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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The available data from three in vitro assays suggest that the substance does not have a genotoxic potential.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 2000-01-19 to 2000-07-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
n/a
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
Cultures of human lymphocytes are primary cell cultures recommended by international regulations for the mammalian chromosome aberration test; they have a stable karyotype with 46 chromosomes and an average cell cycle time of 13-14 hours.
Cultures of human lymphocytes were prepared from whole blood pooled from healthy males donors.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Liver homogenate derived from Aroclor 1254 induced rats (S-9 mix)
Test concentrations with justification for top dose:
Mitotic index analysis:
Test 1 (3-hour treatment without activation): 0, 500, 1000, 1500, 2000, 2500, 2750, 3000, 3250, 3500, 3750 and 4000 µg/mL
Test 1 (3-hour treatment with activation): 0, 625, 1250, 2500, 3500, 4000, 4500 and 5000 µg/mL
Test 2 (20-hr without activation ): 0, 500, 1000, 1250, 1500, 1750, 2000, 2500, 3000, 3500, 4000, 4500 and 5000 µg/mL
Test 2 (3-hour treatment with activation):0, 625, 1250, 2500, 3500, 4000, 4500 and 5000 µg/mL

Metaphase analysis
Test 1 (3-hour treatment without activation): 0, 1000, 2000, 3250 and 3500 µg/mL,
Test 1 (3-hour treatment with activation): 0, 1250, 2500 and 5000 µg/mL,
Test 2 (20-hour without activation ): 0, 1500, 2000 and 2500 µg/mL,
Test 2 (3-hour treatment with activation):0, 1250, 2500 and 4500 µg/mL,
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Culture medium
- Justification for choice of solvent/vehicle: Information supplied by the sponsor stated that the test item was insoluble in water. it was found to form a doseable suspension in culture medium at 10 mg/ml.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
In the first experiment, lymphocyte cultures were exposed to the test or control items (with or without S9 mix) for 3 hours then rinsed. Cells were harvested 20 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles.

The second experiment was performed as follows:
- without S9 mix, cells were exposed continuously to the test or control items until harvest,
- with S9 mix, cells were exposed to the test or control items for 3 hours and then rinsed. Cells were harvested 20 hours after the beginning of treatment.

SPINDLE INHIBITOR (cytogenetic assays): Colcemid at a final concentration of 0.1 µg/ml

STAIN (for cytogenetic assays): Giemsa 10%

NUMBER OF CELLS EVALUATED: 200 metaphases/dose-level

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes, noted when seen
- Determination of endoreplication: yes, noted when seen
Evaluation criteria:
The test substance is considered to cause a positive response if the following conditions are met:
Statistically significant increases (P<0.01) in the frequency of metaphases with aberrant chromosomes (excluding gaps) are observed at one or more test concentration.
The increases exceed the negative control range of the laboratory, taken at the 99% confidence limit.
The increases are reproducible between replicate cultures.
The increases are not associated with large changes in osmolality of the treatment medium or extreme toxicity.
Evidence of a dose-relationship is considered to support the conclusion.

A negative response is claimed if no statistically significant increases in the number of aberrant cells above concurrent control frequencies are
observed, at any dose level.
Statistics:
For each experiment and for each harvest time, the frequency of cells with structural chromosome aberration in treated cultures was compared to that of the vehicle control cultures using Fisher's test.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Test 1:
In the absence of S9 mix, the test item caused a reduction of 5% of the vehicle control value at 5000 µg/ml. Due to steep toxic response at 5000 µg/ml, this test was repeated in order to achieve a concentration causing a decrease in mitotic index closer to 50% of the vehicle control value. In the repeat test, the test item again caused a steep decrease, causing a reduction in the mitotic index to 16% of the vehicle control value at 3000 µg/ml. This test was repeated again and at 3500 µg/ml the test item caused a reduction of 25% to the vehicle control value. One culture at this concentration was considered to have less than 100 metaphases available for chromosome analysis and most of the metaphases were of poor quality. At 3250 µg/ml a reduction of the index mitotic to 91% of the vehicle ontrol value was recorded.
The mitotic indices data obtained in all the three tests showed changes in the toxicity profile which may have been due to the treatment of the test
substance as a suspension. It was decided that as the dose levels at 3250 and 3500 µg/ml were relatively close, slides prepared from these
cultures treated at 3250 µg/ml would be scored as the highest treatment level. The duplicate scoreable culture at 3500 µg/ml was also analysed to
give an indication of chromosome aberrations at this level.
The dose levels selected for the metaphase analysis were 1000, 2000, 3250 and 3500 µg/ml.

In the presence of S9 mix, the test item caused a reduction of the mitotic index to 30% of the vehicle control value at 5000 µg/ml. Due to steep toxic response this test was repeated in order to achieve a concentration causing a decrease in mitotic index closer to 50% of the vehicle control value. In the repeat test, the test item caused a reduction of the mitotic index to 34% of the vehicle control value at 5000 µg/ml. A second repeat test was performed and a reduction to 70% of the control vehicle value at 5000 µg/ml was recorded.
The dose levels selected for metaphase analysis were 1250, 2500 and 5000 µg/ml.

Test 2
In the absence of S9 mix, the test item caused a reduction of the mitotic index to 18% of the vehicle control value at 3000 µg/ml. Due to steep toxic response this test was repeated in order to achieve a concentration causing a decrease in mitotic index closer to 50% of the vehicle control value. The test item failed to cause a significant reduction in the mitotic index and slight bacterial contamination was observed in all cultures, therefore the test was repeated again. In the second repeat test the test item caused a reduction of the mitotic index to 45% of the vehicle control value at 2500 µg/ml.
The dose levels selected for the metaphase analysis were 1500, 2000 and 2500 µg/ml.

In the presence of S9 mix, the test item caused a reduction of the mitotic index to 58% of the vehicle control value at 4500 µg/ml.
The dose levels selected for the metaphase analysis were 1250, 2500 and 4500 µg/ml.

Table 7.6.1.1 Metaphase analysis in Test 1

Substance concentration

(µg/mL)

Cells with aberrations excluding gaps

Cells with aberrations including gaps

Relative cell count

Individual values per 100 cells observed (%)

Mean (%)

Individual values per 100 cells observed (%)

Mean (%)

Method without S9 mix / 3 h. time exposure

0

(culture medium)

0

0

0.0

0

0

0.0

100

1000

0

0

0.0

1

0

0.5

115

2000

0

1

0.5

0

1

0.5

109

3250

1

1

1.0

1

3

2.0

91

3500

2

 

2.0

2

 

2.0

25

0.1

(Mitomycin C)

10

12

11.0***

11

13

12.0***

-

Method with S9 mix / 3 h. time exposure

0

(Culture medium)

0

0

0.0

1

0

0.5

100

1250

0

1

0.5

1

2

1.5

110

2500

1

2

1.5

4

3

3.5

100

5000

0

0

0.0

1

3

2.0

70

6

(Cyclophosphamide))

15

16

15.5***

23

20

21.5***

-

***P<0.001; **P<0.01; otherwise P>0.01

Table7.6.1.2 Metaphase analysis in Test 2

Substance concentration

(µg/mL)

Cells with aberrations excluding gaps

Cells with aberrations including gaps

Relative cell count

Individual values per 100 cells observed (%)

Mean (%)

Individual values per 100 cells observed (%)

Mean (%)

Method without S9 mix / 3 h. time exposure

0

(Culture medium)

2

2

2.0

5

5

5.0

100

1500

2

2

2.0

3

2

2.5

79

2000

2

1

1.5

4

2

3.0

56

2500

1

2

1.5

2

4

3.0

45

0.1

(Mitomycin C)

19

21

20.0***

28

24

26.0***

-

Method with S9 mix / 20 h. time exposure

0

(Culture medium)

1

0

0.5

1

1

1.0

100

1250

2

0

1.0

3

1

2.0

100

2500

2

0

1.0

4

1

2.5

95

4500

2

1

1.5

2

2

2.0

58

6

(Cyclophosphamide))

13

11

12.0***

19

14

16.5***

-

***P<0.001; **P<0.01; otherwise P>0.01

Conclusions:
The substance was not considered to be clastogenic to the human lymphocytes in the in vitro chromosomal aberration assay either in the presence or in the absence of a rat liver metabolizing system.
Executive summary:

An in vitro chromosomal aberration assay was conducted to evaluate the clastogenic potential of the test substance in human lymphocytes according to the OECD Guideline 473 and in compliance withGood Laboratory Practices.


Human lymphocytes, in whole blood culture, were exposed to the test substance both in the absence and presence of S9 mix derived from rat livers. Three hours before the end of the incubation period, cell division was arrested using Colcemid®. The cells were then harvested and slides prepared, so that metaphase cells could be examined for chromosomal damage. On the basis of the mitotic index data obtained from a toxicity test, the following concentrations were selected for two independent tests evaluating the metaphase analysis: 


-        Test 1: 3-h treatment without S9 mix at dose levels of 0, 1000, 2000, 3250 and 3500 µg/mL and 3-h treatment with S9 mix at dose levels of 0, 1250, 2500 and 5000 µg/ml


-        Test 2: 20 -hr treatment without S9 mix at dose levels of: 0, 1500, 2000 and 2500 µg/mL and 3-h treatment with S9 mix at 0, 1250, 2500 and 4500 µg/mL .


Concurrent solvent and positive controls (mitomycin-C (in the absence of S9 mix) and cyclophosphamide (in the presence of S9 mix)) were also included. In both tests, the substance caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations at any concentration in the absence and presence of S9 mix when compared with the vehicle control. Also, no statistically significant increases in the proportion of polyploid or endoreduplicated metaphase cells were observed during metaphase analysis when compared with the vehicle control. All positive control compounds caused statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix.Under the study conditions, the test substance was therefore not considered to be clastogenic to human lymphocytes in the in vitro chromosomal aberration assay.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
from 2000-01-19 to 2000-06-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
Liver homogenate derived from Aroclor 1254 induced rats (S9 mix).
Test concentrations with justification for top dose:
First test
Concentration range (with and without metabolic activation): 5, 15, 50, 150, 500, 1500 and 5000 µg/plate Second test
Concentration range (with and without metabolic activation): 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Solvent used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent: The solubility of the test substance was assessed at 50 mg/ml in DMSO, in which it formed a solution.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-aminoanthracene, 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
The first test, both experiments without S9 mix and the first experiment with S9 mix were performed according to the direct plate
incorporation method. The second experiment with S9 mix was performed according to the preincubation method.

DURATION
- Preincubation period:30 minutes
- Exposure duration: 72 hours

SELECTION AGENT (mutation assays):
- Agar containing traces of histidine , biotin and tryptophan

NUMBER OF REPLICATIONS
- Two independent mutagenicity experiments each using three plates/dose-level

DETERMINATION OF CYTOTOXICITY
- Method: decrease in number of revertant colonies and/or thinning of the bacterial lawn.
Evaluation criteria:
For a test to be considered valid, the mean of the solvent/vehicle control revertant colony numbers for each strain should lie within the 99%
confidence limits of the current historical control range of the laboratory. Alos, the positive control compounds must cause at least a doubling of
mean revertant colony numbers over the negative control.
The mean number of revertant colonies for each treatment group was compared with those obtained for the solvent/vehicle control groups. The
mutagenic activityof a test substance was assessed by applying the following criteria:

- if treatment with a test substance produces an increase in revertant colony numbers of at least twice the concurrent solvent/vehicle controls,
with some evidence of a positive dose-relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S9
mix, the test substance will be considered to show evidence of mutagenic activity in this test system. No statistical analysis will be performed.

- If treatment with a test susbtance does not produce reproductible increases of at least 1.5 times the concurrent solvent/vehicle controls in either
mutation test, the test substance will be considered to show no evidence of mutagenic activity in the test system. No statistical analysis will be
performed.

- If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response given in the first two paragraphs, even after the
additional testing outlined in the mutation test procedure, the test data may be subjected to analysis to determine the statistical significance of any
increases in revertant colony numbers. The statistical procedures used will be those described by Mahon et al (1989) and will usually be analysis of
variance followed by Dunnett's test. Biological significance should always be considered along with statistical significance.
Statistics:
If a clear positive or negative response is not obtained, statistical analysis will be performed. The statistical procedures used will be those described by Mahon et al (1989) and will usually be analysis of variance followed by Dunnett's test.
No statistical analysis will be performed in case of clear positive or negative response as described in the evaluation criteria.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The total colony counts on nutrient agar plates confirmed the viability and high cell density of the culture of the individual organisms.
The mean revertant colony counts for the solvent controls were within the 99% confidence limits of the current historical control range of the
laboratory.

First test
No substancial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to
the test material at any concentrations in either presence or absence of S9 mix. No visible thinning of the background lawn of non-revertant cells was obtained following exposure to the test material.

Second test
No substancial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to
the test material at any concentrations in either presence or absence of S9 mix. No visible thinning of the background lawn of non-revertant cells was obtained following exposure to the test material.

Appropriate positive control chemicals induced substancial increases in revertant colony numbers with all strains, confirming sensitivity of the
cultures and activity of S9 mix.

Table 7.6.1.4: Number of revertants per plate (mean of triplicates) in the absence of metabolic activation (First test)

Test substance concentration

(µg/plate)

TA 1535

TA 1537

WP2uvrA/

PKM101

TA 98

TA 100

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Solvent*

19

2

17

2

128

3

36

2

113

4

5

15

1

19

3

128

7

32

3

109

4

15

14

1

15

4

121

8

34

3

100

11

50

16

4

13

3

125

15

35

4

95

8

150

13

2

16

6

130

8

34

6

97

6

500

12

3

21

1

139

9

34

7

102

8

1500

13

2

15

1

136

9

32

3

105

2

5000

13

2

19

3

122

8

33

2

103

4

Positive control***

410

36

182

16

486

30

536

66

569

8

*Solvent control = DMSO

***Mutagens positive controls: see Table 7.6.1.3

 

 

Table 7.6.1.5: Number of revertants per plate (mean of triplicates) in the presence of metabolic activation (First test)

Test substance concentration

(µg/plate)

TA 1535

TA 1537

WP2uvrA/

PKM101

TA 98

TA 100

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Solvent*

14

2

18

5

149

13

44

5

113

12

5

15

3

19

3

156

1

41

3

104

9

15

16

5

18

2

162

8

43

7

104

10

50

12

3

15

1

154

12

36

7

10

98

150

15

2

14

2

151

16

37

5

95

7

500

15

1

17

4

144

8

42

6

103

6

1500

12

3

13

3

140

8

39

2

104

3

5000

11

3

17

3

142

8

35

3

98

1

Positive control***

94

2

249

24

403

16

752

24

688

61

 

*Solvent control = DMSO

***Mutagens positive controls: see Table 7.6.1.3

 

  

Table 7.6.1.6: Number of revertants per plate (mean of triplicates) in the absence of metabolic activation (Second test)

Test substance concentration

(µg/plate)

TA 1535

TA 1537

WP2uvrA/

PKM101

TA 98

TA 100

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Solvent*

19

2

17

4

125

14

34

2

111

10

50

17

3

14

2

137

11

36

8

107

6

150

17

3

12

2

138

13

35

0

105

9

500

20

6

14

4

138

11

37

7

103

12

1500

18

2

13

3

145

1

28

1

107

16

5000

21

1

11

4

132

12

32

3

99

15

Positive control***

400

48

190

13

548

49

402

9

463

4

*Solvent control = DMSO

***Mutagens positive controls: see Table 7.6.1.3

 

 

Table 7.6.1.7: Number of revertants per plate (mean of triplicates) in the presence of metabolic activation (Second test)

Test substance concentration

(µg/plate)

TA 1535

TA 1537

WP2uvrA/

PKM101

TA 98

TA 100

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Solvent*

17

2

17

4

162

13

38

1

112

9

50

19

5

14

2

169

165

33

4

96

7

150

17

4

15

6

186

165

36

1

96

7

500

20

1

13

3

137

137

35

4

108

18

1500

19

5

15

1

159

153

33

4

98

6

5000

16

3

11

1

147

149

35

3

97

4

Positive control***

102

4

136

15

470

15

535

30

480

2

*Solvent control = DMSO

***Mutagens positive controls: see Table 7.6.1.3

Conclusions:
The test item did not show any mutagenic activity in the bacterial reverse mutation test, either in the presence or in the absence of a rat liver metabolizing system.
Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD guideline N° 471, in compliance with Good Laboratory Practices, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 100 et TA 98) and Escherichia. Coli (WP2uvrA/pKM101) were exposed to the test item at concentrations of 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in two independent experiments both in presence and absence of metabolic activation.

No substantial increases in revertant colony numbers over control count were obtained with any of the tester strains following exposure to the test item at any concentrations in either presence or absence of S9 mix.

The positive control induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

Under these experimental conditions,the substance did not show any mutagenic activity in the bacterial reverse mutation test.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 2009-09-24 to 2009-11-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine Kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI A, 10 and 20
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
Metabolic activation:
with and without
Metabolic activation system:
S9 liver activation system
Test concentrations with justification for top dose:
In the cytotoxicity Range-Finder Experiment six concentrations were tested ranging from 1.18 to 58.0 µg/mL.
In experiment 1 ten concentrations were tested, ranging from 10 to 58 µg/mL.
In experiment 2, six concentrations were tested, ranging from 10 to 58 µg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl formamide (DMF)
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that the test item was soluble in Dimethylformamide (DMF) heated at 80°C at concentrations up to approximately 5.84 mg/mL.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: no data
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 13 days
- Fixation time (start of exposure up to fixation or harvest of cells): 15 days


SELECTION AGENT (mutation assays): resistance to 5-trifluorothymidine (TFT)
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable


NUMBER OF REPLICATIONS: Each treatment, in the absence or presence of S 9, was in duplicate (single cultures only used for positive control treatments).


NUMBER OF CELLS EVALUATED: 100 millions cells


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency


OTHER EXAMINATIONS: no data
Evaluation criteria:
For valid data, the test article was considered to be mutagenic in this assay if:
1. The MF of any test concentration exceeded the sum of the mean control mutant frequency plus GEF
2. The linear trend test was positive.
The test article was considered as positive in this assay if both of the above criteria were met.
The test article was considered as negative in this assay if neither of the above criteria were met.
Results which only partially satisfied the assessment criteria described above were considered on a case-by-case basis.
Statistics:
The statistical significance of the linear trend is assessed by Dunnett's test. The test is one-sided, i.e. negative linear trends are not considered biologically relevant.
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
When tested up to precipitating concentrations in Experiments 1 and 2, no increases in mutant frequency (that exceeded the GEF of 126) were observed at any concentration tested, indicating a negative result. There was no reduction in RTG and no linear trends were observed.Hence, the test item was nut mutagenic in the absence and presence of S-9 when tested up to precipitating concentrations, for 3 hours, in this test system.

Table 7.6.1.4: RTG Values - 3 hour Range-Finder Experiment

Treatment

(µg/mL)

-S-9

% RTG

+S-9

% RTG

0

100

100

1.81

101

125

3.63

86

104

7.25

94

142

14.5

92

81

29.0

89

89

58.0

71

114

Table 7.6.1.5: Summary of mutation data

Experiment 1 (3 Hour Treatment in the absence and presence of S-9)

Treatment

(µg/mL)

-S-9

Treatment

(µg/mL)

+S-9

 

%RTG

MF§

 

%RTG

MF§

0

100

68.33

0

100

83.62

UTC

103

86.58

UTC

104

61.06

10.0

91

86.58

15

99

62.20

15.0

94

72.65

20

112

66.13

20.0

116

71.06

25

82

81.82

25.0

102

79.77

30

104

64.32

30.0

105

68.67

35

88

64.08

35.0

89

108.64

40

107

56.97

40.0

100

87.52

45

97

79.61

45.0

94

81.66

50

90

69.07

50.0

108

80.52

58

80

74.89

58.0

108

76.96

 

 

 

 

 

Linear trend

NS

Linear trend

NS

MMS

 

 

 

B[a]P

 

 

 

15

38

1012.19

2

47

896.47

 

20

20

1652.89

3

27

891.84

 

UTC: untreated culture (culture medium)

Experiment 2 (3 Hour Treatment in the absence and presence of S-9)

Treatment

(µg/mL)

-S-9

Treatment

(µg/mL)

+S-9

 

%RTG

MF§

 

%RTG

MF§

0

100

83.92

0

100

66.55

10.0

88

83.56

10

106

66.33

20.0

87

74.94

20

105

63.10

30.0

98

63.53

30

102

74.17

40.0

100

53.44

40

97

68.86

50.0

95

79.88

50

83

80.76

58.0

96

68.64

58

116

71.25

Linear trend

NS

Linear trend

NS

MMS

 

 

B[a]P

 

 

 

15

51

703.98

2

78

537.58

20

32

1159.11

3

30

1147.18

§                       5-TFT resistant mutants/106viable cells 2 days after treatment

MF                     Mutant frequency

%RTG                 Percentage Relative Total Growth

NS                     Not significant

 

Conclusions:
The test item did not show any mutagenic activity in the mouse lymphoma assay, either in the presence or in the absence of a rat metabolizing system.
Executive summary:

In a mammalian cell gene mutation assay performed according to OECD 476 and in compliance with GLP, the test item diluted in DiMethylFormamide (heated at 80°C) was tested in L5178Y mouse lymphoma cells.

In the cytotoxicity Range-Finder Experiment, six concentrations were tested, in the absence and presence of S9, ranging from 12.5 to 400 µg/mL (limited by solubility in culture medium).

Two independent mutation experiments were performed in the absence and presence of S9. In Experiment 1, ten concentrations, ranging from 10.0 to 58.0 µg/mL for E96096 were tested. In Experiment 2, six concentrations of the test item were tested, ranging from 10.0 to 58 µg/mL.

The positive controls induced the appropriate response and negative controls were valids.

When the test item was tested up to precipitating concentrations in Experiments 1 and 2 no increases in mutant frequency (that exceeded the Global Evaluation Factor of 126) were observed at any concentration tested, indicating a negative result. There was no reduction in Relative Total Growth and no linear trends were observed. Hence, the test item was not mutagenic in the absence and presence of S-9 when tested up to precipitating concentrations, for 3 hours, in this test system.

Under the test consitions employed in this study, the test item did not induce mutation at the tk locus of L5178Y mouse lymphoma cells.

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

Additional information

Gene mutation in bacteria

In a reverse gene mutation assay in bacteria, performed according to the OECD guideline N° 471, in compliance with Good Laboratory Practices, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 100 et TA 98) and Escherichia Coli (WP2uvrA/pKM101) were exposed to the test item at concentrations of 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in two independent experiments both in presence and absence of metabolic activation.

No substantial increases in revertant colony numbers over control count were obtained with any of the tester strains following exposure to the test item at any concentrations in either presence or absence of S9 mix.

The positive control induced the appropriate responses in the corresponding strains.There was no evidence of induced mutant colonies over background.

Under these experimental conditions,the substance did not show any mutagenic activity in the bacterial reverse mutation test.

Gene mutation in mammalian cells

In a mammalian cell gene mutation assay performed according to OECD 476 and in compliance with Good Laboratory Practices, the test item diluted in DiMethylFormamide (heated at 80°C) was tested in L5178Y mouse lymphoma cells.

In the cytotoxicity Range-Finder Experiment, six concentrations were tested, in the absence and presence of S9, ranging from 12.5 to 400 µg/mL (limited by solubility in culture medium).

Two independent mutation experiments were performed in the absence and presence of S9. In Experiment 1, ten concentrations, ranging from 10.0 to 58.0 µg/mL were tested. In Experiment 2, six concentrations of the test item were tested, ranging from 10.0 to 58 µg/mL.

The positive controls induced the appropriate response and negative controls were valids.

When the test item was tested up to precipitating concentrations in Experiments 1 and 2 no increases in mutant frequency (that exceeded the Global Evaluation Factor of 126) were observed at any concentration tested, indicating a negative result. There was no reduction in Relative Total Growth and no linear trends were observed. Hence, the test item was not mutagenic in the absence and presence of S-9 when tested up to precipitating concentrations, for 3 hours, in this test system.

Under the test consitions employed in this study, the test item did not induce mutation at the tk locus of L5178Y mouse lymphoma cells.

Chromosomal aberrations in mammalian cells

An in vitro chromosomal aberration assay was conducted to evaluate the clastogenic potential of the test substance in human lymphocytes according to the OECD Guideline 473 and in compliance with Good Laboratory Practices.

Human lymphocytes, in whole blood culture, were exposed to the test substance both in the absence and presence of S9 mix derived from rat livers. Three hours before the end of the incubation period, cell division was arrested using Colcemid®. The cells were then harvested and slides prepared, so that metaphase cells could be examined for chromosomal damage. On the basis of the mitotic index data obtained from a toxicity test, the following concentrations were selected for two independent tests evaluating the metaphase analysis:  in the Test 1, for 3-h treatment without S9 mix dose levels of 0, 1000, 2000, 3250 and 3500 µg/mL and for 3-h treatment with S9 mix dose levels of 0, 1250, 2500 and 5000 µg/ml ; in the test 2, for 20 -hr treatment without S9 mix dose levels of: 0, 1500, 2000 and 2500 µg/mL and for 3-h treatment with S9 mix dose levels of 0, 1250, 2500 and 4500 µg/mL .

Concurrent solvent and positive controls (mitomycin-C (in the absence of S9 mix) and cyclophosphamide (in the presence of S9 mix)) were also included. In both tests, the substance caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations at any concentration in the absence and presence of S9 mix when compared with the vehicle control. Also, no statistically significant increases in the proportion of polyploid or endoreduplicated metaphase cells were observed during metaphase analysis when compared with the vehicle control. All positive control compounds caused statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix. Under the study conditions, the test substance was therefore not considered to be clastogenic to human lymphocytes in the in vitro chromosomal aberration assay.



Justification for classification or non-classification

Based on the results from three in vitro guideline compliant assays, the substance is not classified for genotoxicity according to regulation (EC) No. 1272/2008 and its subsequent amendments on classification, labeling and packaging (CLP) of substances and mixtures.