Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro genetic toxicity:

Bacterial reverse mutation assay (Ames test / OECD 471): negative with and without metabolic activation (RA from CAS 131459-39-7 and (Q)SAR prediction)

In vitro chromosome aberration test in human lymphocytes (CA / OECD 473): negative with and without metabolic activation (RA from CAS 131459-39-7 and (Q)SAR prediction)

In vitro gene mutation assay in mouse lymphoma cells (MLA / OECD 476): negative with and without metabolic activation (RA from CAS 15834-04-5 and (Q)SAR prediction)

Link to relevant study records

Referenceopen allclose all

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:
8. Dec. 1998 - 19. Jan. 1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see "remarks"
Remarks:
GLP-Guideline study. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (Dec 2012)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.
Justification for type of information:
refer to analogue justification provided in IUCLID section 13
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
GLP compliance:
yes (incl. QA statement)
Remarks:
Safepharm Laboratories Limited, Derby, UK
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon and trp operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: incapable of DNA excision repair (uvrB-); increased permeability of the cell wall (rfa)
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of Sprague-Dawley rats treated with Aroclor 1254
Test concentrations with justification for top dose:
50, 150, 500, 1500, 5000 µg/plate (first and second experiment)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: without S9: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG; 3 µg/plate TA100, 5 µg/plate TA1535, 2 µg/plate WP2uvrA-), 9-Aminoacridine (9-AA; 80 µg/plate TA 1535), 4-Nitroquinoline-1 (4NQO; 0.2 µg/plate TA98)
Positive controls:
yes
Positive control substance:
other: with S9 mix: 2-Aminoanthracene (2AA; 1 µg/plate TA 100, 2 µg/plate TA 1535 and TA 1537, 10 µg/plate WP2uvrA-); Benzo(a)pyrene (BP; 5 µg/plate TA 98)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: inspection of bacterial background lawn, number of revertant colonies
Evaluation criteria:
The test material may be considered to be positive in this test system if the following criteria are met: All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (see table 1); Each positive control value was at least two times the respective vehicle control value for each strain (for historical values see table 2); A minimum of 4 non-toxic dose levels were achieved; The test material should have induced a reproducible, dose-related and statistically significant increase in the revertant count in at least one strain of bacteria.
Statistics:
Dunett's method of linear regression, Kirkland DJ (1989), Statistical Evaluation of Mutagenicity Test Data. UKEMS Sub-comittee on Guidelines for Mutagenicity Testing. Report - Part III - Cambridge University Press
Species / strain:
other: 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, but tested up to precipitating concentrations
Remarks:
oily precipitate at 5000 µg/plate which did not prevent scoring of revertant colonies
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
oily precipitate at 5000 µg/plate which did not prevent scoring of revertant colonies
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: oily precipitate at 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES: the dose range of the test material used in the preliminary toxicity study was 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate. The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-).

COMPARISON WITH HISTORICAL CONTROL DATA: Values of positive and negative controls were in range of historical control data.

Table 3. Test results of experiment 1 (plate incorporation).

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA 100

TA1535

WP2uvrA-

TA98

TA1537

0

74 ± 9.6

21 ± 2.3

22 ± 2.0

23 ± 4.0

8 ± 2.0

50

75 ± 7.1

20 ± 2.1

24 ± 2.1

24 ± 4.0

6 ± 2.0

150

79 ± 13.1

20 ± 6.8

22 ± 1.5

19 ± 2.6

10 ± 1.0

500

76 ± 4.2

15 ± 4.0

23 ± 7.0

21 ± 4.9

9 ± 3.8

1500

71 ± 6.1

20 ± 3.8

18 ± 5.5

21 ± 5.7

10 ± 3.5

5000

73 ± 1.5 P

21 ± 5.5 P

24 ± 6.0 P

21 ± 5.9 P

9 ± 2.5 P

Positive controls, –S9

Name

ENNG

ENNG

ENNG

4NQO

9AA

Concentrations

(μg/plate)

3

5

2

0.2

80

Mean No. of colonies/plate

(average of 3 ± SD)

323 ± 11.7

283 ± 10.0

503 ± 24.2

166 ± 5.3

1027 ± 358.2

+

0

87 ± 1.5

16 ± 1.5

28 ± 2.3

34 ± 6.7

18 ± 0.6

+

50

94 ± 6.1

13 ± 1.2

29 ± 5.8

31 ± 0.6

23 ± 7.4

+

150

88 ± 4.0

13 ± 2.6

25 ± 5.6

35 ± 6.4

19 ± 7.0

+

500

87 ± 3.5

12 ± 1.2

29 ± 3.8

32 ± 9.5

20 ± 5.5

+

1500

81 ± 6.5

10 ± 0.6

30 ± 3.6

33 ± 3.8

15 ± 4.6

+

5000

77 ± 5.1 P

12 ± 0.6 P

30 ± 2.6 P

31 ± 4.7 P

18 ± 3.8 P

Positive controls, +S9

Name

2AA

2AA

2AA

BP

2AA

Concentrations

(μg/plate)

1

2

10

5

2

Mean No. of colonies/plate

(average of 3 ± SD)

920 ± 153.0

198 ± 9.3

555 ± 27.8

511 ± 132.0

196 ± 6.5

ENNG = N-ethyl-N-nitro-N-nitrosoguanidine

4NQO = 4-nitroquinoline-N-oxide

9AA = 9-aminoacridine

2AA = 2 -aminoanthracene

BP = Benzo(a)pyrene

P = Precipitate

Table 4. Test results of experiment 2 (plate incorporation).

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA 100

TA1535

WP2uvrA-

TA98

TA1537

0

103 ± 5.3

26 ± 3.8

31 ± 8.5

29 ± 7.0

12 ± 2.9

50

105 ± 10.8

21 ± 4.9

30 ± 7.4

28 ± 7.1

11 ± 1.0

150

97 ± 7.0

26 ± 1.0

35 ± 2.9

22 ± 1.2

13 ± 3.5

500

104 ± 9.1

25 ± 4.6

34 ± 5.1

26 ± 5.6

15 ± 1.7

1500

110 ± 9.5

23 ± 6.0

25 ± 0.6

27 ± 3.8

13 ± 1.7

5000

112 ± 5.7 P

28 ± 0.6 P

35 ± 2.6 P

20 ± 1.0 P

11 ± 1.5 P

Positive controls, –S9

Name

ENNG

ENNG

ENNG

4NQO

9AA

Concentrations

(μg/plate)

3

5

2

0.2

80

Mean No. of colonies/plate

(average of 3 ± SD)

880 ± 31.5

257 ± 34.4

1191 ± 59.3

166 ± 4.6

1384 ± 263.4

+

0

103 ± 8.6

13 ± 3.6

37 ± 2.1

27 ± 6.2

20 ± 3.8

+

50

92 ± 21.0

12 ± 1.5

33 ± 4.0

27 ± 7.2

20 ± 2.6

+

150

91 ± 7.5

14 ± 1.5

34 ± 2.6

29 ± 4.0

22 ± 2.6

+

500

113 ± 15.0

14 ± 2.3

39 ± 2.1

27 ± 6.7

15 ± 3.2

+

1500

110 ± 7.6

13 ± 2.6

36 ± 3.2

31 ± 4.9

21 ± 3.1

+

5000

97 ± 9.0 P

17 ± 5.8 P

30 ± 2.5 P

28 ± 4.7 P

22 ± 3.0 P

Positive controls, +S9

Name

2AA

2AA

2AA

BP

2AA

Concentrations

(μg/plate)

1

2

10

5

2

Mean No. of colonies/plate

(average of 3 ± SD)

865 ± 43.2

246 ± 7.5

1051 ± 90.0

611 ± 51.5

182 ± 7.0

ENNG = N-ethyl-N-nitro-N-nitrosoguanidine

4NQO = 4-nitroquinoline-N-oxide

9AA = 9-aminoacridine

2AA = 2 -aminoanthracene

BP = Benzo(a)pyrene

P = Precipitate
Conclusions:
negative
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:
01 Feb - 21 Apr 1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions (no data on analytical purity).
Justification for type of information:
refer to analogue justification provided in IUCLID section 13
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
No details on the purity of the test substance
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
No details on the purity of the test substance
GLP compliance:
yes (incl. QA statement)
Remarks:
The Department of Health of the Government of the United Kingdom
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes:
Metabolic activation:
with and without
Metabolic activation system:
Arochlor 1254 induced S9-Mix from male Spraque-Dawley rats
Test concentrations with justification for top dose:
1. Experiment:
4(20) h without S9: 39.06; 78.13; 156.25; 312.5; 625; 1250; 2500; 5000 µg/mL
4(20) h with S9: 39.06; 78.13; 156.25; 312,5; 625; 1250; 2500; 5000 µg/mL

2. Experiment
24 h without S9: 39.06; 78.13; 156.25; 312,5; 625; 1250; 2500; 5000 µg/mL
4(20) h with S9: 39.06; 78.13; 156.25; 312,5; 625; 1250; 2500; 5000 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
in absence of S9-Mix 750 µg/mL
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
in presence of S9-Mix 25 µg/mL
Details on test system and experimental conditions:
Duplicate cultures of human lymphocytes were evaluated for chromosome aberrations at up to three dose levels. For each experiment, whole blood was drawn from the peripheral circulation of a volunteer who had previously been screened for suitability. The cell cycle length of approx. 14 h was determined by BrdU incorporation.
METHOD OF APPLICATION: in medium (Eagle´s minimal essential medium with HEPES buffer supplemented with L-glutamine, Pen/Strep, amphotericin B and 15% FCS)

DURATION
- Exposure duration: Experiment I: 4 h with or without S9 mix.
- Experiment II: Either 4 h with S9 mix or 24 h without S9 mix.
- Expression time (cells in growth medium): 20 h after 4 h exposure to the test substance and 20 h expression time

SPINDLE INHIBITOR: Colcemid (0.1 µg/mL)
STAIN: 5% Giemsa for 5 min

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 200

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
Evaluation criteria:
All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.
Statistics:
The frequency of cells with aberrations and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher´s Exact test.
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
at and above 1250 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
lymphocytes: human
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
at and above 1250 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Medium was "cloudy" from 156.25 µg/mL and a precipitate was seen from 1250 µg/mL without effects on the toxicity response.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes, data were in range with the historical control data.

Table 1: Experiment 1: 4 h treatment without metabolic activation

Treatment group (µg/ml)

Number of cells scored

Total gaps

Chromatid

Chromosome

others

Total abberations

Abberant cells

Breaks

Exchanges

Breaks

Exchanges

x

(+Gaps)

(+Gaps)

(+Gaps)

(+Gaps)

Vehicle control

200

5

0

1

0

1

0

7

2

7

2

1250

200

5

0

0

3

0

0

8

3

8

3

2500

200

2

2

0

0

0

0

4

2

4

2

5000

200

5

4

0

0

1

0

10

5

9

5

EMS 750

200

24

9

5

5

0

0

43

19

35

17

Table 2: Experiment 1: 4 h treatment with metabolic activation

Treatment group (µg/ml)

Number of cells scored

Total gaps

Chromatid

Chromosome

others

Total abberations

Abberant cells

Breaks

Exchanges

Breaks

Exchanges

x

(+Gaps)

(+Gaps)

(+Gaps)

(+Gaps)

Vehicle control

200

4

1

0

0

0

0

5

1

4

1

1250

200

6

4

0

0

0

0

10

4

9

4

2500

200

4

2

0

0

0

0

6

2

5

2

5000

200

2

4

0

1

0

0

7

5

7

5

CP 25

200

13

12

3

2

0

0

30

17

24

15

Table 3: Experiment 2: 20 h treatment without metabolic activation

Treatment group (µg/ml)

Number of cells scored

Total gaps

Chromatid

Chromosome

others

Total abberations

Abberant cells

Breaks

Exchanges

Breaks

Exchanges

x

(+Gaps)

(+Gaps)

(+Gaps)

(+Gaps)

Vehicle control

200

2

2

1

0

0

0

5

3

5

3

1250

200

7

0

0

1

0

0

8

1

8

1

2500

200

0

1

0

0

0

0

1

1

1

1

5000

200

6

0

0

0

0

0

6

0

6

0

EMS 500

200

53

41

13

2

0

0

109

56

74

48

Table 4: Experiment 2: 4 h treatment with metabolic activation

Treatment group (µg/ml)

Number of cells scored

Total gaps

Chromatid

Chromosome

others

Total abberations

Abberant cells

Breaks

Exchanges

Breaks

Exchanges

x

(+Gaps)

(+Gaps)

(+Gaps)

(+Gaps)

Vehicle control

200

0

0

0

2

0

0

2

2

2

2

1250

200

4

2

2

3

0

0

9

5

7

3

2500

200

5

2

0

1

0

0

8

3

8

3

5000

200

3

2

0

2

0

0

7

4

6

3

CP 25

200

10

10

2

0

0

0

22

12

15

11

The test substance was non-clastogenic to human lymphocytes in vitro.

Conclusions:
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:
26 Mar - 11 May 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see "remarks"
Remarks:
Comparable to guideline study with acceptable restrictions (no data on analytical purity).
Justification for type of information:
refer to analogue justification provided in IUCLID section 13
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
thymidine kinase locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 supplemented with 5% (v/v) heat-inactivated horse serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- 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 a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
First experiment: 0.03, 0.1, 0.3, 1, 3, 10, 33, 100 µg/mL (with and without metabolic activation (8%, v/v))
Second experiment: 0.03, 0.1, 0.3, 1, 3, 10, 33, 100 µg/mL (with metabolic activation (12%, v/v)); 0.1, 1, 3, 10, 33, 100, 200, 250 µg/mL (without metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
in the absence of S9-mix 15 and 5 µg/mL for 3 and 24 h treatment period
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
in the presence of S9-mix 7.5 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension

DURATION
- Exposure duration: cells were exposed to the test material for 3 h and 24 h
- Expression time (cells in growth medium): Cells in the final suspension after treatment were counted with the coulter particle counter. For the expression of the mutant phenotype, the cells were separated by 2 centrifugation steps and cultures for 48 h after the treatment period. Cells were plated for the determination of the cloning efficiency and mutation frequency. For the determination of the mutation frequency cells were plated and incubated for 11-12 d. After that, cells were stained for 2 h by adding 0.5 mg/mL MTT (Sigma) to each well. The plates were scored for cloning efficiency and mutation frequency with the naked eye or with the microscope.

SELECTION AGENT (mutation assays): RPMI 1640 supplemented with 20% (v/v) heat-inactivated horse serum and 5 µg/mL trifluorothymidine (TFT).

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency and relative total growth
Evaluation criteria:
Measurement of cytotoxicity by determining the relative cloning efficiency (survival) or relative total growth of the cultures is usually initiated after the treatment period. There are several criteria for determining a positive result, such as a concentration-related, or a reproducible increase in mutant frequency.
Statistics:
The cloning efficiency (CE) was determined as follows:
P(0)= Number of empty wells divided by the total number of wells
CE= P(0)/number of cells plated per well

Relative survival rate (RS): RS= [CE(test)/CE(control)] x 100
Relative total growth (RTG): RTG= RSG x RSday2 / 100
Suspension growth (SG): [Day 0 cell count/1.25x10E005] x [Day 1 cell count/1.25x10E005] x [Day 2 cell count]
Relative suspension growth (RSG): SG(test)/SG(control) x 100

RSday2= CEday2(test) / CEday2(control) x 100

The growth rate (GR) was calculated for the solvent control cultures:
- 3 h treatment: [Day 1 cell count/1.25x105] x [Day 2 cell count/1.25x10E005]
- 24 h treatment: [Day 0 cell count/1.25x105] x [Day 1 cell count/1.25x10E005] x [Day 2 cell count/1.25x10E005]


The mutation frequency was expressed as the number of mutants per 106 viable cells. The plating efficiencies of both mutant and viable cells (CE day2) in the same culture were determined and the mutation frequency (MF) was calculated as follows:

MF= {-ln P(0)/number of cells plated per well)/CE day2 x 10E-006

Small and large colony mutation frequencies were calculated in an identical manner.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
and above (precipitating concentration: 100 µg/mL, tested up to 250 µg/mL)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at and above 100 µg/mL


RANGE-FINDING/SCREENING STUDIES: Yes, cytotoxicity data was obtained by treating cells for 3 h and 24 h, respectively, with a number of increasing test substance concentrations. The highest concentration tested was 200 µg/ml due to poor solubility of the test substance. No toxicity was observed with and without metabolic activation up to and at the maximum dose level tested with 3 h incubation. 24 h incubation resulted in 64% relative suspension growth in the absence of metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes, all controls were in the range of the historical controls.

Table 1: Experiment 1 - 3 hours with and without S9 mix

Dose

(µg/ml)

RSG

(%)

CE day2

(%)

RS day2

(%)

RTG

(%)

mutation frequency x 10-6

 

 

 

 

 

total

Without metabolic activation, 3 h treatment

SC1

100

94

100

100

89

SC2

108

73

0.03

98

101

100

98

63

0.1

92

99

98

90

83

0.3

111

102

101

112

58

1

107

98

97

104

64

3

110

101

100

110

83

10

98

99

98

96

83

33

98

110

109

106

90

100*

74

94

93

69

97

MMS

70

63

63

44

1022

With 8% (v/v) metabolic activation, 3 h treatment

SC1

100

77

100

100

82

SC2

84

87

0.03

96

90

112

107

71

0.1

92

104

129

119

60

0.3

80

108

135

108

55

1

93

105

131

121

69

3

97

90

112

109

65

10

95

84

104

99

71

33

93

81

101

94

91

100*

42

83

103

43

98

CP

20

37

47

9

1107

 

Table 2: Experiment 2 - 3 hours with and 24 hours without S9 mix

Dose

(µg/ml)

RSG

(%)

CE day2

(%)

RS day2

(%)

RTG

(%)

mutation frequency x 10-6

 

 

 

 

 

total

Without metabolic activation, 24 h treatment

SC1

100

102

100

100

62

SC2

104

57

0.1

97

83

80

78

87

1

94

105

102

96

68

3

102

90

87

89

65

10

104

115

111

115

54

33

105

83

80

84

53

100*

102

98

95

97

55

200*

116

104

101

116

52

250*

112

108

105

118

51

MMS

80

81

79

63

631

With 12% (v/v) metabolic activation, 3 h treatment

SC1

100

77

100

100

60

SC2

91

84

0.03

116

58

69

81

108

0.1

97

80

95

93

86

0.3

94

80

95

90

76

1

99

81

97

96

88

3

102

89

106

108

71

10

104

86

103

106

73

33

119

86

103

122

83

100*

105

77

91

96

72

CP

31

54

64

20

814

 RSG: Relative Suspension Growth; CE: Cloning efficiency; RS: Relative Survival; RTG: Relative Total Growth; SC: Solvent Control (DMSO); MMS: Methylmethansulfonate; CP: Cyclophosphamide

*: Precipitation of test substance

Conclusions:
negative
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions (no details on analytical purity given)
Justification for type of information:
refer to analogue justification provided in IUCLID section 13
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
no analytical purity reported
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon (S. typhimurium) and trp operon (E. coli)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male Sprague Dawley rats treated i.p. with a single dose of 500 mg/kg bw Arochlor 1254
Test concentrations with justification for top dose:
Range-finding toxicity study (in TA 100 and WP2 uvrA): 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3330 and 5000 µg/plate, with and without metabolic activation
Main study (all strains): 33.3, 100, 333, 1000, 3330 and 5000 µg/plate, with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
-S9: 2-NF (1 µg/plate, TA 98); SA (2 µg/plate, TA 100 and TA 1535); ICR-191 (2 µg/plate, TA 1537); 4-NQO (1 µg/plate, WP2 uvrA); +S9: BP (2.5 µg/plate, TA 98); 2-AA (2.5-25 µg/plate, TA 100, TA 1535, TA 1537 and WP2 uvrA)
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-aminoanthracene; ICR-191
Remarks:
2-NF: 2-nitrofluorene; SA: sodium azide; 4-NQO: 4-nitroquinoline-N-oxide; BP: benzo(a)pyrene; 2-AA: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 52 ± 4 h

NUMBER OF REPLICATIONS: triplicates each in one experiment

DETERMINATION OF CYTOTOXICITY
- Method: inspection of bacterial background lawn
Evaluation criteria:
The results of the test were considered positive, if the following criteria were met: tester strains TA 98, TA 100 and WP2 uvrA: for a test article to be considered positive, it must produce at least a 2-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate must be accompanied by a dose response to increasing concentrations of the test article; tester strains TA 1535 and TA 1537: for a test article to be considered positive, it must produce at least a 3-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate must be accompanied by a dose response to increasing concentrations of the test article.
Statistics:
Mean values and standard deviations of revertants per plate were calculated.
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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: in the absence of S9 mix, slight precipitation of the test substance was observed in all experiments at concentrations ≥100 µg/plate. In the presence of S9 mix, slight precipitates were noted at ≥333 µg/plate in the preliminary cytotoxicity study and at ≥1000 µg/plate in the main study.

RANGE-FINDING/SCREENING STUDIES: in a preliminary cytotoxicity study, the tester strains TA 100 and WP2 uvrA were treated with the test substance at concentrations ranging from 6.67 to 5000 µg/plate in the presence and absence of metabolic activation (S9 mix). No cytotoxicity was observed in these strains up to the limit dose of 5000 µg/plate, neither with nor without addition of S9 mix.

Table 1. Test results of experiment (plate incorporation)

Bacterial Reverse Mutation Assay, mean revertant colonies/plate (mutation factor) (n=3 ± SD)

EXPERIMENT

S9-Mix

Without

 

Concentration (per plate)

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

SC

25 ± 2

80 ± 9

14 ± 8

3 ± 1

26 ± 3

Test material

 

33.3 µg

19 ± 4

88 ± 5

12 ± 5

4 ± 4

19 ± 2

100 µg

21 ± 7

96 ± 12

11 ± 2

5 ± 4

19 ± 4

333 µg

22 ± 2

92 ± 9

13 ± 2

5 ± 5

23 ± 10

1000 µg

25 ± 6

97 ± 11

25 ± 2

5 ± 3

30 ± 3

3330 µg

22 ± 1

101 ± 3

10 ± 1

3 ± 2

32 ± 2

5000 µg

20 ± 6

85 ± 6

15 ± 3

4 ± 2

27 ± 3

PC

 

2-NF

206 ± 42

-

-

-

-

SA

-

549 ± 71

480 ± 3

-

-

ICR-191

-

-

-

277 ± 33

-

4-NQO

 -

 -

 -

260 ± 43

S9-Mix

 

With

Concentration (per plate)

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

SC

36 ± 2

102 ± 2

19 ± 1

7 ± 1

25 ± 5

Test material

 

33.3 µg

36 ± 7

93 ± 9

15 ± 2

8 ± 4

22 ± 5

100 µg

34 ± 7

97 ± 17

16 ± 4

9 ± 2

22 ± 6

333 µg

34 ± 6

87 ± 6

17 ± 2

9 ± 4

27 ± 3

1000 µg

39 ± 9

94 ± 17

17 ± 3

8 ± 2

26 ± 6

3330 µg

38 ± 8

92 ± 6

16 ± 4

6 ± 3

29 ± 11

5000 µg

34 ± 5

139 ± 5

20 ± 3

3 ± 2

24 ± 4

PC

 

 

 

 

 

BP

471 ± 14

-

-

-

-

2-AA

-

918 ± 296

124 ± 6

171 ± 32

278 ± 24

SC = Solvent control; PC = Positive control substances; SD = standard deviation;

2-NF: 2-nitrofluorene; SA: sodium azide; 4-NQO: 4-nitroquinoline-N-oxide; BP: benzo(a)pyrene; 2-AA: 2-aminoanthracene

Conclusions:
negative
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
refer to analogue justification provided in IUCLID section 13
Reason / purpose for cross-reference:
read-across source
Target gene:
his operon and trp operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: incapable of DNA excision repair (uvrB-); increased permeability of the cell wall (rfa)
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of Sprague-Dawley rats treated with Aroclor 1254
Test concentrations with justification for top dose:
50, 150, 500, 1500, 5000 µg/plate (first and second experiment)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: without S9: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG; 3 µg/plate TA100, 5 µg/plate TA1535, 2 µg/plate WP2uvrA-), 9-Aminoacridine (9-AA; 80 µg/plate TA 1535), 4-Nitroquinoline-1 (4NQO; 0.2 µg/plate TA98)
Positive controls:
yes
Positive control substance:
other: with S9 mix: 2-Aminoanthracene (2AA; 1 µg/plate TA 100, 2 µg/plate TA 1535 and TA 1537, 10 µg/plate WP2uvrA-); Benzo(a)pyrene (BP; 5 µg/plate TA 98)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: inspection of bacterial background lawn, number of revertant colonies
Evaluation criteria:
The test material may be considered to be positive in this test system if the following criteria are met: All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (see table 1); Each positive control value was at least two times the respective vehicle control value for each strain (for historical values see table 2); A minimum of 4 non-toxic dose levels were achieved; The test material should have induced a reproducible, dose-related and statistically significant increase in the revertant count in at least one strain of bacteria.
Statistics:
Dunett's method of linear regression, Kirkland DJ (1989), Statistical Evaluation of Mutagenicity Test Data. UKEMS Sub-comittee on Guidelines for Mutagenicity Testing. Report - Part III - Cambridge University Press
Species / strain:
other: 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, but tested up to precipitating concentrations
Remarks:
oily precipitate at 5000 µg/plate which did not prevent scoring of revertant colonies
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
oily precipitate at 5000 µg/plate which did not prevent scoring of revertant colonies
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: oily precipitate at 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES: the dose range of the test material used in the preliminary toxicity study was 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate. The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-).

COMPARISON WITH HISTORICAL CONTROL DATA: Values of positive and negative controls were in range of historical control data.

Table 3. Test results of experiment 1 (plate incorporation).

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA 100

TA1535

WP2uvrA-

TA98

TA1537

0

74 ± 9.6

21 ± 2.3

22 ± 2.0

23 ± 4.0

8 ± 2.0

50

75 ± 7.1

20 ± 2.1

24 ± 2.1

24 ± 4.0

6 ± 2.0

150

79 ± 13.1

20 ± 6.8

22 ± 1.5

19 ± 2.6

10 ± 1.0

500

76 ± 4.2

15 ± 4.0

23 ± 7.0

21 ± 4.9

9 ± 3.8

1500

71 ± 6.1

20 ± 3.8

18 ± 5.5

21 ± 5.7

10 ± 3.5

5000

73 ± 1.5 P

21 ± 5.5 P

24 ± 6.0 P

21 ± 5.9 P

9 ± 2.5 P

Positive controls, –S9

Name

ENNG

ENNG

ENNG

4NQO

9AA

Concentrations

(μg/plate)

3

5

2

0.2

80

Mean No. of colonies/plate

(average of 3 ± SD)

323 ± 11.7

283 ± 10.0

503 ± 24.2

166 ± 5.3

1027 ± 358.2

+

0

87 ± 1.5

16 ± 1.5

28 ± 2.3

34 ± 6.7

18 ± 0.6

+

50

94 ± 6.1

13 ± 1.2

29 ± 5.8

31 ± 0.6

23 ± 7.4

+

150

88 ± 4.0

13 ± 2.6

25 ± 5.6

35 ± 6.4

19 ± 7.0

+

500

87 ± 3.5

12 ± 1.2

29 ± 3.8

32 ± 9.5

20 ± 5.5

+

1500

81 ± 6.5

10 ± 0.6

30 ± 3.6

33 ± 3.8

15 ± 4.6

+

5000

77 ± 5.1 P

12 ± 0.6 P

30 ± 2.6 P

31 ± 4.7 P

18 ± 3.8 P

Positive controls, +S9

Name

2AA

2AA

2AA

BP

2AA

Concentrations

(μg/plate)

1

2

10

5

2

Mean No. of colonies/plate

(average of 3 ± SD)

920 ± 153.0

198 ± 9.3

555 ± 27.8

511 ± 132.0

196 ± 6.5

ENNG = N-ethyl-N-nitro-N-nitrosoguanidine

4NQO = 4-nitroquinoline-N-oxide

9AA = 9-aminoacridine

2AA = 2 -aminoanthracene

BP = Benzo(a)pyrene

P = Precipitate

Table 4. Test results of experiment 2 (plate incorporation).

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA 100

TA1535

WP2uvrA-

TA98

TA1537

0

103 ± 5.3

26 ± 3.8

31 ± 8.5

29 ± 7.0

12 ± 2.9

50

105 ± 10.8

21 ± 4.9

30 ± 7.4

28 ± 7.1

11 ± 1.0

150

97 ± 7.0

26 ± 1.0

35 ± 2.9

22 ± 1.2

13 ± 3.5

500

104 ± 9.1

25 ± 4.6

34 ± 5.1

26 ± 5.6

15 ± 1.7

1500

110 ± 9.5

23 ± 6.0

25 ± 0.6

27 ± 3.8

13 ± 1.7

5000

112 ± 5.7 P

28 ± 0.6 P

35 ± 2.6 P

20 ± 1.0 P

11 ± 1.5 P

Positive controls, –S9

Name

ENNG

ENNG

ENNG

4NQO

9AA

Concentrations

(μg/plate)

3

5

2

0.2

80

Mean No. of colonies/plate

(average of 3 ± SD)

880 ± 31.5

257 ± 34.4

1191 ± 59.3

166 ± 4.6

1384 ± 263.4

+

0

103 ± 8.6

13 ± 3.6

37 ± 2.1

27 ± 6.2

20 ± 3.8

+

50

92 ± 21.0

12 ± 1.5

33 ± 4.0

27 ± 7.2

20 ± 2.6

+

150

91 ± 7.5

14 ± 1.5

34 ± 2.6

29 ± 4.0

22 ± 2.6

+

500

113 ± 15.0

14 ± 2.3

39 ± 2.1

27 ± 6.7

15 ± 3.2

+

1500

110 ± 7.6

13 ± 2.6

36 ± 3.2

31 ± 4.9

21 ± 3.1

+

5000

97 ± 9.0 P

17 ± 5.8 P

30 ± 2.5 P

28 ± 4.7 P

22 ± 3.0 P

Positive controls, +S9

Name

2AA

2AA

2AA

BP

2AA

Concentrations

(μg/plate)

1

2

10

5

2

Mean No. of colonies/plate

(average of 3 ± SD)

865 ± 43.2

246 ± 7.5

1051 ± 90.0

611 ± 51.5

182 ± 7.0

ENNG = N-ethyl-N-nitro-N-nitrosoguanidine

4NQO = 4-nitroquinoline-N-oxide

9AA = 9-aminoacridine

2AA = 2 -aminoanthracene

BP = Benzo(a)pyrene

P = Precipitate
Conclusions:
negative
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
refer to analogue justification provided in IUCLID section 13
Reason / purpose for cross-reference:
read-across source
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes:
Metabolic activation:
with and without
Metabolic activation system:
Arochlor 1254 induced S9-Mix from male Spraque-Dawley rats
Test concentrations with justification for top dose:
1. Experiment:
4(20) h without S9: 39.06; 78.13; 156.25; 312.5; 625; 1250; 2500; 5000 µg/mL
4(20) h with S9: 39.06; 78.13; 156.25; 312,5; 625; 1250; 2500; 5000 µg/mL

2. Experiment
24 h without S9: 39.06; 78.13; 156.25; 312,5; 625; 1250; 2500; 5000 µg/mL
4(20) h with S9: 39.06; 78.13; 156.25; 312,5; 625; 1250; 2500; 5000 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
in absence of S9-Mix 750 µg/mL
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
in presence of S9-Mix 25 µg/mL
Details on test system and experimental conditions:
Duplicate cultures of human lymphocytes were evaluated for chromosome aberrations at up to three dose levels. For each experiment, whole blood was drawn from the peripheral circulation of a volunteer who had previously been screened for suitability. The cell cycle length of approx. 14 h was determined by BrdU incorporation.
METHOD OF APPLICATION: in medium (Eagle´s minimal essential medium with HEPES buffer supplemented with L-glutamine, Pen/Strep, amphotericin B and 15% FCS)

DURATION
- Exposure duration: Experiment I: 4 h with or without S9 mix.
- Experiment II: Either 4 h with S9 mix or 24 h without S9 mix.
- Expression time (cells in growth medium): 20 h after 4 h exposure to the test substance and 20 h expression time

SPINDLE INHIBITOR: Colcemid (0.1 µg/mL)
STAIN: 5% Giemsa for 5 min

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 200

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
Evaluation criteria:
All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.
Statistics:
The frequency of cells with aberrations and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher´s Exact test.
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
at and above 1250 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
lymphocytes: human
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
at and above 1250 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Medium was "cloudy" from 156.25 µg/mL and a precipitate was seen from 1250 µg/mL without effects on the toxicity response.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes, data were in range with the historical control data.

Table 1: Experiment 1: 4 h treatment without metabolic activation

Treatment group (µg/ml)

Number of cells scored

Total gaps

Chromatid

Chromosome

others

Total abberations

Abberant cells

Breaks

Exchanges

Breaks

Exchanges

x

(+Gaps)

(+Gaps)

(+Gaps)

(+Gaps)

Vehicle control

200

5

0

1

0

1

0

7

2

7

2

1250

200

5

0

0

3

0

0

8

3

8

3

2500

200

2

2

0

0

0

0

4

2

4

2

5000

200

5

4

0

0

1

0

10

5

9

5

EMS 750

200

24

9

5

5

0

0

43

19

35

17

Table 2: Experiment 1: 4 h treatment with metabolic activation

Treatment group (µg/ml)

Number of cells scored

Total gaps

Chromatid

Chromosome

others

Total abberations

Abberant cells

Breaks

Exchanges

Breaks

Exchanges

x

(+Gaps)

(+Gaps)

(+Gaps)

(+Gaps)

Vehicle control

200

4

1

0

0

0

0

5

1

4

1

1250

200

6

4

0

0

0

0

10

4

9

4

2500

200

4

2

0

0

0

0

6

2

5

2

5000

200

2

4

0

1

0

0

7

5

7

5

CP 25

200

13

12

3

2

0

0

30

17

24

15

Table 3: Experiment 2: 20 h treatment without metabolic activation

Treatment group (µg/ml)

Number of cells scored

Total gaps

Chromatid

Chromosome

others

Total abberations

Abberant cells

Breaks

Exchanges

Breaks

Exchanges

x

(+Gaps)

(+Gaps)

(+Gaps)

(+Gaps)

Vehicle control

200

2

2

1

0

0

0

5

3

5

3

1250

200

7

0

0

1

0

0

8

1

8

1

2500

200

0

1

0

0

0

0

1

1

1

1

5000

200

6

0

0

0

0

0

6

0

6

0

EMS 500

200

53

41

13

2

0

0

109

56

74

48

Table 4: Experiment 2: 4 h treatment with metabolic activation

Treatment group (µg/ml)

Number of cells scored

Total gaps

Chromatid

Chromosome

others

Total abberations

Abberant cells

Breaks

Exchanges

Breaks

Exchanges

x

(+Gaps)

(+Gaps)

(+Gaps)

(+Gaps)

Vehicle control

200

0

0

0

2

0

0

2

2

2

2

1250

200

4

2

2

3

0

0

9

5

7

3

2500

200

5

2

0

1

0

0

8

3

8

3

5000

200

3

2

0

2

0

0

7

4

6

3

CP 25

200

10

10

2

0

0

0

22

12

15

11

The test substance was non-clastogenic to human lymphocytes in vitro.

Conclusions:
negative
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
refer to analogue justification provided in IUCLID section 13
Reason / purpose for cross-reference:
read-across source
Target gene:
thymidine kinase locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 supplemented with 5% (v/v) heat-inactivated horse serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- 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 a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
First experiment: 0.03, 0.1, 0.3, 1, 3, 10, 33, 100 µg/mL (with and without metabolic activation (8%, v/v))
Second experiment: 0.03, 0.1, 0.3, 1, 3, 10, 33, 100 µg/mL (with metabolic activation (12%, v/v)); 0.1, 1, 3, 10, 33, 100, 200, 250 µg/mL (without metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
in the absence of S9-mix 15 and 5 µg/mL for 3 and 24 h treatment period
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
in the presence of S9-mix 7.5 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension

DURATION
- Exposure duration: cells were exposed to the test material for 3 h and 24 h
- Expression time (cells in growth medium): Cells in the final suspension after treatment were counted with the coulter particle counter. For the expression of the mutant phenotype, the cells were separated by 2 centrifugation steps and cultures for 48 h after the treatment period. Cells were plated for the determination of the cloning efficiency and mutation frequency. For the determination of the mutation frequency cells were plated and incubated for 11-12 d. After that, cells were stained for 2 h by adding 0.5 mg/mL MTT (Sigma) to each well. The plates were scored for cloning efficiency and mutation frequency with the naked eye or with the microscope.

SELECTION AGENT (mutation assays): RPMI 1640 supplemented with 20% (v/v) heat-inactivated horse serum and 5 µg/mL trifluorothymidine (TFT).

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency and relative total growth
Evaluation criteria:
Measurement of cytotoxicity by determining the relative cloning efficiency (survival) or relative total growth of the cultures is usually initiated after the treatment period. There are several criteria for determining a positive result, such as a concentration-related, or a reproducible increase in mutant frequency.
Statistics:
The cloning efficiency (CE) was determined as follows:
P(0)= Number of empty wells divided by the total number of wells
CE= P(0)/number of cells plated per well

Relative survival rate (RS): RS= [CE(test)/CE(control)] x 100
Relative total growth (RTG): RTG= RSG x RSday2 / 100
Suspension growth (SG): [Day 0 cell count/1.25x10E005] x [Day 1 cell count/1.25x10E005] x [Day 2 cell count]
Relative suspension growth (RSG): SG(test)/SG(control) x 100

RSday2= CEday2(test) / CEday2(control) x 100

The growth rate (GR) was calculated for the solvent control cultures:
- 3 h treatment: [Day 1 cell count/1.25x105] x [Day 2 cell count/1.25x10E005]
- 24 h treatment: [Day 0 cell count/1.25x105] x [Day 1 cell count/1.25x10E005] x [Day 2 cell count/1.25x10E005]


The mutation frequency was expressed as the number of mutants per 106 viable cells. The plating efficiencies of both mutant and viable cells (CE day2) in the same culture were determined and the mutation frequency (MF) was calculated as follows:

MF= {-ln P(0)/number of cells plated per well)/CE day2 x 10E-006

Small and large colony mutation frequencies were calculated in an identical manner.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
and above (precipitating concentration: 100 µg/mL, tested up to 250 µg/mL)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at and above 100 µg/mL


RANGE-FINDING/SCREENING STUDIES: Yes, cytotoxicity data was obtained by treating cells for 3 h and 24 h, respectively, with a number of increasing test substance concentrations. The highest concentration tested was 200 µg/ml due to poor solubility of the test substance. No toxicity was observed with and without metabolic activation up to and at the maximum dose level tested with 3 h incubation. 24 h incubation resulted in 64% relative suspension growth in the absence of metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes, all controls were in the range of the historical controls.

Table 1: Experiment 1 - 3 hours with and without S9 mix

Dose

(µg/ml)

RSG

(%)

CE day2

(%)

RS day2

(%)

RTG

(%)

mutation frequency x 10-6

 

 

 

 

 

total

Without metabolic activation, 3 h treatment

SC1

100

94

100

100

89

SC2

108

73

0.03

98

101

100

98

63

0.1

92

99

98

90

83

0.3

111

102

101

112

58

1

107

98

97

104

64

3

110

101

100

110

83

10

98

99

98

96

83

33

98

110

109

106

90

100*

74

94

93

69

97

MMS

70

63

63

44

1022

With 8% (v/v) metabolic activation, 3 h treatment

SC1

100

77

100

100

82

SC2

84

87

0.03

96

90

112

107

71

0.1

92

104

129

119

60

0.3

80

108

135

108

55

1

93

105

131

121

69

3

97

90

112

109

65

10

95

84

104

99

71

33

93

81

101

94

91

100*

42

83

103

43

98

CP

20

37

47

9

1107

 

Table 2: Experiment 2 - 3 hours with and 24 hours without S9 mix

Dose

(µg/ml)

RSG

(%)

CE day2

(%)

RS day2

(%)

RTG

(%)

mutation frequency x 10-6

 

 

 

 

 

total

Without metabolic activation, 24 h treatment

SC1

100

102

100

100

62

SC2

104

57

0.1

97

83

80

78

87

1

94

105

102

96

68

3

102

90

87

89

65

10

104

115

111

115

54

33

105

83

80

84

53

100*

102

98

95

97

55

200*

116

104

101

116

52

250*

112

108

105

118

51

MMS

80

81

79

63

631

With 12% (v/v) metabolic activation, 3 h treatment

SC1

100

77

100

100

60

SC2

91

84

0.03

116

58

69

81

108

0.1

97

80

95

93

86

0.3

94

80

95

90

76

1

99

81

97

96

88

3

102

89

106

108

71

10

104

86

103

106

73

33

119

86

103

122

83

100*

105

77

91

96

72

CP

31

54

64

20

814

RSG: Relative Suspension Growth; CE: Cloning efficiency; RS: Relative Survival; RTG: Relative Total Growth; SC: Solvent Control (DMSO); MMS: Methylmethansulfonate; CP: Cyclophosphamide

*: Precipitation of test substance

Conclusions:
negative
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
refer to analogue justification provided in IUCLID section 13
Reason / purpose for cross-reference:
read-across source
Target gene:
his operon (S. typhimurium) and trp operon (E. coli)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male Sprague Dawley rats treated i.p. with a single dose of 500 mg/kg bw Arochlor 1254
Test concentrations with justification for top dose:
Range-finding toxicity study (in TA 100 and WP2 uvrA): 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3330 and 5000 µg/plate, with and without metabolic activation
Main study (all strains): 33.3, 100, 333, 1000, 3330 and 5000 µg/plate, with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
-S9: 2-NF (1 µg/plate, TA 98); SA (2 µg/plate, TA 100 and TA 1535); ICR-191 (2 µg/plate, TA 1537); 4-NQO (1 µg/plate, WP2 uvrA); +S9: BP (2.5 µg/plate, TA 98); 2-AA (2.5-25 µg/plate, TA 100, TA 1535, TA 1537 and WP2 uvrA)
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-aminoanthracene; ICR-191
Remarks:
2-NF: 2-nitrofluorene; SA: sodium azide; 4-NQO: 4-nitroquinoline-N-oxide; BP: benzo(a)pyrene; 2-AA: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 52 ± 4 h

NUMBER OF REPLICATIONS: triplicates each in one experiment

DETERMINATION OF CYTOTOXICITY
- Method: inspection of bacterial background lawn
Evaluation criteria:
The results of the test were considered positive, if the following criteria were met: tester strains TA 98, TA 100 and WP2 uvrA: for a test article to be considered positive, it must produce at least a 2-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate must be accompanied by a dose response to increasing concentrations of the test article; tester strains TA 1535 and TA 1537: for a test article to be considered positive, it must produce at least a 3-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate must be accompanied by a dose response to increasing concentrations of the test article.
Statistics:
Mean values and standard deviations of revertants per plate were calculated.
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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: in the absence of S9 mix, slight precipitation of the test substance was observed in all experiments at concentrations ≥100 µg/plate. In the presence of S9 mix, slight precipitates were noted at ≥333 µg/plate in the preliminary cytotoxicity study and at ≥1000 µg/plate in the main study.

RANGE-FINDING/SCREENING STUDIES: in a preliminary cytotoxicity study, the tester strains TA 100 and WP2 uvrA were treated with the test substance at concentrations ranging from 6.67 to 5000 µg/plate in the presence and absence of metabolic activation (S9 mix). No cytotoxicity was observed in these strains up to the limit dose of 5000 µg/plate, neither with nor without addition of S9 mix.

Table 1. Test results of experiment (plate incorporation)

Bacterial Reverse Mutation Assay, mean revertant colonies/plate (mutation factor) (n=3 ± SD)

EXPERIMENT

S9-Mix

Without

 

Concentration (per plate)

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

SC

25 ± 2

80 ± 9

14 ± 8

3 ± 1

26 ± 3

Test material

 

33.3 µg

19 ± 4

88 ± 5

12 ± 5

4 ± 4

19 ± 2

100 µg

21 ± 7

96 ± 12

11 ± 2

5 ± 4

19 ± 4

333 µg

22 ± 2

92 ± 9

13 ± 2

5 ± 5

23 ± 10

1000 µg

25 ± 6

97 ± 11

25 ± 2

5 ± 3

30 ± 3

3330 µg

22 ± 1

101 ± 3

10 ± 1

3 ± 2

32 ± 2

5000 µg

20 ± 6

85 ± 6

15 ± 3

4 ± 2

27 ± 3

PC

 

2-NF

206 ± 42

-

-

-

-

SA

-

549 ± 71

480 ± 3

-

-

ICR-191

-

-

-

277 ± 33

-

4-NQO

 -

 -

 -

260 ± 43

S9-Mix

 

With

Concentration (per plate)

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

SC

36 ± 2

102 ± 2

19 ± 1

7 ± 1

25 ± 5

Test material

 

33.3 µg

36 ± 7

93 ± 9

15 ± 2

8 ± 4

22 ± 5

100 µg

34 ± 7

97 ± 17

16 ± 4

9 ± 2

22 ± 6

333 µg

34 ± 6

87 ± 6

17 ± 2

9 ± 4

27 ± 3

1000 µg

39 ± 9

94 ± 17

17 ± 3

8 ± 2

26 ± 6

3330 µg

38 ± 8

92 ± 6

16 ± 4

6 ± 3

29 ± 11

5000 µg

34 ± 5

139 ± 5

20 ± 3

3 ± 2

24 ± 4

PC

 

 

 

 

 

BP

471 ± 14

-

-

-

-

2-AA

-

918 ± 296

124 ± 6

171 ± 32

278 ± 24

SC = Solvent control; PC = Positive control substances; SD = standard deviation;

2-NF: 2-nitrofluorene; SA: sodium azide; 4-NQO: 4-nitroquinoline-N-oxide; BP: benzo(a)pyrene; 2-AA: 2-aminoanthracene

Conclusions:
negative
Endpoint:
genetic toxicity in vitro, other
Remarks:
QSAR prediction
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Validated QSAR model
Justification for type of information:
The OECD QSAR Toolbox v3.3.5 is a Quantitative Structure-Activity Relationship model that was developed by the Laboratory of Mathematical Chemistry, Bourgas, Bulgaria (http://toolbox.oasis-lmc.org). It contains several different databases with data on chemicals. The model was used to predict the mutagenic potential of the test substance in the database “DNA alerts for AMES, MN and CA by OASIS v.1.3”.
Principles of method if other than guideline:
Prediction based on OECD QSAR Toolbox v3.3.5 of the mutagenic potential of the test substance. The presence of DNA alerts for AMES, MN and CA by OASIS v.1.3 that may indicate mutagenic properties is assessed.
GLP compliance:
no
Species / strain:
other: not applicable
Metabolic activation:
not applicable
Genotoxicity:
other: prediction for mutagenicity: no alert found
Cytotoxicity / choice of top concentrations:
other: not applicable
Vehicle controls validity:
not applicable
Untreated negative controls validity:
not applicable
Positive controls validity:
not applicable
Remarks on result:
no mutagenic potential (based on QSAR/QSPR prediction)
Conclusions:
The test substance is not predicted to have mutagenic or carcinogenic potential.
Endpoint:
genetic toxicity in vitro, other
Remarks:
QSAR prediction
Type of information:
other: (Q)SAR (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Validated QSAR model
Justification for type of information:
The OECD QSAR Toolbox v3.3.5 is a Quantitative Structure-Activity Relationship model that was developed by the Laboratory of Mathematical Chemistry, Bourgas, Bulgaria (http://toolbox.oasis-lmc.org). It contains several different databases with data on chemicals. The model was used to predict the mutagenic potential of the analogue substances in the database “DNA alerts for AMES, MN and CA by OASIS v.1.3”.
Principles of method if other than guideline:
Prediction based on OECD QSAR Toolbox v3.3.5 of the mutagenic potential of the analogue substances. The presence of DNA alerts for AMES, MN and CA by OASIS v.1.3 that may indicate mutagenic properties is assessed.
GLP compliance:
no
Species / strain:
other: not applicable
Metabolic activation:
not applicable
Genotoxicity:
other: prediction for mutagenicity: no alert found
Cytotoxicity / choice of top concentrations:
other: not applicable
Vehicle controls validity:
not applicable
Untreated negative controls validity:
not applicable
Positive controls validity:
not applicable
Remarks on result:
no mutagenic potential (based on QSAR/QSPR prediction)
Conclusions:
The analogue substances are not predicted to have mutagenic potential.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo genetic toxicity:

In vivo micronucleus test in mice (MNT / OECD 474): negative (RA from CAS 68424-31-7)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 May - 08 July 1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see "remarks"
Remarks:
GLP-Guideline study. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (Dec 2012)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.
Justification for type of information:
refer to analogue justification provided in IUCLID section 13
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted in 1983
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories (UK Limited), Margate, Kent, UK
- Age at study initiation: 5-9 weeks for phase I (determination of the maximum tolerated dose) and 7-9 weeks for phase II (Micronucleus test) of the study
- Assigned to test groups randomly: Yes
- Housing: 5 per cage in mobile mouse cage racks, housed per sex
- Diet: Porton Combined Diet, ad libitum
- Water: filtered tap water, ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23
- Humidity (%): 40 - 70
- Air changes (per hr): 25
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Amount of vehicle: 10 mL/kg
Details on exposure:
The study consisted of two phases: in phase I the maximum tolerated dose (MTD) was determined, on the basis of lethality or severe toxicity observed over a four-day observation period following a single intraperitoneal injection. In phase II, male and female animals were weighed and given a single intraperitoneal injection of corn oil (vehicle control), cyclophosphamide (positive control) or test substance prepared in corn oil.
Duration of treatment / exposure:
Single dose
Frequency of treatment:
Single dose
Post exposure period:
24 h and 48 h
Remarks:
Doses / Concentrations:
5000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: i.p.
- Doses / concentrations: 65 mg/kg bw in physiological saline
Tissues and cell types examined:
Monochromatic and polychromatic erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
No deaths or severe adverse effects occurred in Phase I of the study with doses up to 5000 mg/kg bw. This dose was selected as MTD.

TREATMENT AND SAMPLING TIMES: 24 h and 48 h after dosing

DETAILS OF SLIDE PREPARATION: Bone Marrow smears were stained with polychrome methylene blue and eosin

METHOD OF ANALYSIS: 1000 polychromatic erythrocytes were evaluated for micronuclei per slide. In addition, 1000 erythrocytes were counted to determine the percentage of polychromatic erythrocytes in the total erythrocyte population.
Evaluation criteria:
Increase in the incidence of micronucleated polychromatic erythrocytes in any sex or at any time point; Percentage of polychromatic erythrocytes.
Statistics:
The incidence of micronucleated polychromatic erythrocytes and percentage of polychromatic erythrocytes in the erythrocyte sample were considered by analysis of variance regarding each combination of sampling time, dose level and sex as a separate group. Results were examined to determine whether any differences between vehicle control and test substance treated groups were consistent between sexes and across sampling times. Each group mean was compared with the vehicle control group mean at the corresponding sampling time using a one-sided Student´s t-test based on the error mean square in the analysis.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
No statistically or biologically significant increases in the incidence of micronucleated polychromatic erythrocytes over the vehicle control values were seen in either sex at either of the sampling times. Comparison of the percentage of polychromatic erythrocytes showed no significant differences between the female animals treated with the vehicle control or with the test material. A small, but significant decrease was, however, noted in male mice treated with the test material at 5000 mg/kg bw. This small decrease is, however, considered not to be statistically significant compared to the concurrent control values. The positive control induced statistically significant and biologically meaningful increases in micronucleated polychromatic erythrocytes, compared to the vehicle control values, thus demonstrating the sensitivity of the test system to a known clastogen.

Mean incidence of micronucleated polychromatic erythrocytes/1000 polychromatic erythrocytes ± Standard Deviation at two sampling times. n=5

Table 1: Males

Group

Compound

Dose

Mean Incidence

24 h

48 h

11

Vehicle control

(corn oil)

10 mL/kg

0.8 ± 0.8

1.0 ± 1.2

12

Cyclophosphamide

65 mg/kg

24.4 ± 6.0**

 

13

Test substance

5000 mg/kg

0.6 ± 0.6

0.4 ± 0.6

 

Table 2: Females

Group

Compound

Dose

Mean Incidence

24 h

48 h

11

Vehicle control

(corn oil)

10 ml/kg

0.2 ± 0.5

1.4 ± 1.1

12

Cyclophosphamide

65 mg/kg

18.4 ± 7.3**

 

13

 Test substance

5000 mg/kg

0.4 ± 0.9

0.4 ± 0.9

Mean percentage of polychromatic erythrocytes ± Standard Deviation at two sampling times. n=5

Table 3: Males

Group

Compound

Dose

Mean Incidence

24 h

48 h

11

Vehicle control

(corn oil)

10 ml/kg

48.0 ± 5.6

44.3 ± 7.5

12

Cyclophosphamide

65 mg/kg

41.4 ± 4.4*

 

13

 Test substance

5000 mg/kg

42.2 ± 7.0*

43.3 ± 1.9

 

Table 4: Female

Group

Compound

Dose

Mean Incidence

24 h

48 h

11

Vehicle control

(corn oil)

10 ml/kg

41.9 ± 4.8

41.9 ± 1.7

12

Cyclophosphamide

65 mg/kg

45.9 ± 3.49

 

13

 Test substance

5000 mg/kg

46.5 ± 5.8

48.0 ± 5.2

Conclusions:
negative
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
refer to analogue justification provided in IUCLID section 13
Reason / purpose for cross-reference:
read-across source
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories (UK Limited), Margate, Kent, UK
- Age at study initiation: 5-9 weeks for phase I (determination of the maximum tolerated dose) and 7-9 weeks for phase II (Micronucleus test) of the study
- Assigned to test groups randomly: Yes
- Housing: 5 per cage in mobile mouse cage racks, housed per sex
- Diet: Porton Combined Diet, ad libitum
- Water: filtered tap water, ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23
- Humidity (%): 40 - 70
- Air changes (per hr): 25
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Amount of vehicle: 10 mL/kg
Details on exposure:
The study consisted of two phases: in phase I the maximum tolerated dose (MTD) was determined, on the basis of lethality or severe toxicity observed over a four-day observation period following a single intraperitoneal injection. In phase II, male and female animals were weighed and given a single intraperitoneal injection of corn oil (vehicle control), cyclophosphamide (positive control) or test substance prepared in corn oil.
Duration of treatment / exposure:
Single dose
Frequency of treatment:
Single dose
Post exposure period:
24 h and 48 h
Remarks:
Doses / Concentrations:
5000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: i.p.
- Doses / concentrations: 65 mg/kg bw in physiological saline
Tissues and cell types examined:
Monochromatic and polychromatic erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
No deaths or severe adverse effects occurred in Phase I of the study with doses up to 5000 mg/kg bw. This dose was selected as MTD.

TREATMENT AND SAMPLING TIMES: 24 h and 48 h after dosing

DETAILS OF SLIDE PREPARATION: Bone Marrow smears were stained with polychrome methylene blue and eosin

METHOD OF ANALYSIS: 1000 polychromatic erythrocytes were evaluated for micronuclei per slide. In addition, 1000 erythrocytes were counted to determine the percentage of polychromatic erythrocytes in the total erythrocyte population.
Evaluation criteria:
Increase in the incidence of micronucleated polychromatic erythrocytes in any sex or at any time point; Percentage of polychromatic erythrocytes.
Statistics:
The incidence of micronucleated polychromatic erythrocytes and percentage of polychromatic erythrocytes in the erythrocyte sample were considered by analysis of variance regarding each combination of sampling time, dose level and sex as a separate group. Results were examined to determine whether any differences between vehicle control and test substance treated groups were consistent between sexes and across sampling times. Each group mean was compared with the vehicle control group mean at the corresponding sampling time using a one-sided Student´s t-test based on the error mean square in the analysis.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
No statistically or biologically significant increases in the incidence of micronucleated polychromatic erythrocytes over the vehicle control values were seen in either sex at either of the sampling times. Comparison of the percentage of polychromatic erythrocytes showed no significant differences between the female animals treated with the vehicle control or with the test material. A small, but significant decrease was, however, noted in male mice treated with the test material at 5000 mg/kg bw. This small decrease is, however, considered not to be statistically significant compared to the concurrent control values. The positive control induced statistically significant and biologically meaningful increases in micronucleated polychromatic erythrocytes, compared to the vehicle control values, thus demonstrating the sensitivity of the test system to a known clastogen.

Mean incidence of micronucleated polychromatic erythrocytes/1000 polychromatic erythrocytes ± Standard Deviation at two sampling times. n=5

Table 1: Males

Group

Compound

Dose

Mean Incidence

24 h

48 h

11

Vehicle control

(corn oil)

10 mL/kg

0.8 ± 0.8

1.0 ± 1.2

12

Cyclophosphamide

65 mg/kg

24.4 ± 6.0**

 

13

Test substance

5000 mg/kg

0.6 ± 0.6

0.4 ± 0.6

 

Table 2: Females

Group

Compound

Dose

Mean Incidence

24 h

48 h

11

Vehicle control

(corn oil)

10 ml/kg

0.2 ± 0.5

1.4 ± 1.1

12

Cyclophosphamide

65 mg/kg

18.4 ± 7.3**

 

13

 Test substance

5000 mg/kg

0.4 ± 0.9

0.4 ± 0.9

Mean percentage of polychromatic erythrocytes ± Standard Deviation at two sampling times. n=5

Table 3: Males

Group

Compound

Dose

Mean Incidence

24 h

48 h

11

Vehicle control

(corn oil)

10 ml/kg

48.0 ± 5.6

44.3 ± 7.5

12

Cyclophosphamide

65 mg/kg

41.4 ± 4.4*

 

13

 Test substance

5000 mg/kg

42.2 ± 7.0*

43.3 ± 1.9

 

Table 4: Female

Group

Compound

Dose

Mean Incidence

24 h

48 h

11

Vehicle control

(corn oil)

10 ml/kg

41.9 ± 4.8

41.9 ± 1.7

12

Cyclophosphamide

65 mg/kg

45.9 ± 3.49

 

13

 Test substance

5000 mg/kg

46.5 ± 5.8

48.0 ± 5.2

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

Additional information

Justification for read-across

There are no data available on genetic toxicity of Tetraesters of pentaerythritol with heptanoic acid and 3,5,5-trimethylhexanoic acid. In order to fulfil the standard information requirements set out in Annex VII and VIII, 8.4, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006, read-across from structurally related substances was conducted. In accordance with Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met.” In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (grouping or read-across) “to avoid the need to test every substance for every endpoint”. For each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13) and within Chapter 5.1 of the CSR.

 

Genetic toxicity (mutagenicity) in bacteria in vitro

CAS 67762-53-2

Furthermore, a bacterial gene mutation assay (Ames test) was performed with Carboxylic acids, C5-9, tetraesters with pentaerythritol (CAS 67762-53-2) according to OECD TG 471 and in compliance with GLP (Mecchi, 1999). The strains Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA were tested according to the plate incorporation procedure in the absence and presence of a metabolic activation system (Aroclor 1254-induced rat liver S9-mix). The experiment was conducted in 3 repetitions at concentrations from 33.3 to 5000 µg/plate (vehicle: ethanol). Appropriate vehicle and positive controls were included into the study and gave the expected results. No cytotoxicity was observed in a preliminary study using tester strains TA 100 and E. coli WP2 uvrA up to the limit dose of 5000 µg/plate, neither with nor without metabolic activation. Slight precipitation of the test substance was observed with all tester strains at concentrations ≥100 µg/plate (without metabolic activation) and ≥1000 µg/plate (with metabolic activation) in the main study. No increase in the frequency of revertant colonies compared to concurrent negative controls was observed in all strains treated with the test material, neither in the presence nor in the absence of metabolic activation.

Genetic toxicity (cytogenicity) in mammalian cells in vitro

CAS 131459-39-7

An in vitro mammalian chromosome aberration test was conducted with 3,5,5-trimethylhexanoic acid mixed tetraesters with PE and valeric acid (CAS 131459-39-7) in accordance with OECD TG 473 under GLP conditions (Wright, 1999). Human peripheral lymphocytes were cultured and treated with the test material or vehicle (acetone) in the absence or presence of a metabolic activation system (Aroclor 1254-induced rat liver S9-mix) in duplicates at concentrations of 39.06 to 5000 µg/mL for 4 h (with and without S9-mix) and 24 h (without S9-mix). Fixation and staining of the cells were performed 24 hours after start of exposure with the test material. Cytotoxicity was assessed by determination of the mitotic index. Appropriate solvent and positive controls were included in the test and gave the expected results. No cytotoxicity was observed at ay concentration either in the presence or absence of metabolic activation. Precipitation of the test substance was observed at concentrations ≥1250 µg/mL in the presence and absence of metabolic activation (only 4 h exposure). The test substance did not cause a statistically significant, dose-related increase in chromosome aberrations under the tested conditions. Based on the results of the study the test material is considered not to be clastogenic to primary human lymphocytes in this chromosome aberration test in vitro.

Genetic toxicity (mutagenicity) in mammalian cells in vitro

CAS 15834-04-5

An in vitro Mammalian Cell Gene Mutation Test was performed with 2,2-bis[[(1-oxopentyl)oxy]methyl]propane-1,3-diyl divalerate (C5 tetraester with PE) (CAS 15834-04-5) in mouse lymphoma L5178Y cells (heterozygous at the thymidine kinase locus) according to OECD TG 476 and under GLP (Verspeek-Rip, 2010). In the first experiment, the cells were treated with the test substance at concentrations of 0.03, 0.1, 0.3, 1, 3, 10, 33, 100 µg/mL for 3 hours in the presence (8% (v/v) S9-mix) or absence of metabolic activation (phenobarbital/ß-naphthoflavone -induced rat liver S9-mix). In the second experiment, test concentrations of 0.03, 0.1, 0.3, 1, 3, 10, 33, 100 µg/mL were applied with metabolic activation (12% (v/v) S9-mix) for 3 h and 0.1, 1, 3, 10, 33, 100, 200, 250 µg/mL without metabolic activation for 24 hours. The vehicle (DMSO) controls had acceptable mutant frequency values that were within the normal range for the L5l78Y 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. No cytotoxicity was observed at any dose level, whereas precipitation was recorded at concentrations ≥100 µg/mL. No significant increase in the mutation frequency at the TK locus was observed after treatment with the test substance either in the absence or in the presence of S9-mix. Thus, the test substance was considered to be non-mutagenic to L5l78Y cells under the conditions of the test.

 

Genetic toxicity (cytogenicity) in mammals in vivo

CAS 68424-31-7

An in vivo chromosome aberration test (micronucleus assay) with Pentaerythritol tetraesters of n-decanoic, n-heptanoic, n-octanoic and n-valeric acids (CAS 68424-31-7) was performed according to OECD TG 474 and in compliance with GLP (Griffiths, 1992). The test material was administered via intraperitoneal injection to 5 CD-1 mice of each sex at a limit concentrations of 5000 mg/kg bw. Bone marrow cells were freshly isolated 24 and 48 hours post-application. No statistically or biologically significant increases in the incidence of micronucleated polychromatic erythrocytes compared the vehicle control (corn oil) values were seen in either sex at either of the sampling times. Comparison of the percentage of polychromatic erythrocytes showed no significant differences between the female animals treated with the vehicle control or with the test material. A small, but significant decrease was, however, noted in male mice treated with the test material at 5000 mg/kg bw. This small decrease is, however, considered not to be statistically significant compared to the concurrent control values. The positive control induced statistically significant and biologically meaningful increases in micronucleated polychromatic erythrocytes, compared to the vehicle control values, thus demonstrating the sensitivity of the test system to a known clastogen. Based on the results of the study the test substance is negative for the induction of micronuclei under the conditions of the test.

In support of this, OSAR based (OECD Toolbox v3.3.5) prediction of the mutagenic potential of the target and analogue substances were performed using the database “DNA alerts for AMES, MN and CA by OASIS v.1.3”. The presence of DNA alerts may indicate a mutagenic potential. Briefly, no structural alerts for mutagenicity were identified for the main components of the target substance Tetraesters of pentaerythritol with heptanoic acid and 3,5,5-trimethylhexanoic acid and the source substances. On the basis of the outcome of the OECD Toolbox profiling no toxicological concern with respect to mutagenicity was identified.

Overall conclusion for genetic toxicity

The available experimental data from in vitro and in vivo genetic toxicity from several analogue substances does not indicate any mutagenic or clastogenic potential in vitro and in vivo. OSAR based (OECD Toolbox v3.3.5) prediction of the mutagenic potential of the target and analogue substances did not reveal toxicological concerns with respect to mutagenicity. Therefore following the analogue approach, Tetraesters of pentaerythritol with heptanoic acid and 3,5,5-trimethylhexanoic acid is considered to be not mutagenic and clastogenic in vitro.

Justification for classification or non-classification

Based on the analogue read-across approach, the available data on genetic toxicity do not meet the classification criteria according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.