Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance was not mutagenic in the Ames test and not mutagenic in the HPRT test. Furthermore, the test substance did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 74-7718
- Test substance No.: 89/91

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
Target gene:
his
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254
Test concentrations with justification for top dose:
- first test: 20, 100, 500, 2500 and 5000 µg/plate (standard plate test; all test strains)
- second test: 200, 400, 600, 800 and 1000 µg/plate (standard plate test; test strain TA98)
- third test: 20, 100, 500, 2500 and 5000 µg/plate (preincubation test; all test strains)
- fourth test: 4, 20, 100, 500 and 2500 µg/plate (preincubation test; test strain TA98)
Vehicle / solvent:
aqua dest.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: - S9: aminoanthracene (2-AA): all test strains; + S9: methyl-N-nitro-N-nitrosoguanidine (MNNG): TA100 and TA1535, 4-nitro-o-phenylendiamine (NPD): TA98 and 9-aminoacridine chloride monohydrate (AAC): TA1537
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
Plate incorcoration method:
- Exposure duration: ca. 48-72 hours at 37 °C in the dark
Preincubation method:
- Preincubation period: 20 minutes at 37 °C
- Exposure duration: ca. 48-72 hours at 37 °C in the dark

NUMBER OF REPLICATIONS:
- 3 test plates per dose or per control

DETERMINATION OF CYTOTOXICITY
- Decrease in the number of revertants
- Reduced his- or trp- background growth
- Reduction in the titer
Evaluation criteria:
In order to characterized a substance as positive in the Ames test it has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control);
- dose-response relationship;
- reproducibility of the results.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Toxicity:
No bacteriotoxic effect (reduced his- background growth) was observed.

Solubility:
Complete solubility of test substance in aqua dest.

Table 1: Standard Plate Test (TA 98)

Dose

[µg/plate]

TA 98

Mean ± SD

Without S9 mix

Negative control

(aqua dest)

28 ± 5

200

33 ± 12

400

26 ± 4

600

24 ± 5

800

20 ± 2

1000

26 ± 4

10 NPD

1118 ± 125

With S9 mix

Negative control

(aqua dest)

 44 ± 4

200

38 ± 4

400

40 ± 3

600

 36 ± 1

800

 39 ± 3

1000

 30 ± 11

10 2-AA

 1850 ± 26

Table 2: Standard Plate Test (TA 98, TA 100, TA 1535, TA 1537)

Dose

[µg/plate]

TA 98

Mean ± SD

TA 100

Mean ± SD

TA 1535

Mean ± SD

TA1537

Mean ± SD

Without S9 mix

Negative control

(aqua dest)

28 ± 2

140 ± 5

19 ± 6

15 ± 4

20

33 ± 5

129 ± 8

24 ± 3

10 ± 4

100

33 ± 3

131 ± 27

19 ± 1

15 ± 4

500

38 ± 11

99 ± 8

22 ± 5

13 ± 5

2500

26 ± 6

114 ± 6

22 ± 4

10 ± 5

5000

23 ± 7

139 ± 5

23 ± 4

10 ± 3

10 NPD

922 ± 88

 

 

 

100 AAC

 

 

 

430 ± 91

5 MNNG

 

1347 ± 43

1349 ± 273

 

With S9 mix

Negative control

(aqua dest)

41 ± 5

122 ± 13

20 ± 11

14 ± 2

20

57 ± 5

139 ± 6

21 ± 4

15 ± 5

100

54 ± 12

126 ± 4

18 ± 2

15 ± 7

500

62 ± 23

137 ± 16

20 ± 4

21 ± 1

2500

36 ± 9

140 ± 45

19 ± 4

20 ± 8

5000

36 ± 10

117 ± 30

18 ± 1

16 ± 3

10 2-AA

785 ± 62

334 ± 47

379 ± 53

96 ± 15

Table 3: Preincubation Test (TA 98)

Dose

[µg/plate]

TA 98

Mean ± SD

Without S9 mix

Negative control

(aqua dest)

26 ± 2

4

23 ± 2

20

26 ± 2

100

21 ± 2

500

27 ± 1

2500

26 ± 2

10 NPD

1120 ± 98

With S9 mix

Negative control

(aqua dest)

41 ± 4

4

39 ± 6

20

34 ± 13

100

35 ± 13

500

38 ± 6

2500

33 ± 4

10 2-AA

1247 ± 87

Table 4: Preincubation Test (TA 98, TA 100, TA 1535, TA 1537)

Dose

[µg/plate]

TA 98

Mean ± SD

TA 100

Mean ± SD

TA 1535

Mean ± SD

TA 1537

Mean ± SD

Without S9 mix

Negative control

(aqua dest)

24 ± 4

104 ± 11

14 ± 3

12 ± 2

20

39 ± 2

117 ± 16

15 ± 3

12 ± 2

100

36 ± 3

95 ± 15

16 ± 2

14 ± 1

500

38 ± 6

95 ± 5

16 ± 6

9 ± 4

2500

39 ± 9

115 ± 13

19 ± 3

13 ± 2

5000

31 ± 2

118 ± 11

14 ± 2

11 ± 2

10 NPD

1320 ± 131

 

 

 

100 AAC

 

 

 

313 ± 36

5 MNNG

 

1343 ± 81

1317 ± 6

 

With S9 mix

Negative control

(aqua dest)

35 ± 3

113 ± 19

17 ± 6

12 ± 1

20

59 ± 20

126 ± 12

18 ± 2

14 ± 2

100

51 ± 10

148 ± 18

19 ± 1

11 ± 1

500

49 ± 4

129 ± 18

14 ± 2

14 ± 5

2500

48 ± 5

126 ± 21

17 ± 2

20 ± 3

5000

46 ± 3

135 ± 10

17 ± 2

19 ± 1

10 2-AA

865 ± 241

886 ± 63

167 ± 11

141 ± 22

2 -AA: 2 -aminoanthracene

MNNG: N-methyl-N-nitro-N-nitroso-guanidine

NPD: 4 -nitro-o-phenylendiamine

AAC: 9 -aminoacridine chloride monohydrate

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
GLP compliance:
yes (incl. certificate)
Remarks:
Harlan Cytotest Cell Research GmbH, In den Leppsteinswiesen 19, 64380 Rossdorf, Germany
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 11-0041
- Expiration date of the lot/batch: 18 June 2012

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


Target gene:
HPRT (hypoxanthine-guanine phosphoribosyl transferase)
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Before freezing, the level of spontaneous mutants was depressed by treatment with HAT medium. Each batch is screened for mycoplasma contamination and checked for karyotype stability and spontaneous mutant frequency.
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-naphthoflavone induced S9 mix
Test concentrations with justification for top dose:
30.9, 61.9, 123.8, 247.5, 495.0, 990.0 µg/mL (see 'Any other information on materials and methods incl. tables')
Vehicle / solvent:
Deionised water. The final concentration of deionised water in the culture medium was 10 % v/v.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation; Final concentration: 0.15 mg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation, Final concentration: 1.1 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 for about 8 days.
- Exposure duration: The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

NUMBER OF REPLICATIONS/ NUMBER OF CELLS EVALUATED:
Approximately 1.5E+06 (single culture) and 5E+02 cells (in duplicate) were seeded in plastic culture flasks. The cells were grown for 24 hours prior to treatment. Three or four days after treatment 1.5E+06 cells per experimental point were sub-cultivated in medium. Following the expression time of 7 days five cell culture flasks were seeded with about 3 - 5E+05 cells each in medium containing 6-thioguanine. Two additional flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability.

DETERMINATION OF CYTOTOXICITY
In a pre-test the colony forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test item was observed and compared to the controls. Toxicity of the test item is indicated by a reduction of the cloning efficiency (CE).
Evaluation criteria:
- A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
- A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.

A positive response is described as follows:
- A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
- The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.

However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance was considered together.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
The range finding pre-experiment was performed using a concentration range of 7.7 to 990 µg/mL (≈10 mM) to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation. No relevant toxic effect occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no relevant shift of pH up to the maximum concentration of the test item.
- Effects of osmolality: no relevant shift of osmolarity up to the maximum concentration of the test item.
- Precipitation: no precipitation occurred up to the highest concentration with and without metabolic activation following 4 and 24 hours treatment.

An increase of the induction factor reaching or exceeding the threshold of three times the mutation frequency of the corresponding solvent control was observed in the first culture of the first experiment without metabolic activation at 61.9 and 123.8 µg/mL and in the first culture of the second experiment 123.8 and 247.5 µg/mL. However, the increases were judged as biologically irrelevant fluctuations since they were based on a rather low mutation frequency of the solvent controls of just 5.9 (experiment I) and 7.7 colonies per 1 E+06 cells. Furthermore, the effects were not reproduced in the parallel cultures under identical experimental conditions.  

DOSE RESPONSE RELATIONSHIP
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of the mutation frequency. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.  

COMPARISON WITH HISTORICAL CONTROL DATA:
The numbers of mutant colonies per 10E+6 cells found in the solvent controls falls within the laboratory historical control data.

Table 1: Summary of results

 

Conc.

[µg/mL]

S9 mix

Relative cloning efficiency I

[%]

Relative cell density

[%]

Relative cloning efficiency II

[%]

Mutant colonies/

10^6 cells

Induction factor

Relative cloning efficiency I

[%]

Relative cell density

[%]

Relative cloning efficiency II

[%]

Mutant colonies/ 10^6 cells

Induction factor

Experiment I/

4h treatment

 

 

Culture I

Culture II

Solvent control with water

 

-

100

100

100

5.9

1.0

100

100

100

20.2

1.0

Positive control (EMS)

150.0

-

97.4

88.3

95.8

72.6

12.4

106.0

116.0

98.5

118.2

5.8

Test item

30.9

-

95.6

 

 

 

 

106.9

 

 

 

 

Test item

61.9

-

99.6

100.8

98.4

21.7

3.7

96.3

100.9

94.6

9.1

0.4

Test item

123.8

-

96.4

87.8

91.7

19.4

3.3

105.1

106.7

93.1

16.5

0.8

Test item

247.5

-

99.0

78.2

92.9

3.9

0.7

110.5

98.3

92.2

8.2

0.4

Test item

495.0

-

97.4

83.9

97.7

6.1

1.0

106.6

103.2

90.1

10.4

0.5

Test item

990.0

-

99.2

86.5

87.0

14.6

2.5

98.4

97.3

91.5

8.3

0.4

Solvent control with water

 

+

100.0

100.0

100.0

5.4

1.0

100.0

100.0

100.0

6.5

1.0

Positive control (DMBA)

1.1

+

27.9

133.9

99.8

537.8

99.1

35.9

146.1

74.2

1333.9

203.7

Test item

30.9

+

101.8

Culture was not continued#

98.6

Culture was not continued#

Test item

61.9

+

95.8

111.1

99.5

7.5

1.4

100.4

63.3

90.6

12.4

1.9

Test item

123.8

+

98.2

82.2

89.6

8.4

1.5

93.0

89.0

98.6

16.1

2.5

Test item

247.5

+

97.0

83.2

84.7

12.0

2.2

92.4

99.6

89.6

14.3

2.2

Test item

495.0

+

97.7

78.7

89.0

6.8

1.3

93.9

176.4

96.6

8.2

1.3

Test item

990.0

+

95.6

74.6

100.8

8.2

1.5

94.7

113.9

92.9

16.8

2.6

Experiment II/

24 h treatment

 

 

Culture I

Culture II

Solvent control with water

 

-

100.0

100.0

100.0

7.7

1.0

100.0

100.0

100.0

8.5

1.0

Positive control (EMS)

150.0

-

101.5

73.9

93.8

151.6

19.6

99.2

91.7

84.8

188.8

22.1

Test item

30.9

-

98.2

Culture was not continued#

96.9

Culture was not continued#

Test item

61.9

-

99.4

106.4

80.3

13.7

1.8

97.2

74.4

89.4

9.5

1.1

Test item

123.8

-

100.0

105.7

89.2

33.1

4.3

97.6

51.8

99.8

10.9

1.3

Test item

247.5

-

98.0

122.1

95.0

23.5

3.0

97.0

72.6

100.8

6.0

0.7

Test item

495.0

-

98.6

132.6

89.7

13.4

1.7

100.5

73.9

92.5

17.8

2.1

Test item

990.0

-

101.9

122.2

103.2

9.3

1.2

97.6

54.4

87.5

9.0

1.0

Experiment II/

4 h treatment

 

 

Culture I

Culture II

Solvent control with water

 

+

100.0

100.0

100.0

9.2

1.0

100.0

100.0

100.0

14.3

1.0

Positive control (DMBA)

1.1

+

43.1

90.5

83.8

530.1

57.6

34.6

99.6

94.2

676.7

47.4

Test item

30.9

+

97.7

Culture was not continued#

96.4

Culture was not continued#

Test item

61.9

+

93.5

111.8

112.1

8.0

0.9

93.7

102.4

87.4

20.9

1.5

Test item

123.8

+

97.0

96.6

103.4

9.0

1.0

95.4

113.7

89.1

11.5

0.8

Test item

247.5

+

95.0

94.7

97.0

5.6

0.6

86.3

108.7

93.2

9.6

0.7

Test item

495.0

+

91.8

102.0

96.4

9.1

1.0

90.0

109.1

98.0

15.6

1.1

Test item

990.0

+

90.4

84.1

92.3

10.0

1.1

96.2

99.0

105.3

13.7

1.0

# Culture was not continued since a minimum of only four analysable concentrations is required.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
26 September 2014
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
ENVIGO CRS GmbH, In den Leppsteinwiesen 19, 64380 Roßdorf
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and batch No.of test material: 16960025
- Expiration date of the lot/batch: 28 February 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Solubility and stability of the test substance in the solvent/vehicle: Stable in water over 4 days at room temperature

Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Healthy non-smoking donors not receiving medication.
- Suitability of cells: Human lymphocytes are the most common cells in the micronucleus test and have been used successfully for a long time in in vitro experiments. They show stable spontaneous micronucleus frequencies at a low level.
- Sex, age and number of blood donors if applicable: One male donor (23 years old) for experiment I and one male donor (33 years old) for experiment II.
- Whether whole blood or separated lymphocytes were used: Whole blood were used
- Methods for maintenance in cell culture: Human lymphocytes were stimulated for proliferation by the addition of the mitogen PHA to the culture medium for a period of 48 hours.
Blood cultures were established by preparing an 11% mixture of the whole blood medium within 30 hrs after blood collection.

MEDIA USED
- Type and identity of media including CO2 concentration: The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/ streptomycin (100 U/mL/ 100 µg/mL), the mitogen PHA (3 µg/mL), 10% FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL). All incubations were done at 37 °C with 5.5 % CO2 in humidified air.
Cytokinesis block (if used):
Cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Concentrations used: 6.3, 11.0, 19.2, 33.6, 58.8, 103, 180, 315, 551 and 965 µg/mL
The highest applied concentration in this study (965 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight and the purity (89.2%) of the test item and with respect to the current OECD Guideline 487.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionized water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without metabolic activation: Mitomycin (MMC) and demecolcin; With metabolic activation: Cyclophosphamide (CPA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Cells were fixed with ice-cold mixture of methanol and glacial acetic acid (19 parts plus 1 part, respectively). The cells were stained with Giemsa.

NUMBER OF CELLS EVALUATED: 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides, except for the positive control in Experiment I without S9 mix, where only 500 binucleated cells per culture were evaluated due to strong clastogenic effects.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: The micronuclei were counted in cells showing a clearly visible cytoplasm area.

DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis-block proliferation index (CBPI)
Evaluation criteria:
The micronucleus assay will be considered acceptable if it meets the following criteria:
a) The rate of micronuclei in the solvent controls falls within the historical laboratory control data range.
b) The rate of micronuclei in the positive controls is statistically significant increased.
c) The quality of the slides must allow the evaluation of a sufficient number of analyzable cells.

A test item can be classified as non-clastogenic and non-aneugenic if:
− the number of micronucleated cells in all evaluated dose groups is in the range of the historical laboratory control data and
− no statistically significant or concentration-related increase of the number of micronucleated cells is observed in comparison to the respective solvent control.

A test item can be classified as clastogenic and aneugenic if:
− the number of micronucleated cells is not in the range of the historical laboratory control data and
− either a concentration-related increase in three test groups or a statistically significant increase in the number of micronucleated cells is observed.
If the above mentioned criteria for the test item are not clearly met, the test item will be classified as equivocal or a confirmatory experiment may be performed. However, results may remain questionable regardless of the number of times the experiment is repeated.
An increase in the number of micronucleated mononucleate cells may indicate that the test item has aneugenic potential.
Statistics:
Statistical significance was confirmed by the Chi square test (α < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.
Key result
Species / strain:
lymphocytes: human
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: Summary of results

Exp.

Preparation interval

Test item concentration [µg/mL]

Proliferation index CBPI

Cytostasis [%]*

Micronucleated cells [%]**

Exposure period 4 hrs without S9 mix

I

40 hrs

Solvent controla)

2.04

 

0.55

 

 

Positive controlb)#

1.58

44.2

30.30s)

 

 

315

2.02

1.4

0.35

 

 

551

2.03

0.7

0.60

 

 

965

2.00

3.8

0.65

Exposure period 20 hrs without S9 mix

II

40 hrs

Solvent controla)

2.12

 

0.65

 

 

Positive controlc)

1.74

34.4

5.40s)

 

 

315

2.11

1.3

0.70

 

 

551

2.08

3.3

0.30

 

 

965

2.04

7.3

0.55

Exposure period 4 hrs with S9 mix

I

40 hrs

Solvent controla)

1.99

 

0.40

 

 

Positive controld)

1.64

35.3

3.40s)

 

 

315

2.02

n.c.

0.05

 

 

551

2.06

n.c.

0.10

 

 

965

2.01

n.c.

0.35

II

40 hrs

Solvent controla)

2.15

 

0.80

 

 

Positive controle)

2.11

3.9

4.10s)

 

 

315

2.00

13.0

0.50

 

 

551

2.03

10.6

0.50

 

 

965

2.00

13.1

0.60

* For the positive control groups and the test item treatment groups the values are related to the solvent controls

** The number of micronucleated cells was determined in a sample of 2000 binucleated cells

# The number of micronucleated cells was determined in a sample of 1000 binucleated cells.

s) The number of micronucleated cells is statistically significantly higher than corresponding control values

n.c. Not calculated as the CBPI is equal or higher than the solvent control value

a) Deionized water 10 %

b) Mitomycin C (MMC) 1.0 µg/mL

c) Demecolcin 125 ng/mL

d) CPA 15.0 µg/mL

e) CPA 12.5 µg/mL

Conclusions:
Under the experimental conditions chosen in this study, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, the test item is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The substance was tested for its mutagenic potential in an Ames test performed under GLP and according to OECD guideline 471 in Salmonella typhimurium strains TA1535, TA100, TA1537 and TA98 (BASF AG, 1991). A standard plate and a preincubation test were performed both with and without metabolic activation (Aroclor induced rat liver S-9 mix). In the standard plate test all test strains were exposed to 20 - 5000 µg/plate and in the preincubation test strain TA1535, TA100 and TA1537 were exposed to 20 - 5000 µg/plate and strain TA98 was exposed to 4 - 5000 µg/plate. Complete solubility of the test substance in aqua dest. was observed and no bacteriotoxic effect was observed. An increase in the number of his+ revertants was not observed both in the standard plate test and in the preincubation test either without S-9 mix or after the addition of a metabolising system. According to the results of the present study, the test substance is not mutagenic in the Ames test under the experimental conditions used.

Supportingly, the substance was not mutagenic in an Ames test using Salmonella typhimurium strain his G-46 (Seiler, 1979).

A GLP study was performed to investigate the potential of the test substance to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster, according to OECD 476, (Harlan, 2012). The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours.The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The highest concentration of the pre-experiment and the main experiments (990 µg/mL) was equal to a molar concentration of about 10 mM. The test item was dissolved in deionised water. No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. Appropriate reference mutagens (EMS and DMBA), used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.

To assess the potential of the test item to induce micronuclei in human lymphocytes, an in vitro micronucleus assay according to OECD guideline 487 was performed (BASF, 31M0054/08X085, 2016). Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure periods were 4 hours with S9 mix and 20 hours without S9 mix. The cells were prepared 40 hours after start of treatment with the test item. In each experimental group two parallel cultures were analysed. 1000 binucleate cells per culture were evaluated for cytogenetic damage on coded slides, except for the positive control in Experiment I without S9 mix, where only 500 binucleated cells per culture were evaluated due to strong clastogenic effects. In Experiment II the cells were treated with test item concentrations of 58.8, 103, 180, 315, 551 and 965 µg/mL. In addition to these concentrations, cells were treated with test item concentrations of 6.3, 11.0, 19.2 and 33.6 µg/mL in Experiment I. To determine a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is described as % cytostasis. In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. No precipitation occurred and no osmolality and pH changes were observed. In the absence and presence of S9 mix, no relevant increase in the number of micronucleated cells was observed after treatment with the test item. In conclusion, the test item did not induce micronuclei in human lymphocytes under the test conditions chosen. Thus, the test item is considered to be non-mutagenic (non-clastogenic and non-aneugenic), when test up to the highest required concentration.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP).