72 hours. Signs were observed at all other dose levels. One or more animals had a decrease in body tone at 4, 24, 48 and 72 hours in all test groups. Additional signs included abnormal gait at 72 hours in one female in each of the 1500, 4000 and 5000 mg/kg group. Body drop was observed at 72 hours in one male in the 3000 mg/kg group, in one male at 4000 mg/kg and in two males in the 5000 mg/kg group. No other signs were observed and no mortality occurred in the dose range study.

RESULTS OF DEFINITIVE STUDY
No statistically significant increases in the incidence of micronucleated PCE were detected in animals administered the test material, at any of the sacrifice times evaluated. No statistically significant differences were observed in the PCE/NCE ratio in any group of animals administered the test material.
Animals treated with the positive control gave a statistically significant increase in the incidence of micronucleated PCE and a statistically significant depression of the PCE/NCE ratio.
The study fulfils the criteria of a valid test.

PHARMACOTOXIC EFFECTS OF TREATMENT IN DEFINITIVE STUDY
Immediately after dose administration, one male dosed with the test material exhibited decreased body tone. At 4, 24, 48 and 72 hours several animals exhibited decreased body tone and/or abdominal gait. Body drop was observed in a few animals at 48 hours and in two animals at 72 hours. No mortality occurred in this study.
No signs were observed in the animals administered the positive or negative controls.

Table 1: Micronucleated PCE per 1000 Polychromatic erythrocytes per animal

Animal number

Sex

Controls

Test material

Corn oil (48 hours)

TEM (30 hours)

5000 mg/kg

10 mL/kg

0.5 mg/kg

30 hours

48 hours

72 hours

1

M

0

48

0

0

0

2

M

0

46

0

0

0

3

M

0

97

1

1

0

4

M

1

52

0

0

2

5

M

0

42

0

0

0

6

F

0

47

0

0

0

7

F

0

65

1

0

0

8

F

0

40

0

0

1

9

F

0

74

0

1

0

10

F

0

45

2

0

0

Mean ± S.D.

0.10 ± 0.32

55.60 ± 18.01

0.40 ± 0.70

0.20 ± 0.42

0.30 ± 0.67

t value

-

9.744**

1.236

0.600

0.848

**Denotes statistical significance at P0.01

Conclusions:
Under the conditions of the study, the test material is not considered to be a clastogenic agent.
Executive summary:

An assay was conducted to evaluate the potential of the test material to induce micronuclei in mice following intraperitoneal administration using methodology equivalent to the standardised guideline OECD 474 under GLP conditions.

In a preliminary dose range finding test the test material was administered intraperitoneally at dose levels of 500, 1500, 3000, 4000 and 5000 mg/kg of bodyweight. Although some clinical signs were observed (decreased body tone, abnormal gait and body drop) the 5000 mg/kg of bodyweight dose was selected for the definitive study.

In the micronucleus test three groups of ten animals (5 males and 5 females per group) were given single doses of the test material at 5000 mg/kg in corn oil by intraperitoneal injection and sacrificed at 30, 48 or 72 hours. Similar groups (5 males and 5 females) were dosed with positive (triethylenemelamine) or vehicle (corn oil) controls and sacrificed at 30 and 48 hours respectively. Slides were prepared from the bone marrow of the femora and stained. Coded slides were scored for the number of polychromatic erythrocytes (PCE) with micronuclei in 1000 PCE per animal. The ratio of polychromatic to normochromatic erythrocytes was determined for each animal.

No statistically significant increases in the incidence of micronucleated PCE were detected in animals administered the test material, at any of the sacrifice times evaluated. No statistically significant differences were observed in the PCE/NCE ratio in any group of animals administered the test material.

Animals treated with the positive control gave a statistically significant increase in the incidence of micronucleated PCE and a statistically significant depression of the PCE/NCE ratio.The study fulfils the criteria of a valid test.

The test material was negative in the micronucleus test at 5000 mg/kg of bodyweight. These findings are based upon the inability of the test material to produce a significant increase in the incidence of micronuclei per 1000 polychromatic erythrocytes per animal in the treated groups versus the vehicle control group. 

Under the conditions of the study, the test material is not considered to be a clastogenic agent.

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

Additional information

This endpoint is addresses with two key in vitro studies (Ames test and chromosome aberration assay) and one key in vivo study (mouse micronucleus). Two further supporting studies are also provided which investigated the potential of the test material to be mutagenic in the presence of UV light.

IN VITRO

Wisher, 2017 (Ames test)

A bacterial reverse mutation assay was carried out in accordance with the standardised guidelines OECD 471, EU Method B13/14, US EPA OCSPP 870.5100 and the testing published by the Japanese METI, MHLW and JMAFF under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test material in DMSO using both the Ames plate incorporation and pre-incubation methods at up to eight 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 Experiment 1 was predetermined and was 1.5 to 5000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test material formulations. The dose range was amended following the results of Experiment 1 and was 15 to 5000 μg/plate. Bacteria were also exposed to vehicle controls and appropriate positive controls.

The vehicle 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.

In Experiment 1 there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method). Similarly there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). A test material precipitate (cream-coloured and particulate in appearance) was noted at and above 1500 μg/plate; this observation did not prevent the scoring of revertant colonies.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation in Experiment 1. Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation in Experiment 2. A small, statistically significant increase in TA100 revertant colony frequency was observed in the absence of S9-mix at 150 μg/plate in the second mutation test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at 150 μg/plate were within the in-house historical untreated/vehicle control range for the tester strain.

Under the conditions of this study the test material was considered to be non-mutagenic.

Murli 1991 (Human whole blood lymphocyte chromosomal aberration)

A chromosomal aberration assay using human lymphocytes was carried out using methodology equivalent to the standardised guideline OECD 473 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Whole blood lymphocytes from a single human donor were used for the study. The test material was dissolved in ethanol which was then diluted in the medium for exposure to the cells.

A range finding test was performed to determine the concentrations to be used for testing, both with and without metabolic activation. Without metabolic activation, mitotic index was evaluated in cultures dosed with 167, 501, 1670, and 5010 µg/mL. No reduction in mitotic index was evident; therefore a 24 hour harvest was selected for testing concentrations of 315, 630, 1260, 2510, and 5010 µg/mL in the non-activation aberrations assay. The results from this assay were confirmed in independently conducted assays, testing concentrations of 313, 625, 1250, 2500 and 5000 µg/mL with 24 and 48 hour harvests.

With metabolic activation, mitotic index was evaluated in cultures dosed with 50.1, 167, 501, 1670 and 5010 µg/mL. No reduction in mitotic index was evident; therefore a 24 hour harvest was selected for testing concentrations of 315, 630, 1260, 2510, and 5010 µg/mL in the aberrations assay with metabolic activation. The results from this assay were confirmed in independently conducted assays, testing concentrations of 313, 625, 1250, 2500, and 5000 µg/mL with 24 and 48 hour harvests.

Each concentration was tested in duplicate both with and without metabolic activation, in the form of S-9 mix. Negative (medium), solvent (ethanol) and positive controls (mitomycin C or cyclophosphamide) were also tested concurrently.

Following exposure the cells were harvested, fixed and stained with 5 % Giemsa solution. 100 cells, if possible, from each replicate culture at four test material dose levels and controls were analysed for chromosome aberrations. 25 cells were evaluated for the positive control.

No significant increase in the number of cells with chromosomal aberrations was observed at the concentrations analysed either with or without metabolic activation. Positive and negative controls were deemed to be valid.

Under the conditions of this study, the test material is considered to be negative for inducing chromosomal aberrations in cultured human whole blood lymphocyte cells with and without metabolic activation.

Dean (1993, E.coli UV light)

The photo- mutagenic potential of the test material was investigated using the Escherichia coli strain WP2 under GLP conditions. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was dissolved in DMSO and the bacteria exposed in both the presence and absence of UV light using the plate incorporation method. Following a range-finding experiment, the dose levels selected for the assays were 8.0, 40.0, 200.0, 1000.0 and 5000.0 µg/plate for Experiment 1 and 62.5, 125.0, 250.0, 500.0 and 1000.0 µg/plate for Experiment 2.

Negative controls comprised treatments with sterile DMSO, either un-irradiated or exposed to the chosen doses of unfiltered or glass-filtered UV light. Further negative controls comprised the photoactivating positive control, 8-methoxypsoralen (8-MOP), but in the absence of light treatment. The positive control chemicals were 8-MOP in the presence of light and 4-nitroquinoline1-oxide (NQO) in the absence of light.

In Experiment 1, precipitation of the test material was seen at 5000 and 1000 µg/plate. No significant increase in revertant colonies was seen at any dose when treated in the absence or presence of UV light.

For the second experiment, in which a top, precipitating dose of 1000 µg/plate and reduced dose intervals were used, no significant increase in the number of mutants was seen for any light treatment.

Negative control (solvent with or without light treatment, 8-MOP without light, test material without light) plates all gave low mean numbers of revertants. The mean number of revertants on the positive control (NQO, 8-MOP with light) plates were significantly elevated.

Under the conditions of the study the test material caused no reproducible increase in the reversion of E.coli WP2 to tryptophan independence when exposed to 2 doses of unfiltered and glass filtered UV light. It is concluded that the test material is not photomutagenic.

Dean (1993, CHO UV light)

The test material was investigated for its potential to cause gene mutations in cultured CHO cells with and without ultraviolet light under GLP conditions. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was tested for toxicity in CHO cells in a range-finder experiment using final concentrations of 7.813, 15.63, 31.25, 62.5, 125, 250 and 500 µg/mL. The top three doses showed persistent precipitate. Mitotic inhibition was seen at concentrations of 62.5 µg/mL and above and 200 µg/mL was selected as the top dose concentration for the main study.

Part of Main study 1 was repeated in Main study 2 in order to achieve levels of mitotic inhibition consistent with demonstrating toxicity at the highest doses scored. For main study 1 final concentrations ranged from 3.125 to 200 µg/mL and for main study 2 from 10 to 100 µg/mL of test material in DMSO. Negative controls were treated with the solvent and 8-methoxypsoralen (8-MOP) but without UV irradiation. The positive controls were 8-MOP with irradiation and 4-nitroquinoline 1-oxide (NQO).

Concentrations of 50 µg/mL test material and above showed precipitation of the test material in the culture medium. Treatment of cultures with concentrations up to 50 µg/mL of test material in the presence of 200 mJ/cm2 UVA, 32 mJ/cm2 UVB (unfiltered) or 60 µg/mL in the presence of 700 mJ/cm2 UVA (glass filtered) did not show significant increases in the incidence of chromosome aberrations in CHO cells, compared with concurrent controls.

In the main study experiments the un-irradiated solvent control cultures showed frequencies of chromosome aberrations within the historical control ranges, as did the un-irradiated 8-MOP control and the un-irradiated test material cultures. The positive control treatments (NQO and 8-MOP plus light) induced large increases in the incidence of chromosome aberrations indicating that the test system was functioning.

Under the conditions of this study, when treated up to a precipitating concentration which also significantly reduced the mitotic index, the test material did not induce structural aberrations at a frequency significantly greater than the concurrent control values following exposure to unfiltered or glass filtered UV-light. It is concluded that the test material is not photoactivated to a clastogenic form.

IN VIVO

San Sebastian (1987, Micronucleus test))

An assay was conducted to evaluate the potential of the test material to induce micronuclei in mice following intraperitoneal administration using methodology equivalent to the standardised guideline OECD 474 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

In a preliminary dose range finding test the test material was administered intraperitoneally at dose levels of 500, 1500, 3000, 4000 and 5000 mg/kg of bodyweight. Although some clinical signs were observed (decreased body tone, abnormal gait and body drop) the 5000 mg/kg of bodyweight dose was selected for the definitive study.

In the micronucleus test three groups of ten animals (5 males and 5 females per group) were given single doses of the test material at 5000 mg/kg in corn oil by intraperitoneal injection and sacrificed at 30, 48 or 72 hours. Similar groups (5 males and 5 females) were dosed with positive (triethylenemelamine) or vehicle (corn oil) controls and sacrificed at 30 and 48 hours respectively. Slides were prepared from the bone marrow of the femora and stained. Coded slides were scored for the number of polychromatic erythrocytes (PCE) with micronuclei in 1000 PCE per animal. The ratio of polychromatic to normochromatic erythrocytes was determined for each animal.

No statistically significant increases in the incidence of micronucleated PCE were detected in animals administered the test material, at any of the sacrifice times evaluated. No statistically significant differences were observed in the PCE/NCE ratio in any group of animals administered the test material.

Animals treated with the positive control gave a statistically significant increase in the incidence of micronucleated PCE and a statistically significant depression of the PCE/NCE ratio.The study fulfils the criteria of a valid test.

The test material was negative in the micronucleus test at 5000 mg/kg of bodyweight. These findings are based upon the inability of the test material to produce a significant increase in the incidence of micronuclei per 1000 polychromatic erythrocytes per animal in the treated groups versus the vehicle control group. 

Under the conditions of the study, the test material is not considered to be a clastogenic agent.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to genetic toxicity.