Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on a bacterial reverse mutation assay according to OECD Guideline 471 the test substance was considered to be non-mutagenic with and without metabolic activation (reference 7.6.1-1).

Based on an in vitro mammalian cell gene mutation assay (V79/HPRT) according to OECD Guideline 476 the test substance did not show a mutagenic potential (reference 7.6.1-2).

Based on chromosomal aberration test in human lymphocytes according to OECD guideline 473 the test item is considered to be non-clastogenic (reference 7.6.1-3)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
19 July 2017 - 28 August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
cf. Sofuni, 1993
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
ß-Naphthoflavone/Phenobarbital induced rat liver S9
Test concentrations with justification for top dose:
5, 15.8, 50, 158, 500, 1580 and 5000 µg/plate
top dose: maximum recommended concentration according to OECD guideline 471
Vehicle / solvent:
- Vehicle/solvent used: ultrapure water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
sodium azide
other: daunomycin, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 2 days

NUMBER OF REPLICATIONS: 3 (two independent experiments)

DETERMINATION OF CYTOTOXICITY
- Method: counting numbers of revertant colonies
Evaluation criteria:
A test material was to be defined as positive or mutagenic in this assay if
- the assay is considered valid and
- a biologically relevant increase in the mean number of revertants above a threshold of 2¬fold (TA 98, TA 100, WP2 uvrA) or 3-fold (TA 1535, TA 1537) as compared to the concurrent negative controls is observed
- an increase exceeding the threshold at only one concentration is considered as biologically meaningful if reproduced in a second independent experiment - a concentration-dependent increase is considered biologically meaningful if the threshold is exceeded at more than one concentration

A test material is defined as negative or non-mutagenic in this assay if
- the assay is considered valid and
- none of the above mentioned criteria are met
Key result
Species / strain:
S. typhimurium TA 1535
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
Key result
Species / strain:
S. typhimurium TA 1537
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
Key result
Species / strain:
S. typhimurium TA 98
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
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at 5000 µg/plate, only at the beginning of the experiment

Table 1: Summary 1st Series

Metabolic Activation

Test Material

Concentr. [µg/plate]

Revertants per plate (Mean ± SD)

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

Without Activation

H2O

 

25 ± 6

125 ± 9

17 ± 3

8 ± 2

29 ± 6

Test item

5.00

26 ± 5

128 ± 9

22 ± 5

7 ± 4

34 ± 5

15.8

36 ± 9

139 ± 19

18 ± 1

10 ± 5

37 ± 6

50.0

22 ± 3

137 ± 14

15 ± 3

9 ± 3

36 ± 5

158

20 ± 6

134 ± 21

18 ± 6

8 ± 1

41 ± 5

500

25 ± 4

131 ± 14

20 ± 2

9 ± 4

39 ± 6

1580

27 ± 4

145 ± 9

19 ± 6

12 ± 4

33 ± 1

5000

22 ± 7B

171 ± 13B

22 ± 4B

6 ± 1B

40 ± 8B

DAUN

1.00

226 ± 27

 

 

 

 

NaN3

2.00

 

1725 ± 52

906 ± 14

 

 

9-AA

50.0

 

 

 

1235 ± 401

 

NQO

2.00

 

 

 

 

1975 ± 85

With Activation

H2O

 

41 ± 5

138 ± 6

19 ± 3

10 ± 4

35 ± 6

Test item

5.00

28 ± 1C

143 ± 21

19 ± 3

11 ± 3

43 ± 3

15.8

29 ± 10

149 ± 9

18 ± 15

10 ± 0

33 ± 3

50.0

35 ± 5

136 ± 14

14 ± 7

9 ± 6

49 ± 4

158

31 ± 4

152 ± 6

18 ± 4

9 ± 3

36 ± 6

500

35 ± 5

192 ± 19

16 ± 4

8 ± 3

37 ± 1

1580

31 ± 8

170 ± 22

20 ± 1

4 ± 2

47 ± 4

5000

25 ± 4B

146 ± 19B

15 ± 1B

4 ± 3B

33 ± 2B

2-AA

2.00

969 ± 226

1720 ± 57

 

 

 

2-AA

5.00

 

 

153 ± 28

351 ± 18

 

2-AA

10.0

 

 

 

 

377 ± 34

 Key to Positive Controls

NaN3    Sodium azide

2-AA     2-Aminoanthracene

9-AA     9-Aminoacridine

DAUN   Daunomycin

NQO     4-Nitroquinoline-N-oxide

Key to Plate Postfix Codes

B          Precipitation at beginning of experiment

C          Contaminated

 

Table 1: Summary 2nd Series

Metabolic Activation

Test Material

Concentr. [µg/plate]

Revertants per plate (Mean ± SD)

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

Without Activation

H2O

 

31 ± 11

125 ± 6

20 ± 5

8 ± 3

44 ± 6

Test item

158

22 ± 5

127 ± 3

29 ± 5

8 ± 1

49 ± 4

500

37 ± 4

152 ± 7

21 ± 4

7 ± 2

33 ± 4

1580

24 ± 7

125 ± 14

27 ± 5

8 ± 3

47 ± 9

2810

24 ± 2

135 ± 17

21 ± 6

8 ± 1

50 ± 6

5000

20 ± 3B

138 ± 11B

26 ± 11B

7 ± 4B

51 ± 11B

DAUN

1.00

265 ± 4

 

 

 

 

NaN3

2.00

 

1721 ± 43

989 ± 2

 

 

9-AA

50.0

 

 

 

804 ± 190

 

NQO

2.00

 

 

 

 

1988 ± 166

With Activation

H2O

 

40 ± 3

122 ± 12

17 ± 4

11 ± 5

47 ± 11

Test item

158

30 ± 5

140 ± 17

17 ± 5

9 ± 3

51 ± 9

500

39 ± 9

157 ± 15

19 ± 5

11 ± 3

47 ± 10

1580

35 ± 6

154 ± 12

15 ± 8

11 ± 5

44 ± 5

2810

25 ± 3

142 ± 12

11 ± 6

4 ± 2

41 ± 4

5000

29 ± 3B

128 ± 16B

11 ± 8B

8 ± 2B

43 ± 4B

2-AA

2.00

349 ± 55

781 ± 57

 

 

 

2-AA

5.00

 

 

162 ± 21

349 ± 59

 

2-AA

10.0

 

 

 

 

309 ± 7

 Key to Positive Controls

NaN3    Sodium azide

2-AA     2-Aminoanthracene

9-AA     9-Aminoacridine

DAUN   Daunomycin

NQO     4-Nitroquinoline-N-oxide

Key to Plate Postfix Codes

B           Precipitation at beginning of experiment

 

 

Table 3: Historical Data

Negative Controls

Strain

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

S9 Mix

Without

With

Without

With

Without

With

Without

With

Without

With

Compound

Solvent

Solvent

Solvent

Solvent

Solvent

Solvent

Solvent

Solvent

Solvent

Solvent

Total Plates

234

234

229

234

170

170

190

190

190

189

Number of Values

45

45

44

45

29

29

34

34

34

34

Minimum

19

20

94

90

15

6

4

6

22

28

Maximum

52

58

152

151

38

28

11

15

39

45

Mean

38

44

120

125

25

21

8

9

30

37

Standard Deviation

7.3

7.6

12.4

12.5

5.8

4.2

1.6

1.9

5.2

4.4

Positive Controls

Strain

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

S9 Mix

Without

With

Without

With

Without

With

Without

With

Without

With

Compound

DAUN

2-AA

NaN3

2-AA

NaN3

2-AA

9-AA

2-AA

NQO

2-AA

Total Plates

117

117

115

117

85

85

95

95

95

95

Number of Values

45

45

44

45

29

29

34

34

34

34

Minimum

84

112

821

437

351

43

247

88

872

187

Maximum

779

3015

2376

3429

2149

758

1485

705

2275

696

Mean

285

816

1524

1411

869

186

673

299

1704

375

Standard Deviation

155.6

532.0

238.7

736.6

279.1

126.1

291.1

174.9

337.2

140.5

 

Conclusions:
The test substance was considered to be non-mutagenic with and without metabolic activation in bacteria.
Executive summary:

The present study was conducted to investigate the test material for its mutagenic potential in a bacterial reverse gene mutation assay in the absence and presence of a rat liver metabolizing system (S9 mix).

The investigations for the mutagenic potential of the test item were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 1535 and TA 1537 and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from rats pretreated with (3-Naphthoflavone/Phenobarbital was used. In this study, two experimental series were performed. The S9 mix used contained 10% S9 in the 1st and 20% S9 in the 2nd series, respectively.

Solvent and positive control treatments were included for all strains. The mean numbers of revertant colonies were all within acceptable ranges for solvent control treatments, or were clearly elevated by positive control treatments, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.

Following treatment of all bacteria tester strains with the test item in the absence and presence of S9 mix, no relevant increases in revertant numbers were observed. It was concluded that with and without addition of S9 mix as the exogenous metabolizing system, the test item was not mutagenic under the experimental conditions described.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
08 June 2017 - 23 August 2017
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:
2016-07-29
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2008-05-30
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

CELLS USED
- Source of cells: Laboratory for Mutagenicity Testing; Techni-cal University, 64287 Darmstadt, Germany)
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years. Especially the high proliferation rate and a good cloning efficiency of untreated cells (as a rule more than 50%) both necessary for the appropriate performance of the study, recommend the use of this cell line.
- Doubling time: 12 - 16 h
- Methods for maintenance in cell culture: Thawed stock cultures were propagated at 37 °C in 75 cm² plastic flasks. About 2-3×10E6 cells were seeded into each flask with 15 mL of MEM (minimal essential medium) containing Hank’s salts supplemented with 10 % foetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1 %). The cells were sub-cultured once or twice weekly. All incubations were done at 37 °C with 1.5 % carbon dioxide (CO2) in humidified air.
- Modal number of chromosomes: 22

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: For seeding of the cell cultures the complete culture medium was MEM (minimal essential medium) containing Hank’s salts, neomycin (5 μg/mL), 10 % FBS, and amphotericin B (1 %). During treatment no FBS was added to the medium. For the selection of mutant cells the complete medium was supplemented with 11 μg/mL 6-thioguanine. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/Beta-Naphtoflavone induced Rat liver S9
Test concentrations with justification for top dose:
The maximum concentration was limited by phase separation visible at the end of the treatment time (4 hours), in accordance with the OECD 476.
Experiment I
4 hours treatment without S9 mix: 0.13; 0.24; 0.31; 0.40 (PS) µL/mL
4 hours treatment with S9 mix: 0.13; 0.24; 0.31; 0.40 (PS) µL/mL

Experiment IA:
without S9 mix: 0.13; 0.24; 0.31; 0.40 (PS); 0.52 (PS) µL/mL

PS= Phase Separation visible to the unaided eye at the end of treatment

Vehicle / solvent:
Solvent/vehicle: deionized water, final concentration in culture medium 10 %
Justification for choice of solvent/vehicle: Solubility and relatively low cytotoxicity in accordance with OECD 476
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
300 µg/mL, without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
2.3 µg/mL, with metabolic activation
Details on test system and experimental conditions:
Two independent experiments were performed. In experiment I the treatment duration was 4 hours with and without metabolic activation. Due to equivocal results in experiment I in the absence of S9 mix, this part of the experiment was repeated under identical experimental conditions (reported as Experiment IA). Two parallel cultures were used throughout the assay.

METHOD OF APPLICATION: in medium
- Cell density at seeding: Two to four days after sub-cultivation stock cultures were trypsinized at 37 °C for approximately 5 to 10 minutes. Then the enzymatic digestion was stopped by adding complete culture medium with 10 % FBS and a single cell suspension was prepared. The trypsin concentration for all sub-culturing steps was 0.2 % in saline. Prior to the trypsin treatment the cells were rinsed with PBS. Approximately 0.7 to 1.2×10E7 were seeded in plastic flasks. The cells were grown for 24 hours prior to treatment.

DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation, Experiment IA: 4 hours without metabolic activation
- Expression time (cells in growth medium): 72 hours
- Selection time: 10 days

SELECTION AGENT (mutation assays): 6-Thioguanine

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: >1.5x10E6

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:
A test item is classified as positive if it induces a concentration-related increase of the mutant frequency exceeding the historical solvent control range.
A test item producing no concentration-related increase of the mutant frequency above the historical solvent control range is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces with at least one of the concentrations in both parallel cultures a mutation frequency that exceeds the historical negative and solvent control data range (95 % confidence interval limits).
The increase should be significant and dose dependent as indicated by statistical analysis (linear regression, least squares).
Statistics:
A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated 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.
A t-Test was performed using a validated test script of “R”, a language and environment for statistical computing and graphics, to evaluate an isolated increase of the mutation frequency at a test point exceeding the 95 % confidence interval. Again a t-test is judged as significant if the p-value (probability value) is below 0.05. However, both, biological and statistical significance were 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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Pre-experiment: Not effected (pH 7.45 in the solvent control versus pH 7.01 at 2.0 µL/mL)
- Effects of osmolality: Pre-experiment: No relevant increase (284 mOsm in the solvent control versus 318 mOsm at 2.0 µL/mL)
- Evaporation from medium: Not examined
- Phase separation: determined at 0.40 µL/mL with and without metabolic activation
- Other confounding effects: None


RANGE-FINDING/SCREENING STUDIES:
According to the current OECD Guideline for Cell Gene Mutation Tests at least four analysable concentrations should be used in two parallel cultures. For freely-soluble and non-cytotoxic test items the maximum concentration should be 2 mg/mL, 2 µL/mL or 10 mM, whichever is the lowest. For cytotoxic test items the maximum concentration should result in approximately 10 to 20 % relative survival or cell density at subcultivation and the analysed concentrations should cover a range from the maximum to little or no cytotoxicity. Relatively insoluble test items should be tested up to the highest concentration that can be formulated in an appropriate solvent as solution or homogenous suspension. These test items should be tested up to or beyond their limit of solubility. Precipitation or phase separation should be evaluated at the beginning and at the end of treatment by the unaided eye.
The pre-experiment was performed in the presence and absence of metabolic activation. Test item concentrations between 0.04 µL/mL and 2 µL/mL were used. The highest concentration was chosen with respect to the current OECD Guideline 476.
In the pre-experiment toxic effects were observed after 4 hours treatment at 0.65 µL/mL and above in the absence of S9 mix and at 2 µL/mL in the presence of S9 mix.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 hours) before the test item was removed. Phase separation occurred at 1.14 µL/mL and above in the presence and absence of metabolic activation.
There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.
The concentrations used in the main experiment were selected based on the data of the pre-experiment. The maximum concentration was 1.14 µL/mL. The individual concentrations were spaced by a factor of 1.3.
To overcome problems with possible deviations in toxicity the main experiment was started with more than four concentrations.


COMPARISON WITH HISTORICAL CONTROL DATA: Complies


ADDITIONAL INFORMATION ON CYTOTOXICITY:
In experiment I no relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50 % in both cultures occurred up to the maximum concentration with and without metabolic activation.
In experiment IA no relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50 % in both cultures occurred up to the maximum concentration without metabolic activation.
Summary Table

Culture I

 

 

 

 

relative

relative

rel. adjusted

mutant

95 %

 

conc.

S9

cloning

cell

cloning

colonies/

confidence

 

µL/mL

PS

mix

efficiency I

density

efficiency I

10E6

interval

 

 

 

 

%

%

%

cells

 

Experiment I without S9 mix

 

 

 

 

 

 

 

 

Solvent control with water

-

100.0

100.0

100.0

9.7

1.7 - 30.2

Positive control (EMS)

300.0 µg/mL

-

92.7

81.7

75.7

325.5

1.7 - 30.2

Test item

0.13

-

73.0

125.0

91.3

25.4

1.7 - 30.2

Test item

0.24

-

80.7

87.3

70.5

30.9

1.7 - 30.2

Test item

0.31

-

91.1

83.3

75.9

34.5

1.7 - 30.2

Test item

0.40

PS

-

95.5

80.0

76.4

32.5

1.7 - 30.2

Test item

0.52

PS

-

#

#

#

#

#

Test item

0.67

PS

-

#

#

#

#

#

Test item

0.88

PS

-

#

#

#

#

#

Test item

1.14

PS

-

#

#

#

#

#

Experiment I with S9 mix

 

 

 

 

 

 

 

 

Solvent control with water

+

100.0

100.0

100.0

11.2

2.0 - 29.4

Positive control (DMBA)

2.3 µg/mL

+

84.2

93.9

79.0

145.6

2.0 - 29.4

Test item

0.13

+

93.6

78.2

73.2

33.6

2.0 - 29.4

Test item

0.24

+

97.9

75.0

73.4

14.9

2.0 - 29.4

Test item

0.31

+

95.2

89.8

85.5

30.3

2.0 - 29.4

Test item

0.40

PS

+

108.8

83.6

90.9

16.8

2.0 - 29.4

Test item

0.52

PS

+

#

#

#

#

#

Test item

0.67

PS

+

#

#

#

#

#

Test item

0.88

PS

+

#

#

#

#

#

Test item

1.14

PS

+

#

#

#

#

#

Experiment IA without S9 mix

 

 

 

 

 

 

 

 

Solvent control with water

-

100.0

100.0

100.0

32.1

1.7 - 30.2

Positive control (EMS)

300.0 µg/mL

-

99.3

104.8

104.0

247.9

1.7 - 30.2

Test item

0.13

-

100.3

72.5

72.8

9.6

1.7 - 30.2

Test item

0.24

-

101.1

93.6

94.6

17.9

1.7 - 30.2

Test item

0.31

-

100.4

88.5

88.9

16.9

1.7 - 30.2

Test item

0.40

PS

-

101.2

68.4

69.2

16.6

1.7 - 30.2

Test item

0.52

PS

-

96.4

77.6

74.8

31.5

1.7 - 30.2

Test item

0.67

PS

-

#

#

#

#

#

Test item

0.88

PS

-

#

#

#

#

#

Test item

1.14

PS

-

#

#

#

#

#

 

Culture II

 

 

 

 

relative

relative

rel. adjusted

mutant

95%

 

conc.

S9

cloning

cell

cloning

colonies/

confidence

 

µL/mL

PS

mix

efficiency I

density

efficiency I

10 exp. 6

interval

 

 

 

 

%

%

%

cells

Experiment I without S9 mix

 

 

 

 

 

 

 

 

Solvent control with water

-

100.0

100.0

100.0

18.4

1.7 - 30.2

Positive control (EMS)

300.0 µg/mL

-

89.2

77.5

69.1

317.8

1.7 - 30.2

Test item

0.13

-

102.4

86.9

88.9

24.6

1.7 - 30.2

Test item

0.24

-

100.0

79.8

79.8

19.1

1.7 - 30.2

Test item

0.31

-

96.3

80.6

77.7

31.3

1.7 - 30.2

Test item

0.40

PS

-

92.8

63.7

59.1

21.7

1.7 - 30.2

Test item

0.52

PS

-

#

#

#

#

#

Test item

0.67

PS

-

#

#

#

#

#

Test item

0.88

PS

-

#

#

#

#

#

Test item

1.14

PS

-

#

#

#

#

#

Experiment I with S9 mix

 

 

 

 

 

 

 

 

Solvent control with water

+

100.0

100.0

100.0

18.4

2.0 - 29.4

Positive control (DMBA)

2.3 µg/mL

+

92.4

98.0

90.5

179.6

2.0 - 29.4

Test item

0.13

+

95.4

86.7

82.7

22.6

2.0 - 29.4

Test item

0.24

+

97.5

77.0

75.1

16.2

2.0 - 29.4

Test item

0.31

+

98.1

97.5

95.7

17.5

2.0 - 29.4

Test item

0.40

PS

+

84.5

87.4

73.8

26.6

2.0 - 29.4

Test item

0.52

PS

+

#

#

#

#

#

Test item

0.67

PS

+

#

#

#

#

#

Test item

0.88

PS

+

#

#

#

#

#

Test item

1.14

PS

+

#

#

#

#

#

Experiment IA without S9 mix

 

 

 

 

 

 

 

 

Solvent control with water

-

100.0

100.0

100.0

27.0

1.7 - 30.2

Positive control (EMS)

300.0 µg/mL

-

98.7

72.1

71.1

185.5

1.7 - 30.2

Test item

0.13

-

103.7

86.6

89.8

26.0

1.7 - 30.2

Test item

0.24

-

101.6

110.7

112.5

21.0

1.7 - 30.2

Test item

0.31

-

102.6

79.9

82.0

19.6

1.7 - 30.2

Test item

0.40

PS

-

102.1

78.5

80.2

21.0

1.7 - 30.2

Test item

0.52

PS

-

107.4

68.9

74.0

25.9

1.7 - 30.2

Test item

0.67

PS

-

#

#

#

#

#

Test item

0.88

PS

-

#

#

#

#

#

Test item

1.14

PS

-

#

#

#

#

#

 CE = Cloning efficiency

PS = phase separation visible to the unaided eye at the end of treatment

# culture was not continued to avoid analysis of too many concentrations with phase separation

Statistical analysis:

Linear Regression Analysis

 

experimental group

p-value

Experiment I, culture I without S9 mix

0.033s

Experiment I, culture II without S9 mix

0.508

Experiment I, culture I with S9 mix

0.799

Experiment I, culture II with S9 mix

0.520

Experiment IA, culture I without S9 mix

0.895

Experiment IA, culture II without S9 mix

0.467

T-Test

 

experimental group

p-value

Experiment I without S9 mix, 0.2 µL/mL

0.010s

Experiment I without S9 mix, 0.3 µL/mL

0.000s

Experiment I without S9 mix, 0.4 µL/mL

0.008s

Experiment I with S9 mix, 0.1 µL/mL

0.002s

Experiment I, with S9 mix, 0.3 µL/mL

0.040s

Experiment IA without S9 mix, 0.52 µL/mL

0.675

S = significant (p< 0.05)

Conclusions:
In an in vitro mammalian cell gene mutation assay (V79/HPRT) according to OECD Guideline 476, the test substance did not show a mutagenic potential.
Executive summary:

The study was performed to investigate the potential of tetrachloro-μ-hydroxy(μ-methacrylato-O:O')dichromium to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster according to OECD Guideline 476. The treatment period was 4 hours at concentrations of 0.13, 0.24, 0.31, and 0.40 µL/mL with and without metabolic activation (S9 mix, Phenobarbital/beta-naphthoflavone induced rat liver). No relevant and reproducible increase in mutant colony numbers/10E6 cells was observed in the main experiments up to the maximum concentration. Appropriate reference mutagens, 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. Under the experimental conditions reported the test substance did not induce gene mutations at the HPRT locus in V79 cells.Therefore, it is considered to be non-mutagenic in this HPRT assay.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
31 May 2017 - 05 September 2017
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:
2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: Chromosome Aberration Test in mammalian cells
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: healthy non-smoking donors
- Suitability of cells: Yes. The lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation with PHA and to positive control substances.
- Sex, age and number of blood donors: Blood Experiment I: female donor (26 years old), Experiment II: female donor (31 years old)
- Whether whole blood or separated lymphocytes were used : whole blood was used

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Dulbecco's Modified Eagles Medium/Ham's F12; 5.5 % CO2
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
With metabolic activation:
Experiment I: 0.01, 0.02, 0.04, 0.07, 0.12, 0.21, 0.37, 0.65, 1.14, 2.00 µL/mL

Without metabolic activation:
Experiment I: 0.01, 0.02, 0.04, 0.07, 0.12, 0.21, 0.37, 0.65, 1.14, 2.00 µL/mL
Experiment II: 0.04, 0.07, 0.12, 0.21, 0.37, 0.65, 1.14, 2.00 µL/mL
Vehicle / solvent:
- Vehicle/solvent used: Deionized water
- Justification for choice of solvent/vehicle: solubility and low cytotoxicity in accordance with OECD Guideline 473
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in culture medium

DURATION
- Exposure duration: 4 hours (+/- S9 mix) and 22 hours (- S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: about 1.5

NUMBER OF CELLS EVALUATED: 150 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: in 250 metaphase cells per culture
- Determination of endoreplication: in 250 metaphase cells per culture

Evaluation criteria:
Evaluation of the slides was performed according to the standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik" using microscopes with 100 x oil immersion objectives. Cytotoxicity is characterized by the percentages of mitotic suppression in comparison with the controls by counting 1000 cells per culture in duplicate. 150 well-spread metaphases were evaluated per culture for structural aberrations. Only metaphases containing a number of centromeres equal to a number of 46 ± 2 were included in the analysis. Breaks, fragments, deletions, exchanges and chromosomal disintegrations are recorded as structural chromosomal aberrations. Gaps were recorded as well, but they are not included in the calculation of the aberration rates since gaps are achromatic lesions of unknown biological relevance for which a clear relationship to treatment cannot be established. In addition, the number of polyploid cells in 250 metaphase cells per culture (% polyploid metaphases) was evaluated.
Statistics:
Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).
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 examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Other confounding effects: Phase separation was observed in Experiment I at 0.65 µL/mL and above in the absence of S9 mix and at 0.37 µL/mL and above in the presence of S9 mix. In Experiment II, phase separation was observed at 1.14 µL/mL and above in the absence of S9 mix.

RANGE-FINDING/SCREENING STUDIES: A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: mitotic index

Table 1: Summary of results

 Exp.

Prepa-

ration
interval 

 Test item
concen-

tration
in µL/mL

 Poly-

ploid
cells
in %

Endo-

mitotic

cells in % 

Mitotic

indices

in %

of control 

Aberrant cells

in %

incl. gaps*


 Aberrant cells

in %

excl. gaps*


 Aberrant cells

in % 

carrying exchanges

Exposure period 4 hrs without S9 mix

I

22 hrs

Solvent control1

0.0

0.0

100.0

0.3

0.3

0.0

 

 

Positive control2

n.d.

n.d.

71.8

9.3

9.3S

1.7

 

 

0.21

0.0

0.0

87.5

1.3

1.3

0.3

 

 

0.37

0.2

0.0

88.7

0.0

0.0

0.0

 

 

0.65PS

0.2

0.0

94.2

0.7

0.7

0.0

Exposure period 22 hrs without S9 mix

II

22 hrs

Solvent control1

0.2

0.0

100.0

1.3

1.0

0.0

 

 

Positive control3

n.d.

n.d.

44.3

17.3

17.0S

3.7

 

 

0.37

0.0

0.0

100.0

1.7

1.3

0.0

 

 

0.65

0.0

0.0

87.3

1.0

1.0

0.0

 

 

1.14PS

0.0

0.0

94.5

0.3

0.0

0.0

Exposure period 4 hrs with S9 mix

I

22 hrs

Solvent control1

0.4

0.2

100.0

1.7

1.7

0.0

 

 

Positive control4

n.d.

n.d.

77.4

10.7

10.3S

0.7

 

 

0.12

0.2

0.0

87.9

1.0

1.0

0.0

 

 

0.21

0.4

0.0

109.9

2.3

2.3

0.0

 

 

0.37PS

0.6

0.2

109.3

1.7

1.7

0.0

*       Including cells carrying exchanges

n.d.  Not determined

PS    Phase separation occurred at the end of treatment

S       Aberration frequency statistically significant higher than corresponding control values

1       Deionized water 10.0 % (v/v)

2       EMS: 825µg/mL

3       EMS: 550 µg/mL

4       CPA: 7.5 µg/mL

Conclusions:
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro.
Therefore, the test item is considered to be non-clastogenic in this chromosome aberration test.
Executive summary:

The test item, dissolved in deionized water, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation (S9 mix) according to OECD guideline 473.

Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure period was 22 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item. In each experimental group two parallel cultures were analysed. 150 metaphases per culture were evaluated for structural chromosomal aberrations. 1000 cells were counted per culture for determination of the mitotic index. The highest treatment concentration in this study, 2.00 μL/mL was chosen with respect to the OECD Guideline for in vitro mammalian cytogenetic tests. 

Phase separation was observed in Experiment I at 0.65 μL/mL and above in the absence of S9 mix and at 0.37 μL/mL and above in the presence of S9 mix. In Experiment II, phase separation was observed at 1.14 μL/mL and above in the absence of S9 mix. No relevant influence on osmolarity or pH was observed. In both experiments, in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation. In this study, neither with nor without metabolic activation, clastogenicity was observed at the concentrations evaluated. In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (0.0 – 0.6 %) as compared to the rates of the solvent controls (0.0 – 0.4 %). In both experiments, either EMS (550 or 825 μg/mL) or CPA (7.5 μg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro.

Therefore, the test item is considered to be non-clastogenic in this chromosome aberration test.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Bacterial Reverse Mutation Test (reference 7.6.1-1)

A study according to OECD guideline 471 was conducted to investigate the test material for its mutagenic potential in a bacterial reverse gene mutation assay in the absence and presence of a rat liver metabolizing system (S9 mix). The investigations for the mutagenic potential of the test item were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 1535 and TA 1537 and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from rats pretreated with (3-Naphthoflavone/Phenobarbital was used. In this study, two experimental series were performed. The S9 mix used contained 10% S9 in the 1st and 20% S9 in the 2nd series, respectively. Solvent and positive control treatments were included for all strains. The mean numbers of revertant colonies were all within acceptable ranges for solvent control treatments, or were clearly elevated by positive control treatments, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used. Following treatment of all bacteria tester strains with the test item in the absence and presence of S9 mix, no relevant increases in revertant numbers were observed. It was concluded that with and without addition of S9 mix as the exogenous metabolizing system, the test item was not mutagenic under the experimental conditions described.

HPRT (reference 7.6.1-2)

The study was performed to investigate the potential of tetrachloro-μ-hydroxy(μ-methacrylato-O:O')dichromium to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster according to OECD Guideline 476. The treatment period was 4 hours at concentrations of 0.13, 0.24, 0.31, and 0.40 µL/mL with and without metabolic activation (S9 mix, Phenobarbital/beta-naphthoflavone induced rat liver). No relevant and reproducible increase in mutant colony numbers/10E6 cells was observed in the main experiments up to the maximum concentration. Appropriate reference mutagens, 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. Under the experimental conditions reported the test substance did not induce gene mutations at the HPRT locus in V79 cells. Therefore, it is considered to be non-mutagenic in this HPRT assay.

Chromosomal aberration test (reference 7.6.1-3)

The test item, dissolved in deionized water, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation (S9 mix) according to OECD guideline 473. Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure period was 22 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item. In each experimental group two parallel cultures were analysed. 150 metaphases per culture were evaluated for structural chromosomal aberrations. 1000 cells were counted per culture for determination of the mitotic index. The highest treatment concentration in this study, 2.00 μL/mL was chosen with respect to the OECD Guideline for in vitro mammalian cytogenetic tests. Phase separation was observed in Experiment I at 0.65 μL/mL and above in the absence of S9 mix and at 0.37 μL/mL and above in the presence of S9 mix. In Experiment II, phase separation was observed at 1.14 μL/mL and above in the absence of S9 mix. No relevant influence on osmolarity or pH was observed. In both experiments, in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation. In this study, neither with nor without metabolic activation, clastogenicity was observed at the concentrations evaluated. In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (0.0 – 0.6 %) as compared to the rates of the solvent controls (0.0 – 0.4 %).In both experiments, either EMS (550 or 825 μg/mL) or CPA (7.5 μg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro. Therefore, the test item is considered to be non-clastogenic in this chromosome aberration test.

Justification for classification or non-classification

Classification, Labeling, 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. The results indicate that the test substance is non-mutagenic. Based on available data on genetic toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EC) No 2017/776.