Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Negative results in Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537 and E. coli WP2 uvr A pKM 101 with and without metabolic activation (OECD 471).
Negative results in mammalian chromosomal aberration test in human lymphocytes (OECD 473).
Negative results in mammalian gene mutation test in L5178Y mouse lymphoma cells (OECD 476).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 Jun - 7 Jul 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes: cultured peripheral human lymphocytes
Details on mammalian cell type (if applicable):
- Type and identity of media:
RPMI 1640 supplemented with
- 10% fetal calf serum (FCS)
- penicillin / streptomycin (20 IU/mL / 20 µg/mL)
- 2 mM glutamine
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
First experiment
3 h treatment: 18.3, 36.6, 73.1, 146.3, 292.5, 585, 1170 and 2340 µg/mL without metabolic activation (for mitotic index data)
3 h treatment: 292.5, 585 and 1170 µg/mL with and without metabolic activation

Second experiment
21 h treatment: 146.3, 585, 1170 and 2340 µg/mL without metabolic activation
3 h treatment: 585, 1170 and 2340 µg/mL with metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle and exposure concentrations: the test substance was found to form a dosable suspension in DMSO at 100 mg/mL. On dosing at 1% (v/v) into aqueous tissue culture medium, giving a final concentration of 1000 µ/mL, a precipitate was observed. A precipitate was also observed at 125 µg/mL, but was soluble after 3 h incubation. Concentrations with high ionic strength and osmolality may cause chromosomal aberrations (Galloway et al., 1987). Therefore, concentrations greater than 5000 µg/mL or 10 mM are not used in this test system.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
cyclophosphamide, 25 µg/mL in water (3 h exp), +S9; mitomycin C, 0.8 µg/mL in water (3 and 21 h exp), -S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 3 and 21 h
- Fixation time (start of exposure up to fixation or harvest of cells): 3 h treatment: 21 h; 21 h treatment: 21 h
SPINDLE INHIBITOR (cytogenetic assays): colcemid (Sigma) 0.1 µg/mL medium
STAIN (for cytogenetic assays): Giemsa 10% (v/v) in buffered water (pH 7.2)
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: 100 per culture
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 1000 cells (except for positive control treated cultures; only dose level causing a decrease in mitotic index of at least 50% of the solvent control or if there was no decrease, the maximum concentration was used as the highest dose level for the metaphase analysis)
OTHER EXAMINATIONS:
- Determination of polyploidy: yes
Evaluation criteria:
A test substance was considered positive (clastogenic) in the chromosome aberration test if:
It induced a dose-related statistically significant increase (p < 0.01) in the frequency of metaphases with aberrant chromosomes (excluding gaps) at one or more test concentrations.
The increases exceed the negative control range of the laboratory, taken at the 99% confidence limit.
The increases are reproducible between replicate cultures.
The increases are not associated with large changes in osmolality of the treatment medium or extreme toxicity.
Evidence of a dose-relationship is considered to support the conclusion.
A negative response is claimed if no statistically significant increases in the number of aberrant cells above concurrent control frequencies are observed, at any dose level.
A further evaluation may be carried out if the above criteria for a positive or a negative response are not met.
Statistics:
Fisher´s test, p < 0.01, p < 0.001
Species / strain:
lymphocytes: cultured peripheral human lymphocytes
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:
COMPARISON WITH HISTORICAL CONTROL DATA:
In the second experiment (-S9), the test material caused a small statistically significant increase in the proportion of cells with chromosomal aberrations at 1170 µg/mL in comparison to the solvent control. This increase, to 3.5% (p < 0.01), lied just outside the upper 99% confidence limit of the historical control range (2.5%). However, it lied just within the upper limit (3.68%) when not applying the 99% level. To further investigate this response, a higher dose level (2340 µg/mL) was subsequently analysed. As no response was seen at this higher dose level, the increase was not considered indicative of a clastogenic response.

Table 1. Results of Experiment 1 and 2.

Test item

Concentration

Mitotic Index

Aberrant cells in %

 

in µg/mL

in %

with gaps

without gaps

Experiment 1
Exposure period 3 h, fixation time 21 h, without S9 mix

DMSO

 

100

0

0

MMC

0.8

-

20***

20***

Test substance

292.5

86

0

0

585

53

2

2

1170

48

0.5

0.5

Exposure period 3 h, fixation time 21 h, with S9 mix

DMSO

 

100

0,5

0,5

CP

25

-

34***

34***

Test substance

585

91

1

1

1170

66

1

1

2340

49

1

1

Experiment 2
Exposure period 21 h, fixation time 21 h, without S9 mix

DMSO

 

100

0.5

0

MMC

0.8

-

30***

30***

Test substance

146.3

90

3

1

585

51

4

2

1170

51

5**

3.5**

2340

56

4

2

Exposure period 3 h, fixation time 21 h, with S9 mix

DMSO

 

100

0,5

0.5

CP

25

-

1

0.5

Test substance

585

93

1

0.5

1170

89

1,5

1

2340

62

27***

25***

MMC: Mitomycin C; CP: Cyclophosphamide (positive controls)

**: p < 0.01, ***: p < 0.001

Table 2. Mitotic index data of Experiment 1 and 2.

Concentration of test substance (µg/mL)

 

DMSO

18.3

36.6

73.1

146.3

292.5

585

1170

2340

Exposure period 3 h, fixation time 21 h, without S9 mix

Relative mitotic index (%)

100

107

90

96a

78a

86a

53a

48b

43b

Exposure period 3 h, fixation time 21 h, with S9 mix

Relative mitotic index (%)

100

89

82

79a

90a

87a

91a

66b

49b

Exposure period 21 h, fixation time 21 h, without S9 mix

Relative mitotic index (%)

100

n.d.

n.d.

86

90a

48a

51a

51b

56b

Exposure period 3 h, fixation time 21 h, with S9 mix

Relative mitotic index (%)

100

n.d.

n.d.

n.d.

n.d.

84a

93b

89b

62b

a: Precipitate apparent on dosing, not apparent at end of treatment (3 h)

b: Precipitate apparent on dosing, still apparent at end of treatment (3 h)

Conclusions:
Interpretation of results: negative
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 Jul - 11 Dec 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom
Type of assay:
mammalian cell gene mutation assay
Target gene:
TK locus
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, 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)
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone
Test concentrations with justification for top dose:
Preliminary toxicity test
With and without S9 mix: 9.14, 18.28, 36.56, 73.13, 146.25, 292.5, 585, 1170 and 2340 µg/mL (4 h)
Without S9 mix: 9.14, 18.28, 36.56, 73.13, 146.25, 292.5, 585, 1170 and 2340 µg/mL (24 h)
Experiment I
Without S9 mix: 0.63, 1.25, 2.5, 5, 10, 20, 40 and 60 µg/mL (4 h)
With S9 mix: 20, 40, 60, 80, 100, 120, 140 and 160 µg/mL (4 h)
Experiment II
Without S9 mix: 0.63, 1.25, 2.5, 5, 10, 20, 30, 40 and 60 µg/mL (24 h)
With S9 mix: 10, 20, 40, 60, 80, 100, 120 and 140 µg/mL (4 h)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: following solubility checks, the test item was accurately weighed and formulated in dimethyl sulphoxide (DMSO).
- Other: the solvent acetone was used as the vehicle control.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: cyclophosphamide, 2 µg/mL, with S9; ethylmethanesulphonate, 400 µg/mL (4 h), 150 µg/mL (24 h), without S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
1st experiment: 4 h exposure with and without S9 mix.
2nd experiment: 4 h exposure with S9 mix and 24 h without S9 mix.
- Expression time (cells in growth medium): On Day 2 of the experiment, the cells were counted, diluted to 10E4 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL 5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium.
- Selection time (if incubation with a selection agent): 10-14 days

SELECTION AGENT (mutation assays): 4 µg/mL TFT

NUMBER OF REPLICATIONS: duplicates each in two independent experiments in 96-well microtitre plates

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency and relative total growth

OTHER EXAMINATIONS:
- Other: small and large colonies were differentiated, as small colonies represent large genetic changes involving chromosome 11b (indicative of clastogenic activity).
Evaluation criteria:
For a test item 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. Following discussions at an International Workshop on Genotoxicity Test Procedures in Plymouth, UK, 2002 it was felt the IMF must exceed some value based on the global background MF for each method (agar or microwell). This Global Evaluation Factor (GEF) value was set at 126 x 10E-6 for the microwell method. Therefore, any test item dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126 x 10E-6 and demonstrates a positive linear trend will be considered positive.
Statistics:
The experimental data was analysed using a computer program (Mutant 240C, York Electronic Research) which follows the statistical guidelines recommended by the UKEMS.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at ≥ 20 µg/mL (4 h, -S9); ≥ 40 µg/mL (4 h, +S9) and ≥ 10 µg/mL (24 h, -S9)
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: there was no marked change in pH when the test item was dosed into media.
- Effects of osmolality: the osmolality did not increase by more than 50 mOsm in the solubility test.
- Precipitation: a greasy oily precipitate was observed at and above 585 µg/mL in the preliminary toxicity test.

RANGE-FINDING/SCREENING STUDIES: In all three of the exposure groups (with and without S9-mix (4 h) and without S9-mix (24 h)) there was evidence of marked dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls. From 146.25 µg/mL no or hardly any cell survival was observed during short term exposure. Without S-9 mix (24 h), 0% RSG was observed from 73.13 µg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA:
- HIstorical control values of the vehicle control aceton:
with metabolic acitivation 80.37-193.81 mutants per 1E+06 surviving cells
without metabolic acitivation 89.57-169.64 mutants per 1E+06 surviving cells

 Table 1: Experiment I - 4 h exposure - Without Metabolic Activation

Concentration

Relative Total Growth [%]

RTG

Mutants per 1E+06 surviving cells

[µg/mL]

0 (aceton)

100

1

149.83

0.63

107

1.16

127.46

1.25

112

0.95

176.83

2.5

98

0.89

159.43

5

99

0.95

157.31

10

84

0.8

150.74

20

60

0.56

192.46

40

1

-

-

60

0

-

-

EMS, 400

79

0.51

1060.03

EMS = Ethylmethanesulphonate

Table 2: Experiment I - 4 h exposure - With Metabolic Activation

Concentration

Relative Total Growth [%]

RTG

Mutants per 1E+06 surviving cells

[µg/mL]

0 (aceton)

100

1

183.23

20

92

1.03

164.05

40

81

0.87

175.58

60

69

0.68

167.72

80

53

0.57

185.44

100

21

0.21

232.47

120

6

0.03

364.1

140

2

-

-

160

2

-

-

CP, 2

49

0.25

1631.36

CP = Cyclophosphamide

Table 3: Experiment II - 24 h Exposure - Without Metabolic Activation

Concentration

Relative Total Growth [%]

RTG

Mutants per 1E+06 surviving cells

[µg/mL]

0 (aceton)

100

1

122.09

0.63

96

1.02

107.09

1.25

84

0.85

115.48

2.5

70

0.9

94.05

5

57

0.89

66.83

10

31

0.5

135.92

20

19

0.34

149.46

40

6

-

-

60

1

-

-

EMS, 400

38

0.37

1285.3

EMS = Ethylmethanesulphonate

Table 4: Experiment II - 4 h exposure - With Metabolic Activation

Concentration

Relative Total Growth [%]

RTG

Mutants per 1E+06 surviving cells

0 (aceton)

100

1

91.27

10

92

0.93

123.48

20

83

0.84

92.73

40

70

0.56

68.92

60

44

0.33

88.43

80

11

0.05

129.9

100

1

-

-

120

1

-

-

140

1

-

-

CP, 2

64

0.42

1112.23

CP = Cyclophosphamide

Conclusions:
Interpretation of results: negative
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 Feb - 07 Feb 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japan Ministry of Agriculture, Forestry and Fisheries. (1985) NohSan No. 4200
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon (for S. typhimurium strains)
trp operon (for E. coli strain)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254.
Test concentrations with justification for top dose:
First experiment: 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation
Second experiment: 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test substance at 50 mg/mL was not miscible in water. At 50 mg/mL in DMSO, the test substance was miscible. Therefore DMSO (AnalR grade, Lot no. 9899176468, Fisher Scientific, Loughborough, UK) was used.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide (0.5 µg/plate in DMSO, -S9, TA 1535 and TA 100); 2-Aminoacridine (30 µg/plate in DMSO, -S9, TA 1537); 2-Nitrofluorene (1 µg/plate in DMSO, -S9, TA 98); 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (0.05 µg/plate in DMSO, -S9, WP2uvr A/pKM101)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2 and 10 µg/plate in DMSO, +S9, TA 1535 and WP2uvr A/pKM101); Benzo[a]pyrene (5 µg/plate in DMSO, +S9, TA 1537, TA 98 and TA 100)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation, first experiment), preincubation (second experiment)

DURATION
- Exposure duration: 72 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments (except the concentrations 5 and 15 µg/plate were tested only once)

DETERMINATION OF CYTOTOXICITY
- Method: inspection of the bacterial background lawn

Evaluation criteria:
The mean number of revertant colonies for all treatment groups is compared with those obtained for the solvent control groups. The mutagenic activity of a test substance is assessed by applying the following criteria:
If treatment with a test substance produces an increase in revertant colony numbers of at least twice the concurrent solvent controls, with some evidence of a positive dose-relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S9 mix, it is considered to show evidence of mutagenic activity in this system.
If treatment with a test substance does not produce reproducible increases of at least 1.5 times the concurrent solvent controls, in either mutation test, it is considered to show no evidence of mutagenic activity in this test system.
If the results obtained fail to satisfy the criteria for a clear positive or negative response, additional testing may be performed. Should an increase in revertant colony numbers then be observed which satisfies the criteria, the substance is considered to show evidence of mutagenic activity in this system.
If no clear positive response can be obtained, the test data may be subjected to analysis to determine the statistical significance of any observed increases in revertant colony numbers. The statistical procedures used will be the analysis of variance followed by Dunnett´s test. Biological significance should always be considered along with statistical significance.
Statistics:
The statistical procedures used will be the analysis of variance followed by Dunnett´s test.
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Table 1. Results of Experiment 1 (plate incorporation).

With or without S9-Mix

Test substance concentration

Mean number of revertant colonies per plate

(μg/plate)

(average of 3 plates ± Standard deviation)

 

Base-pair substitution type

Frameshift type

 

TA 1535

TA100

E coli

TA98

TA1537

Solvent

19 ± 2

112 ± 3

185 ± 9

19 ± 2

10 ± 4

5

17 ± 3

90 ± 5

161 ± 7

18 ± 4

10 ± 2

15

12 ± 2

94 ± 6

219 ± 3

23 ± 0

9 ± 1

50

11 ± 1

100 ± 11

186 ± 4

24 ± 1

6 ± 4

150

13 ± 3

105 ± 2

193 ± 16

19 ± 4

9 ± 2

500

15 ± 2

83 ± 5

192 ± 7

21 ± 3

6 ± 1

1500

18 ± 4

93 ± 9

194 ± 22

19 ± 1

7 ± 2

5000

15 ± 1

98 ± 19

184 ± 8

17 ± 4

5 ± 3

Positive controls, –S9

Name

NaAz

NaAz

AF-2

NF

9AC

Concentrations (μg/plate)

0.5

0.5

0.05

1

30

Mean No. of colonies/plate (average of 3 ± SD)

146 ± 4

454 ± 15

1911 ± 18

191 ± 31

67 ± 5

+

Solvent

15 ± 2

122 ± 5

185 ± 11

27 ± 1

12 ± 4

+

5

11 ± 4

109 ± 14

191 ± 23

27 ± 2

9 ± 2

+

15

14 ± 3

112 ± 8

197 ± 18

26 ± 3

13 ± 2

+

50

16 ± 3

107 ± 1

202 ± 18

30 ± 5

12 ± 1

+

150

16 ± 2

116 ± 16

207 ± 19

25 ± 8

11 ± 3

+

500

18 ± 4

122 ± 11

216 ± 20

21 ± 3

12 ± 0

+

1500

19 ± 6

114 ± 6

184 ± 10

30 ± 2

13 ± 2

+

5000

15 ± 0

93 ± 4

153 ± 22

18 ± 3

9 ± 2

Positive controls, –S9

Name

2AA

BP

2AA

BP

BP

Concentrations (μg/plate)

2

5

10

5

5

Mean No. of colonies/plate (average of 3 ± SD)

211 ± 9

814 ± 38

1856 ± 26

304 ± 20

113 ± 9

NaAz = Sodium azide

9AC = 9-Aminoacridine

2AA = 2-Aminoanthracene

BP = Benzo(a)pyrene

NF = 2-Nitrofluorene

AF-2 = 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

Table 2. Results of Experiment 2 (preincubation).

With or without S9-Mix

Test substance concentration

Mean number of revertant colonies per plate

(μg/plate)

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA 1535

TA100

E coli

TA98

TA1537

Solvent

13 ± 5

94 ± 14

176 ± 7

25 ± 3

11 ± 2

50

11 ± 5

84 ± 7

186 ± 6

20 ± 2

11 ± 3

150

14 ± 5

91 ± 9

178 ± 9

20 ± 2

7 ± 2

500

15 ± 5

97 ± 11

204 ± 2

19 ± 4

6 ± 1

1500

14 ± 4

99 ± 9

146 ± 11

24 ± 2

7 ± 4

5000

15 ± 3

96 ± 6

179 ± 13

27 ± 5

9 ± 1

Positive controls, –S9

Name

NaAz

NaAz

AF-2

NF

9AC

Concentrations (μg/plate)

0.5

0.5

0.05

1

30

Mean No. of colonies/plate (average of 3 ± SD)

165 ± 18

476 ± 16

2170 ± 56

209 ± 18

192 ± 21

+

Solvent

16 ± 3

93 ± 9

176 ± 10

28 ± 3

14 ± 3

+

50

17 ± 1

79 ± 2

195 ± 12

25 ± 2

12 ± 2

+

150

16 ± 2

86 ± 11

185 ± 8

25 ± 7

12 ± 4

+

500

14 ± 4

89 ± 6

199 ± 12

28 ± 2

11 ± 2

+

1500

13 ± 2

72 ± 13

146 ± 36

32 ± 1

10 ± 3

+

5000

11 ± 2

80 ± 19

158 ± 6

21 ± 4

13 ± 3

Positive controls, –S9

Name

2AA

BP

2AA

BP

BP

Concentrations (μg/plate)

2

5

10

5

5

Mean No. of colonies/plate (average of 3 ± SD)

124 ± 32

726 ± 4

1515 ± 15

288 ± 3

100 ± 5

NaAz = Sodium azide

9AC = 9-Aminoacridine

2AA = 2-Aminoanthracene

BP = Benzo(a)pyrene

NF = 2-Nitrofluorene

AF-2 = 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

Conclusions:
Interpretation of results: negative
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A bacterial gene mutation assay (Ames test) was performed with 2-ethylhexyl benzoate following OECD guideline 471 in compliance with GLP. The tested strains were Salmonella typhimurium TA 98, TA 100, TA 1537 and TA 1538 and E. coli WP2 uvr A pKM 101 (Kitching, 2000).

The experiment was performed according to the plate incorporation and preincubation procedure at concentrations from 5-5000 µg/plate with and without a metabolic activation system. Cytotoxicity was determined by inspection of the bacterial background lawn.

The included positive and negative controls in the experiments showed the expected results and were therefore considered as valid. No increase in the number of revertant colonies was noted in any of the bacterial strains, with and without metabolic activation system.

No cytotoxicity was observed up to the highest dose tested.

Furthermore, a short abstract of an Ames test with 2-ethylhexyl benzoate is available. The study was performed in the tester strains TA 98 and TA 100 equivalent to OECD guideline 471 (Callander, 2001).

Concentrations of 100-5000 µg/plate of the test material were evaluated with and without metabolic activation.

In both strains, a negative result with and without metabolic activation system was noted.

In summary, the test substance did not induce mutations in the bacterial mutation tests in the absence and presence of a metabolic activation system in any of the strains tested.

An in-vitro mammalian chromosome aberration test was conducted with 2-ethylhexyl benzoate in accordance with OECD guideline 473 under GLP conditions (Akhurst, 2000).

The induction of structural chromosome aberrations was evaluated in human lymphocytes, in-vitro incubated for 3 and 21 h with and without a metabolic activation system. Concentrations of 18.3-2340 µg/mL of the test substance in the vehicle DMSO were applied.

The used negative and positive controls showed the expected results and were considered valid.

In the experiments both with and without metabolic activation, a systematic influence of the test substance was observed, which led to a reduction in the mitotic index. In the second experiment without metabolic activation, the test material caused a small statistically significant increase in the proportion of cells with chromosomal aberrations at 1170 µg/mL in comparison to the solvent control. No response was seen at the higher dose level of 2340 µg/mL, therefore, the increase was not considered indicative of a clastogenic response. Thus, no statistically and biologically significant increase in the incidence of chromosome aberrations was observed under the conditions of the study.

Under the conditions of the study, the test substance did not show clastogenic activity in this chromosomal aberration test performed in human lymphocytes in-vitro.

An in vitro mammalian cell gene mutation study was carried out with 2-ethylhexyl benzoate according to OECD guideline 476 under GLP conditions (Brown, 2013). Gene mutations in the thymidine kinase locus were investigated in L5178Y mouse lymphoma cells in the presence and absence of a metabolic activation system. Concentrations of the first experiment without metabolic activation for an exposure time of 4 h ranged from 0.63-60 µg/mL and with metabolic activation (4 h) from 20-160 µg/mL. The second experiment was conducted with concentrations of 0.63-60 µg/mL without metabolic activation (24 h) and from 10-140 µg/mL with metabolic activation (4 h). The vehicle and positive controls in the study showed the expected results and were within the range of historical control data. Marked dose-related reductions in the Relative Suspension Growth of cells treated with the test item at concentrations from ≥ 20 µg/mL (4 h, -S9 -mix), ≥ 40 µg/mL (4 h, +S9 -mix) and ≥ 10 µg/mL (24 h, +S9-mix) were observed. There was no significant increase in the number of forward mutations at the thymidine kinase locus of L5178Y mouse lymphoma cells treated with the test material, neither in the presence nor in the absence of a metabolic activation system. Under the conditions of the study 2-ethylhexyl benzoate did not show gene mutation activity in L5178Y mouse lymphoma cells in vitro.


Justification for classification or non-classification

The available data on the genetic toxicity of 2-ethylhexyl benzoate do not meet the criteria for classification according to Regulation (EC) 1272/2008, and the data are therefore conclusive but not sufficient for classification.