Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 June - 11 August 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study in accordance with international guidelines.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
thymidine kinase locus (Tk1)
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Cleansed and for mycoplasma contamination screened stocks of cells were stored in liquid nitrogen in the cell bank of LAUS GmbH to allow a continuous stock of cells, which guarantees similar parameters of the experiment and reproducible characteristics of the cells.
Cleansed and for mycoplasma contamination screened cells were thawed 6 - 7 days prior to treatment and cultivated in RPMI 1640 complete culture medium with 5 % HS in cell culture flasks at 37.0 ± 1.5 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2. After thawing, the cells were again screened for mycoplasma contamination before they were used in the experiments.
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from male rats, treated with Aroclor 1254
Test concentrations with justification for top dose:
nominal concentrations in pre-test [µL/mL] 5.00 2.50 1.25 0.63 0.31 0.16 0.08
nominal concentrations in experiment I +S9 [µL/mL] 5.00 0.63* 0.31 0.16 0.08 0.04 0.02*
nominal concentrations in experiment I -S9 [µL/mL] 5.00 0.63* 0.31 0.16 0.08 0.04 0.02*
nominal concentrations in experiment II +S9 [µL/mL] 5.00 0.63* 0.31 0.16 0.08 0.04 0.02*
nominal concentrations in experiment II +S9 [µL/mL] 5.00 0.63* 0.31* 0.16 0.08 0.04 0.02
Details on test system and experimental conditions:
Test System
The L5178Y is a murine T-cell lymphoma cell line, which grows as single or aggregated round cells in suspension. This cell line is characterized by a high sensitivity to chemical mutagens, by a high proliferation rate (doubling time 10-12 hours in stock cultures), a high cloning efficiency (CE) and a stable spontaneous mutant frequency. The L5178Y consists of a stable karyotype and shows a diploid chromosome number (40 ± 2). The cells were purchased by ATCC (Wesel, Germany) and were sold under the name L5178Y TK+/- clone (3.7.2C) [TK+/- (clone 3.7.2C)] (ATCC® CRL-9518™).

Cell Cultures
Cleansed and for mycoplasma contamination screened stocks of cells were stored in liquid nitrogen in the cell bank of LAUS GmbH to allow a continuous stock of cells, which guarantees similar parameters of the experiment and reproducible characteristics of the cells.
Cleansed and for mycoplasma contamination screened cells were thawed 6 - 7 days prior to treatment and cultivated in RPMI 1640 complete culture medium with 5 % HS in cell culture flasks at 37.0 ± 1.5 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2. After thawing, the cells were again screened for mycoplasma contamination before they were used in the experiments.


Species / strain:
mouse lymphoma L5178Y cells
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:
In total three experiments were performed of which one was invalid (experiment I) and had to be repeated. Finally, two independent valid experiments, using two parallel cultures each (replicates) were performed. Experiment I was performed with and without metabolic activation and a treatment period of 4 h. Experiment II was performed with 24 h treatment without metabolic activation. The third experiment was performed with 4 h treatment with metabolic activation.
The following concentrations of the test item were investigated in experiments I and II: 5.00 µL/mL, 0.63 µL/mL, 0.31 µL/mL, 0.16 µL/mL, 0.08 µL/mL, 0.04 µL/mL, 0.02 µL/mL.
The nominal and real test item concentrations were identical in this study. Precipitation of the test item was visible at the concentration 5 µL/mL. in both experiments in both ap-proaches. In the test item concentration 0.63 µl/mL and all further dilutions no precipitation was visible with the unaided eye during treatment but during the washing and counting step the test item clumped with the cells and those agglutinations could not be broken up also by intensively resuspension. Accordingly, the counts in those concentrations were all extremely low in comparison to the other concentrations and the controls.
MMS (19.5 µg/mL in experiment I and 12.5 µg/mL in experiment II) and CPA (4.5 µg/mL) were used as positive controls.
In experiment I (+S9 and –S9) a cytotoxic effect was observed in the concentration 0.63 µL/mlL. In experiment II (+S9 and –S9), again a cytotoxic effect was observed in the concentration 0.63 µL/mL (+S9 and -S9) as well as 0.31 µL/mL (-S9). According to the OECD 476 guideline those concentrations, that showed a cytotoxic effect (RTG <20 %) cannot be analysed for mutagenicity. Therefore, these concentrations were not considered in evaluation of the mutant frequencies.
The mutant frequencies for the solvent control DMSO in experiment I and II (+ S9 and –S9) were in the range of 50 – 170 colonies per 106 cells (experiment I: +S9: 128 and -S9: 127; experiment II: +S9: 116, -S9: 82). The mutation frequencies of the solvent controls NaCl 0.9 % (experiment I 127, experiment II 96) and MMS (experiment I 150, experiment II 119) were also in the normal range between 50 – 170 colonies per 106 cells. The positive controls MMS and CPA showed a distinct increase in total mutant frequency of more than 300 in comparison to the respective solvent control.
In all tested concentrations of the test item no substantial and reproducible dose depend-ent increase in mutant colony numbers was observed in both main experiments (see table 8-a and figures 8-a to 8-d). No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item. The mutation frequency did not reach or exceed the threshold of 126 above the corresponding solvent control.

Main results from experiment I and II (Mean values of both cultures)

Content

Relative Total Growth [%]

Mutants per 106Cells

 

Experiment I

Experiment II

Experiment I

Experiment II

 

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

Solvent Control of Test Item

-

-

-

-

129

127

116

82

Solvent Control of Positive Control

-

-

-

-

148

150

96

119

Positive Control

54.8

65.3

47.9

76.5

433

452

498

421

Test Item 5.00 µL/mL

98.1

90.7

102.7

88.1

155

166

150

127

Test Item 0.63 µL/mL

0.1

0.2

0.2

0.0

-

-

-

-

Test Item 0.31 µL/mL

49.1

42.1

77.0

0.0

163

168

163

-

Test Item 0.16 µL/mL

65.0

69.6

87.9

20.2

177

161

166

136

Test Item 0.08 µL/mL

74.6

82.4

86.9

58.8

176

179

218

134

Test Item 0.04 µL/mL

86.7

77.9

100.8

87.0

146

217

170

134

Test Item 0.02 µL/mL

104.9

89.9

84.1

92.0

*

*

*

150

- Cultures could not be evaluated for mutagenicity because of cytotoxicity.

* Cultures were not continued since only 4 analysable concentrations are required by the guideline.

Note: The threshold (number of mutant colonies per 106cells of the respective solvent control plus 126) was 255 (+S9) and 253 (-S9) in Experiment I as well as 242 (+S9) and 208 (-S9) in Experiment II.

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the L5178Y Tk+/- cell line in the absence and the presence of metabolic activation.
The recorded data in this study declare the test item as a non mutagen.
Executive summary:

This study was performed to investigate the potential of the substance to induce mutations at thethymidine kinase locus(Tk1) on chromosome 11 and/or structural chromosomal aberrations in mouse lymphoma L5178YTk+/-cells.

The assay was performed in a pre-test and in two independent main experiments (experiment I and II) whereby the first experiment I was invalid and had to be repeated. The results of the invalid experiment will not be included in this report but will be archived with the raw data. Therefore, in total one pre-test and three experiments were performed.

The pre-test was done to detect a potential cytotoxic effect of the test item. Based on the results of this test the concentrations for the main experiments were determined.

Experiment I was performed with and without metabolic activation (liver enzyme S9 fraction / “liver S9 mix from male rats, treated with Aroclor 1254”) and a treatment period of 4 h. Experiment II was performed with a treatment period of 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.

The highest nominal concentration that was used in the experiment was 5 µL/mL.

Not all tested concentrations could be evaluated for mutagenicity. Some of them induced a cytotoxic reaction and had to be excluded from the evaluation of the mutagenicity. However in all analysable concentrations no substantial and reproducible dose dependent increase in mutant colony numbers was observed in both experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From June 30 to July 10, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study in accordance with OECD TG 471
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium TA 102
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 97
Species / strain / cell type:
S. typhimurium TA 98
Metabolic activation:
with and without
Metabolic activation system:
S9-mix, rat liver S9-mix induced by Aroclor 1254
Test concentrations with justification for top dose:
The following nominal concentrations were prepared for the experiment 1:
5000 µg/plate, 1500 µg/plate, 500 µg/plate, 150 µg/plate and 50 µg/plate.
The following nominal concentrations were prepared for the experiment 2:
5000 µg/plate, 2500 µg/plate, 1250 µg/plate, 625 µg/plate, 313 µg/plate, 156 µg/plate and 78 µg/plate.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 4-Nitro-1,2-phenylene diamine, 2-Amino-anthracene
Details on test system and experimental conditions:
Salmonella typhimurium (all strains used) were obtained from TRINOVA BioChem (batch of the bacteria strains: TA97a: 4904D, TA98: 4903D, TA100: 4902D, TA102: 4872D, TA1535: 4908D) and were stored as lyophilisates in the fridge at 2-8 °C.

8 h before the start of each experiment, one lyophilisate per strain to be used was taken from the fridge to inoculate a culture vessel containing nutrient broth. After incubation over night at 37 ± 1 °C, the cultures were used in the experiment. During the test, the cultures were stored at room temperature as to prevent changes in the titre.
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:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
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
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:
valid
Positive controls validity:
valid
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:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 97
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
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

First experiment

The mean revertant values of the 3 replicates are presented in the following table

Mean Revertants Experiment 1

Strain

TA97a

TA98

TA100

TA102

TA1535

Induction

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

H2O

Mean

257

161

9

13

116

118

243

235

11

11

sd

100.7

2.3

1.5

2.5

4.2

3.5

16.2

11.5

1.7

2.6

DMSO

Mean

168

145

10

16

120

125

205

216

11

12

sd

24.3

42.0

0.6

1.5

5.0

4.7

18.9

21.2

4.2

1.7

Positive
Controls*

Mean

447

451

395

54

587

683

1053

917

193

64

sd

84.0

54.6

25.7

11.8

28.4

15.1

43.1

230.0

53.3

7.4

f(I)

2.66

3.11

39.50

3.38

5.06

5.46

5.14

4.25

17.55

5.33

5000 µg/plate

Mean

103

135

11

12

107

119

160

193

16

16

sd

8.3

40.5

2.3

2.0

4.7

2.5

21.2

10.1

1.0

0.0

f(I)

0.61

0.93

1.10

0.75

0.89

0.95

0.78

0.89

1.45

1.33

1500 µg/plate

Mean

145

160

14

11

114

114

233

197

16

13

sd

39.5

64.4

0.6

1.2

10.1

10.7

64.7

41.6

0.6

2.0

f(I)

0.86

1.10

1.40

0.69

0.95

0.91

1.14

0.91

1.45

1.08

500 µg/plate

Mean

131

137

9

9

116

128

207

140

14

11

sd

40.1

33.3

1.7

1.7

15.6

3.0

11.5

21.2

2.6

0.0

f(I)

0.78

0.94

0.90

0.56

0.97

1.02

1.01

0.65

1.27

0.92

150 µg/plate

Mean

99

108

13

10

109

107

169

255

11

13

sd

6.1

4.0

2.9

3.0

14.7

18.0

16.7

44.1

1.7

1.7

f(I)

0.59

0.74

1.30

0.63

0.91

0.86

0.82

1.18

1.00

1.08

50 µg/plate

Mean

123

121

10

10

111

123

223

215

11

14

sd

43.1

19.7

0.0

0.6

20.1

11.0

24.4

16.7

1.7

2.9

f(I)

0.73

0.83

1.00

0.63

0.93

0.98

1.09

1.00

1.00

1.17

Conclusions:
Interpretation of results (migrated information):
negative

No mutagenicity effects were observed in 5 different strains with and without metabolic activation.
Executive summary:

Two valid experiments was performed

First Experiment:

5 concentrations of the test item, dissolved in DMSO (ranging from 50 to 5000 µg/plate) were used. 5 genetically changed strains ofSalmonella typhimurium(TA97a, TA98, TA100, TA102 and TA1535) were exposed to the test item both in the presence and in the absence of a metabolic activation system (S9-mix, rat liver S9-mix induced by Aroclor 1254) for 48 h, using the plate incorporation method.

None of the concentrations caused a significant increase in the number of revertant colonies in the tested strains. The test item did not show any mutagenic effects in the first experiment.

The test item showed no precipitates on the plates in all tested concentrations.

No signs of toxicity towards the bacteria could be observed.

The sterility control and the determination of the titre did not show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation.

Second Experiment:

To verify the results of the first experiment, a second experiment was performed, using 7  concentrations of the test item (ranging from 78 to 5000 µg/plate) and a modification in study performance (pre-incubation method).

The test item did not show mutagenic effects in the second experiment, either.

The test item showed no precipitates on the plates in all tested concentrations.

No signs of toxicity towards the bacteria could be observed.

The sterility control and the determination of the titre did not show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 17 Jun 2015 to 14 Sept 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study in accordance with international guidelines
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
A cytotoxicity of 55 ± 5% could not be achieved in the 1st and the 3rd experiment without metabolic activation. This is considered uncritical, because the test item showed chromosomal aberrations independent from cytotoxicity.
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: human
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Experiment 1: 5, 0.16, 0.08 and 0.04 µL/mL
Experiment 2: invalid, not reported.
Experiment 3: 5, 0.32, 0.08 and 0.02 µL/mL

Vehicle / solvent:
DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
6.2 Test System
6.2.1 Specification
Human whole blood treated with anti-coagulant (heparin).
6.2.2 Blood Collection
Blood samples were obtained from healthy donors who neither smoke nor receive medication. The following donors were chosen for the experimental part:
Table 6.2 a Blood donors
Experimental part Exposure date Donor Examined for chromo-somal damage
Pre-experiment without S9 17. Jun. 15  female
32 years yes ≙ experiment I
without S9
Pre-experiment with S9 17. Jun. 15  female
32 years yes ≙ experiment I
with S9
Experiment III without S9 22. Jul. 15  female
24 years yes

6.3 Solvent Controls
DMSO was used as solvent control for the test item.
Medium without FCS was used as solvent control for the positive control EMS.
0.9% NaCl was used as solvent control for the positive control CPA.
Evaluation criteria:
Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded separately and reported, but they were not included in the total aberration frequency. Chromosome aberrations were scored according to the classification of ISCN 2013. At least 150 well spread metaphases per culture (positive controls: at least 100 metaphases due to strong clastogenicity) were scored for cytogenetic damage. Only metaphases with 46 +- 2 centromer regions were included in the analysis.
The number of aberrant metaphase cells in each treatment group was compared with the corresponding solvent control value using Fisher’s exact test resp. chi-square-test at the five per cent level (p <0.05).
7.3.2 Classification
A test item is classified as non-mutagenic if:
• None of the test item concentrations shows a statistically significant increase of cells with structural chromosomal aberrations compared with the concurrent negative con-trol.
• No dose-related increase of cells with chromosomal aberrations can be observed.
• The obtained values lie within the range of the historical negative control data.
A test item is classified as mutagenic if:
• At least one of the test item concentrations shows a statistically significant increase of cells with structural chromosomal aberrations compared with the concurrent negative control.
• A dose-related increase of cells with chromosomal aberrations can be observed.
• Any of the obtained values lies outside the historic negative control data.

Species / strain:
lymphocytes: Human
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
lymphocytes: human
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
a statistically significant increase of structural chromosomal aberrations was observed in 3 concentrations of the test item (5, 0.32 and 0.08 µL/mL) in the 3rd experiment and in 1 concentration (0.04 µL/mL) in the 1st experiment with metabolic activation. Furthermore, chromatid exchanges could be observed in both independent experiments (experiment I without metabolic activation: 5 µL/mL; experiment I with metabolic activation: 5, 0.16 and 0.08 µL/mL; experiment III without metabolic activation: 5 and 0.32 µL/mL). This type of aberration is practically never seen in untreated cultures and therefore is also a hint, that the test item has mutagenic properties.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: Experiment I

Results

1.1     Summary of Results Cytotoxicity

1.1.1    Experiment I without S9 - Cytotoxicity

Experiment I without and with metabolic activation was performed using an exposure period of 4 h, culture harvest time was 23 h.The results of the cytotoxicity test of experiment I are presented in the following table:

 Results Cytotoxicity Experiment I without S9

Treatment

Mitotic indices in %

of the solvent control

Experiment I: exposure period 4 h without S9

 

Positive control EMS 300 µg/mL

56.9

 

Positive control EMS 600 µg/mL

108.1

 

Test item 5 µL/mL

101.3

 

Test item 2.5 µL/mL

0.4

 

Test item 1.25 µL/mL

0.0

 

Test item 0.63 µL/mL

7.0

 

Test item 0.32 µL/mL

21.8

 

Test item 0.16 µL/mL

41.5

 

Test item 0.08 µL/mL

64.6

 

Test item 0.04 µL/mL

77.7

 

Experiment I: exposure period 4 h with S9

 

Positive control CPA 25 µg/mL

32.9

 

Positive control CPA 35 µg/mL

32.5

 

Test item 5 µL/mL

113.1

 

Test item 2.5 µL/mL

90.4

 

Test item 1.25 µL/mL

0.0

 

Test item 0.63 µL/mL

0.4

 

Test item 0.32 µL/mL

7.6

 

Test item 0.16 µL/mL

35.9

 

Test item 0.08 µL/mL

59.4

 

Test item 0.04 µL/mL

86.9

 

 

1.1.2    Experiment III without S9 - Cytotoxicity

Experiment III without metabolic activation was performed using an exposure period of 24 h, culture harvest time was 24 h. The results of the cytotoxicity test of experiment III without S9 are presented in the following table:

  Results Cytotoxicity Experiment III without S9

Treatment

Mitotic indices in %

of the solvent control

Experiment III: exposure period 24 h without S9

 

Positive control EMS 300 µg/mL

37.9

 

Positive control EMS 600 µg/mL

n.e.*

 

Test item 5 µL/mL

71.4

 

Test item 0.63 µL/mL

31.4

 

Test item 0.32 µL/mL

52.1

 

Test item 0.16 µL/mL

101.4

 

Test item 0.08 µL/mL

94.8

 

Test item 0.04 µL/mL

104.8

 

Test item 0.02 µL/mL

87.2

 

*n.e.= not evaluated because of very strong cytotoxicity

 


 

1.2     Summary of Results Genotoxicity

1.2.1    Experiment I – Genotoxicity

In the experimental part with metabolic activation, 1 concentration of the test item (0.04 µL/mL) showed a statistically significant (p < 0.05) increase of structural chromosomal aberrations. In a total of 4 cultures chromatid exchanges were observed. This can be assessed as a strong hint, that the test item has mutagenic properties, because this type of aberration is practically never seen in untreated cultures.

The results of experiment I are presented in the following table:

   Results Genotoxicity Experiment I

Treatment

Aberrant cells in %

Inclusive gaps*

Exclusive gaps*

with exchanges

Experiment I: exposure period 4 h without S9

Solvent control serum free medium RPMI 1640

5.7

1.3

--

Solvent control DMSO

5.7

2.7

0.3 (cx1))

Positive control EMS 300 µg/ml

51.6

40.4S

22.0

Test item 5 µL/mL

7.0

2.7

0.3(ex2))

Test item 0.16 µL/mL

6.3

2.0

--

Test item 0.08 µL/mL

9.0

2.3

--

Test item 0.04 µL/mL

7.7

2.0

--

Experiment I: exposure period 4 h with S9

Solvent control DMSO

3.0

1.3

--

Solvent control for the positive control NaCl 0.9 %

4.7

1.7

--

Positive control CPA 25 µg/ml

59.0

45.5S

16.5

Test item 5 µL/mL

6.3

3.0

0.3(ex2))

Test item 0.16 µL/mL

9.7

2.7

0.3(ex2))

Test item 0.08 µL/mL

5.7

3.0

0.3(ex2))

Test item 0.04 µL/mL

8.0

4.0S

--

*Inclusive cells carrying exchanges

SAberration frequency statistically significant higher than the corresponding control values

1)chromosome exchange (e.g. translocation)2)chromatid exchange

Note: in this table, the mean values are stated. Data for the individual cultures are listed in the annex, see chapter14.2, page31.

1.2.2    Experiment III

The 3 highest evaluated concentrations of the test item showed statistically relevant increases of structural chromosomal aberrations. Again, chromatid exchanges could be found in 2 cultures.

The results of experiment III are presented in the following table:

Results Genotoxicity Experiment II

Treatment

Aberrant cells in %

Inclusive gaps*

Exclusive gaps*

with exchanges

Experiment III: exposure period 24 h without S9

Solvent control serum free medium RPMI 1640

4.0

0.3

--

Solvent control DMSO

2.0

0.3

--

Positive control EMS 300mg/mL

57.0

38.0S

15.0

Test item 5 µL/mL

7.0

2.3S

0.3(ex1))

Test item 0.32 µL/mL

8.7

3.3S

0.3(ex1))

Test item 0.08 µL/mL

7.0

2.0S

--

Test item 0.02 µL/mL

5.7

1.7

--

*Inclusive cells carrying exchanges

SAberration frequency statistically significant higher than the corresponding control values

1)chromatid exchange

Conclusions:
Interpretation of results (migrated information):
positive

The test item is considered to possess mutagenic properties, as a statistically significant increase of structural chromosomal aberrations was observed in both experiments, with and without metabolic activation. Furthermore, chromatid exchanges could be observed in both independent experiments.
Executive summary:

3 experiments were performed. The 1stand the 3rdexperiment were valid.The 2ndexperiment was not valid, because the positive control caused complete cytotoxicity.This experiment is not reported in this report, the raw data are kept in the test facility in the GLP- archive.

This study was performed to assess the mutagenic potential of the substance to induce structural chromosomal aberrations in human lymphocytes culturedin vitroin the absence and the presence of an exogenous metabolic activation system (liver S9 mix from male rats, treated with Aroclor 1254).

The test item (whitish waxy pastilles) was melted at 50 °C and the series of dilutions was prepared with pre-heated DMSO (50 °C). 50 µl of the pure test item as well as 50 µL of the resp. solution to be tested was added to the cultures, corresponding to a maximum concentration of 5 µL/mL in the test.

Human lymphocytes, in whole blood culture, were stimulated to divide by addition of phytohaemagglutinin and exposed to medium control, solvent control, test item and positive control both in the presence and the absence of S9 mix derived from rat livers. 3 h before the end of cultivation, cell division was arrested using colcemid, the cells were harvested and slides were prepared. Then, the metaphase cells were examined for chromosomal damage.

The following schedule was observed:

 

Exp. I

Exp. III*

Metabolic activation

without S9 mix

with S9 mix

without S9 mix

Exposure period

4 h

4 h

24 h

Expression time in growth medium

19 h

19 h

--

Culture harvest time

23 h

23 h

24 h

Concentrations selected for metaphase analysis

5, 0.16, 0.08 and 0.04 mL/mL

5, 0.16, 0.08 and 0.04 mL/mL

5, 0.32, 0.08

and

0.02mL/mL

* performed because the results of experiment I were equivocal and experiment II was invalid

In both experiments, all cell cultures were set up in duplicates. In order to assess the toxicity of the test solution to cultured human lymphocytes; the mitotic index was calculated for all cultures treated with medium control, solvent control, test item and positive control. On the basis of the data from the mitotic index, the concentrations (see table 3-a, page7) were selected for metaphase analysis.

In both experiments, toxicity was detected in the intermediate concentrations of the test item. In the 1stexperiment with metabolic activation, a statistically significant increase of structural chromosomal aberrations was detected in the concentration 0.04 µL/mL. In the 3rdexperiment, a statistically significant increase of structural chromosomal aberrations was observed in 3 tested concentrations (5 µL/mL, 0.32 µL/mL and 0.08 µL/mL). Furthermore, biologically relevant types of aberrations were detected at several evaluated concentrations in all independent experiments.

All positive control compounds caused large, statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system.

In conclusion, under the experimental conditions reported, the substance has the ability to induce structural chromosomal aberrations in human lymphocytesin vitro.

The test item is considered as “mutagenic under the conditions of the test”.

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

Genetic toxicity in vivo

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From April 11 to May 25, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The study was not planned under REACH strategy. It was performed for other regulatory purposes.
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: Mammalian Erythrocyte Micronucleus Test
Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
NMRI mice (SPF) were used as the test system. These mice are recommended by international
guidelines (e.g. OECD, EC). Females were nulliparous and non-pregnant. The animals were provided
by Charles River, Sulzfeld, Germany.
Young adult animals were selected (6 weeks old at the start of treatment). The total number of animals
used in the dose range finding study was 3 and in the main study 25. In the micronucleus main study
5 male mice were treated per sampling time in each treatment group.
The body weights of the mice at the start of the treatment in the main study were within 20% of the sex
mean. The mean body weights were 34.3 ± 1.3 g and the range was 32 - 37 g. The mice were
identified by a unique number on the tail written with a marker pen. The animals were allocated at
random to the treatment groups.
The acclimatisation period was at least 6 days before the start of treatment under laboratory
conditions.
On arrival and at the start of the treatment, all animals were clinically examined to ensure selected
animals were in a good state of health.
The animals were housed in room number A0.18.
Conditions
A controlled environment was maintained in the room with optimal conditions of approximately 10 air
changes per hour, a temperature of 21.0 ± 3.0°C (actual range: 21.4 - 22.2°C), a relative humidity of
40 - 70% (actual range: 39 - 56%) and a 12 hour light/12 hour dark cycle. Due to e.g. cleaning
procedures, temporary deviations from the light/dark cycle (with a maximum of 4 hours) and the
minimum\maximum level for humidity (with max. 13%) occurred. Based on laboratory historical data
these deviations are considered not to affect the study integrity.
Accommodation
The animals were group housed (maximum 5 animals per sex per cage) in labelled Macrolon cages
(type MIII height 180 mm, length 380 mm and width 220 mm) containing sterilised sawdust as bedding
material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany). A
shelter (disposable paper corner home, MCORN 404, Datesand Ltd, USA) and paper bedding (Envirodri,
Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom) was provided as cage-enrichment.
Diet
The animals had free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH,
Soest, Germany).
Water
The animals had free access to tap-water.
Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was
performed according to facility standard procedures. There were no findings that could interfere with
the study.
Route of administration:
oral: gavage
Vehicle:
The test item was suspended in propylene glycol (Merck, Darmstadt, Germany). The specific gravity of
propylene glycol is 1.036 g/ml.
Details on exposure:
Test item concentrations were treated with ultra-sonic waves and
heating to approximately 40 - 55ºC to obtain a homogeneous suspension. Test item concentrations
were dosed within 4 hours after preparation.
Frequency of treatment:
Two treatments were performed, administered at a 24-hour interval. The test item was administered as
a split dose, i.e., two treatments on the same day separated by no more than 2-3 hours, to facilitate
administering a large volume necessary due to limited solubility of the test item.
Post exposure period:
48 h
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 males for each dose group.
Control animals:
yes, concurrent vehicle
Positive control(s):
The positive control used in the micronucleus test was cyclophosphamide (CP; CAS no. 50-18-0;
Baxter B.V., Utrecht, The Netherlands) dissolved in physiological saline (Eurovet Animal Health,
Bladel, the Netherlands) dosed as a single oral intubation of 40 mg/kg body weight.
The route of administration of the positive control was the same as that of the test item.
Tissues and cell types examined:
Bone marrow erythrocytes
Details of tissue and slide preparation:
Bone marrow was sampled 48 hours after the first dosing. The animals were sacrificed by cervical
dislocation. Both femurs were removed and freed of blood and muscles. Both ends of the bone were
shortened until a small opening to the marrow canal became visible. The bone was flushed with
approximately 2 ml of fetal calf serum (Invitrogen Corporation, Breda, The Netherlands). The cell
suspension was collected and centrifuged at 216 g for 5 min.
The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells
in the sediment were carefully mixed with the remaining serum. A drop of the cell suspension was
placed on the end of a clean slide, which was previously immersed in a 1:1 mixture of 96% (v/v)
ethanol (Merck, Darmstadt, Germany)/ether (Merck) and cleaned with a tissue. The slides were
marked with the study identification number and the animal number. The drop was spread by moving a
clean slide with round-whetted sides at an angle of approximately 45° over the slide with the drop of
bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100% methanol (Merck)
and air-dried overnight. At least two slides were prepared per animal.
The slides were automatically stained using the "Wright-stain-procedure" in a HEMA-tek slide stainer
(Hematek 3000, Siemens Healthcare, Den Haag, the Netherlands). This staining is based on Giemsa.
The dry slides were automatically embedded in a 1:10 mixture of xylene (Klinipath, Duiven, The
Netherlands)/pertex (Klinipath) and mounted with a coverslip in an automated coverslipper (Leica
Microsystems B.V., Rijswijk, The Netherlands).
To prevent bias, all slides were randomly coded before examination. An adhesive label with the study
identification number and code was stuck over the marked slide. At first the slides were screened at a
magnification of 100 x for regions of suitable technical quality, i.e. where the cells were well spread,
undamaged and well stained. Slides were scored at a magnification of 1000 x. The number of
micronucleated polychromatic erythrocytes was counted in at least 4000 polychromatic erythrocytes
(with a maximum deviation of 5%). The ratio of polychromatic to normochromatic erythrocytes was
determined by counting and differentiating at least the first 1000 erythrocytes at the same time.
Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were
calculated.
Evaluation criteria:
A test item is considered positive in the micronucleus test if all of the following criteria are met:
a) At least one of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase
in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent
negative control
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test item is considered negative in the micronucleus test if:
a) None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in the
frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative
control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are within the 95% control limits of the negative historical control data range.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The mean number of micronucleated polychromatic
erythrocytes scored in Reaction product of: stearyl-diethanol-amine with C16-C18 saturated fatty acids
treated groups were compared with the corresponding vehicle control group.
No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the
bone marrow of test item treated animals compared to the vehicle treated animals.
All animals treated with Reaction product of: stearyl-diethanol-amine with C16-C18 saturated fatty
acids exhibited both group mean and individual MN PCE which were comparable with both the
concurrent vehicle control and the laboratory’s historical vehicle control data.
The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control
animals was within the within the 95% control limits of the distribution of the historical negative control
database.
Cyclophosphamide, the positive control item, induced a statistically significant increase in the number
of micronucleated polychromatic erythrocytes. In addition, the number of
micronucleated polychromatic erythrocytes found in the positive control animals was within the 95%
control limits of the distribution of the historical positive control database. Hence, all criteria for an
acceptable assay were met.
The animals of the groups which were treated with the test item showed no decrease in the ratio of
polychromatic to normochromatic erythrocytes, which indicated a lack of toxic effects of this test item
on the erythropoiesis. The animals of the group treated with cyclophosphamide showed an expected
decrease in the ratio of polychromatic to normochromatic erythrocytes, demonstrating toxic effects on
erythropoiesis.
The concentrations analysed in the formulations were in agreement with target concentrations (i.e.
mean accuracies between 85% and 115%). No test item was detected in the vehicle control samples.
The formulations were homogeneous (i.e. coefficient of variation ≤ 10%).
Analysis of formulations after storage yielded a relative difference of ≤ 10%.
Based on the above, the formulations were found to be stable during storage at room temperature
under normal laboratory light conditions for at least 4 hours.
Conclusions:
It is concluded that this test is valid and that Reaction product of: stearyl-diethanol-amine with C16-
C18 saturated fatty acids is not clastogenic or aneugenic in the bone marrow of male mice up to a
dose of 2000 mg/kg (the maximum recommended dose in accordance with current regulatory
guidelines) under the experimental conditions described.
Executive summary:

Micronucleus test in bone marrow cells of the mouse with Reaction product of: stearyl-diethanol-amine with C16-C18 saturated fatty acids. Reaction product of: stearyl-diethanol-amine with C16-C18 saturated fatty acids was tested in the Micronucleus Test in mice, to evaluate its genotoxic effect in developing erythrocytes (polychromatic erythrocytes) in the bone marrow.

Reaction product of: stearyl-diethanol-amine with C16-C18 saturated fatty acids consisted of slightly yellow pastilles. The test item was suspended in propylene glycol. The concentrations analysed in the formulations of were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). No test item was detected in the vehicle control samples. The formulations were homogeneous (i.e. coefficient of variation ≤ 10%). Analysis of formulations after storage yielded a relative difference of ≤ 10%. Based on this, the formulations were found to be stable during storage at room temperature under normal laboratory light conditions for at least 4 hours. In the dose range finding study males were dosed once via oral gavage 2000 mg Reaction product of: stearyl-diethanol-amine with C16-C18 saturated fatty acids per kg body weight. The animals showed no treatment related clinical signs or mortality after dosing. In the main study male animals were dosed twice via oral gavage with vehicle or with 2000 and 1000 mg Reaction product of: stearyl-diethanol-amine with C16-C18 saturated fatty acids per kg body weight. A positive control group was dosed once via oral gavage with 40 mg cyclophosphamide (CP) per kg body weight. In total 5 treatment groups were used, each consisting of 5 animals. No treatment related clinical signs or mortality were noted in any animal treated with the test item or control animals receiving vehicle or cyclophosphamide. Bone marrow was sampled 48 hours after the first dosing. No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with the test item compared to the vehicle treated animals. The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the 95% control limits of the distribution of the historical negative control database. Cyclophosphamide, the positive control item, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. In addition, the number of micronucleated polychromatic erythrocytes found in the positive control animals was within the 95% control limits of the distribution of the historical positive control database. Hence, all criteria for an acceptable assay were met. The groups that were treated with the test item showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test item on erythropoiesis. The group that was treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle control, demonstrating toxic effects on erythropoiesis. It is concluded that Reaction product of: stearyl-diethanol-amine with C16-C18 saturated fatty acids is not clastogenic or aneugenic in the bone marrow of male mice up to a dose of 2000 mg/kg (the maximum recommended dose in accordance with current regulatory guidelines) under the experimental conditions described.

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

Additional information

Justification for classification or non-classification

In vitro testing of the target substance has produced the following results:

- OECD 471: gene mutation test in bacteria (Ames test): negative

- OECD 476: gene mutation assay in mammalian cells: negative

- OECD 473: in vitro chromosome aberration test: positive

According to ECHA’s Guidance on the Application of the CLP Criteria (Version 4.1), “in vitro results can only lead to a Category 2 mutagen classification in a case where there is support by chemical structure activity relationship to known germ cell mutagens.” The target substance is a UVBC with the main component 2-[(2-hydroxyethyl)octadecylamino]ethyl stearate and has the following composition:

- Acids (C16/C18);

- ethoxylated amines (C16/C18);

- Monoester of the ethoxylated amines (C16/C18); and

- Diester of the ethoxylated amines (C16/C18).

There are no known chemical structure activity relationships to known germ cell mutagens for these components.

While there is one positive in vitro result there are no known chemical structure activity relationships to known germ cell mutagens. Therefore, the target substance is not classified for germ cell mutagenicity according to the criteria of Regulation (EC) No. 1272/2008 (as amended).

This conclusion is supported by an in vivo study performed for different regulatory purposes. The negative result coming from the mammalian erythrocytes micronucleous test (no effects on mice after oral gavage at 2000 mg/kg) confirms that the substance should not be classified as mutagen.