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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria (OECD 471, Ames): negative with and without metabolic activation in S. typhimurium strains TA 97a, TA 98, TA 100, TA 102 and TA 1535.

Chromosome aberration in mammalian cells (OECD 473, CA): negative with and without metabolic activation in cultured peripheral human lymphocytes

Gene mutation in mammalian cells (OECD 476, HGPRT): negative with and without metabolic activation in Chinese Hamster V79 cells

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 - 27 March 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The lack of some identifiers of the test substance given in the study report is caused by the status of the test substance at the time of the test period. At that time the test substance was under development with regard to its detailed composition. At a later stage the test substance was identified as UVCB according to REACH regulation.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Str. 7, 55116 Mainz, Germany
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
other: hisG46, uvrB, rfa
Species / strain / cell type:
S. typhimurium, other: TA 97a
Additional strain / cell type characteristics:
other: his D6610, uvrB, pKM 101, rfa
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
other: hisD3052, uvrB, pKM 101, rfa
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
other: hisG46, uvrB, pKM101, rfa
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
other: hisG428, pKM101, rfa
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
First experiment (plate incorporation method): 502, 151, 50, 15, 15, 5, 1.5 µg/plate
Second experiment (pre-incubation method): 498, 149, 50, 15, 5 µg/plate
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent:
DMSO was chosen as solvent, because the test item was completely soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants. For solution of the test item the stock solution was used. Each solution was membrane filtrated to accomplish sterility.
Untreated negative controls:
yes
Remarks:
Water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 4-Nitro-o-phenylenediamine; 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION:
First experiment: in agar (plate incorporation)
Second experiment: preincubation method

DURATION
- Preincubation period: 10 h at 37 °C
- Exposure duration: 48 h at 37 °C
- Expression time (cells in growth medium): Befor estarting the experiment cell titre should give a density of 10 9 cells/mL at the least.

SELECTION AGENT (mutation assays): Depending on deficinecy of the individual strain used: ampicillin or ampicillin in combination with tetracyclin were added. TA 1535 was incubated without addition of antibiotica

NUMBER OF REPLICATIONS: Per strain and dose four strains with and four plates without S9 mix were used.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity control was performed by adding 500, 1500 and 500 µg/plate of the test item to the overnight culture diluted in sodium chloride solution with and without S9 on maximal soft agar. Normal growth was observed at a concentration not exceeding 500 µg/plat. Therefor this concentration was chosen as highest concentration in the test.

CONTROL TESTS TO CONFIRM COMPLIANCE OF THE RESULTS OBTAINED IN THE STUDY
- Genotype Confirmation. Genotype confirmation is performed once a term.
- Histidine requiremen: Each strain was streaked on a biotin and a histidine-biotin-plate, using a sterilized wire loop.
- Ampicillin-Resistance (pKM 101) resp. ampicillin-tetracycline-resistance (pAQ1): The strains were streaked on ampicillin agar, TA102 on ampicillin-tetracycline agar. TA1535 was taking the function of control strain, since it is not ampicillin resistant.
- UV-sensitivity (uvrB): Two plates were streaked with the five strains, and one half of the plate covered with aluminium foil so that one half of each streak was protected against light. The plates were irradiated for 8 seconds with a germicidal lamp (254 nm, 30W), keeping a distance of 33 cm. Incubation over night at 37 °C followed.
- Crystal violet sensitivity (deep rough): For each strain, two plates were used. 0.1 mL of bacteria suspension were mixed with 2 mL Top-Agar and poured on nutrient agar. Sterile paper discs (Æ9 mm), each soaked with 10 µl of crystal violet solution (0.1%) were placed into the middle of each plate, followed by incubation over night.
- Spontaneous revertants: Four replicates, with/without S9, for each solvent which was used in the test.
- Determination of Titre: The titre was determined by dilution of the overnight culture using sodium chloride solution and placing 0.1 mL on maximal-soft agar. It should give a density of 109cells/mL (at the least).
- Toxicity Control: Performed analogously to the titre control with the maximum dose of test item with and without S9 on maximal-soft agar.
- Sterility Control: Performed analogously to the test with solvent only and S9 (without adding bacteria) on top agar.
- Positive Controls: Using diagnostic mutagens, four replicates were prepared. The stock solutions of the substances were diluted to effect an application volume of 0.1 mL/plate.
Without metabolic activation system
4-Nitro-1,2-phenylene diamine Concentration per plate: 80 µg; strains 97a, 98 and 102.
Sodium azide Concentration per plate: 6 µg; strains 100 and 1535.
With metabolic activation system
Benzo(a)pyrene Concentration per plate: 40 µg; strain 98.
2-Aminoanthracene (2-AA) Concentration per plate: 3 µg; strains 97a, 100, 102 and 1535.
Evaluation criteria:
A test item can be considered to have mutagneic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor > = 2) in at least one strain can be observed. A concentration - related increase over the range tested can also be taken as a sign of mutagenetic activity.
Statistics:
The colonies were counted visually, the numbers were recorded. A spreadsheet software (microsoft excel) was used to calculate mean values and standard deviations as well as the increase factor of revertant induction.
Key result
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
Remarks:
Exp. 2 highest concentration
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium, other: TA 97a
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
Exp. 2 highest concentration
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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 nor precipitates, but tested up to recommended limit concentrations
Remarks:
Exp. 2 highest concentration
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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 nor precipitates, but tested up to recommended limit concentrations
Remarks:
Exp. 2 highest concentration
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
Exp. 2 highest concentration
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: see result on cytotoxicity test
COMPARISON WITH HISTORICAL CONTROL DATA:
In the following table, the mean of the spontaneous revertants and positive controls of all performed experiments in the year 2009 is stated (Number of experiments: 3) in compari-son with the experiments performed within this study.

Strain TA97a TA98 TA100 TA102 TA1535
Induction - S9 + S9 - S9 + S9 - S9 + S9 - S9 + S9 - S9 + S9
H2O Mean 105 105 7 8 119 112 215 225 11 13
Min 92 99 6 7 107 100 165 205 8 10
Max 124 109 9 10 130 122 261 237 14 16
Exp 1 92 108 6 10 120 113 220 237 8 10
Exp 2 124 109 9 7 107 100 165 205 14 14
DMSO Mean 123 95 7 8 121 123 247 223 10 11
Min 106 86 6 5 111 115 219 162 8 9
Max 133 108 8 10 138 131 269 253 13 13
Exp 1 133 86 6 8 111 115 219 253 8 12
Exp 2 129 90 7 10 115 131 269 162 9 9
Pos. Contr. Mean 1001 1001 1001 1001 1001 1001 1001 1001 1001 1001
Min 1001 1001 1001 1001 1001 1001 1001 1001 1001 1001
Max 1001 1001 1001 1001 1001 1001 1001 1001 1001 1001
Exp 1 1001 1001 1001 1001 1001 1001 1001 1001 1001 1001
Exp 2 1001 1001 1001 1001 1001 1001 1001 1001 1001 1001
In this table, “> 1000” is represented by “1001”.


ADDITIONAL INFORMATION ON CYTOTOXICITY:
The toxicity of the following concentrations were tested: 5005, 1502 and 501 µg/plate.
Per strain, four plates were incubated with the corresponding dose of the test item on maximal soft agar.
16.1 Experimental Parameters
Date of treatment 11. March 2009
Concentrations tested 5005 / 1502 / 501µg/plate
Incubation time 48 hours
Incubation temperature 37 °C
Tester strains TA97a, TA98, TA100, TA102, TA1535
Method Plate incorporation method
In the two higher concentrations (5003 and 1502 µg/plate), the test item showed cytotoxic-ity towards all the strains. In the lower concentration (501 µg/plate), normal growth oc-curred.
On the base of these results, the first experiment was performed with 500 µg/plate as maximum dose for all strains.


Table 1: Mean revertants Experiment 1

Strain

TA97a

TA98

TA100

TA102

TA1535

Induction

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

H20

Mean

92

108

6

10

120

113

220

237

8

10

sd

32.0

23.5

1.7

3.8

7.2

4.4

46.5

43.4

3.7

2.4

DMSO

Mean

133

86

6

8

111

115

219

253

8

12

sd

31.7

27.0

1.0

3.2

14.5

26.3

22.2

19.1

2.4

2.4

 

Pos.Contr.

Mean

1001

1001

1001

1001

1001

1001

1001

1001

1001

1001

sd

0

0

0

0

0

0

0

0

0

0

f(I)

7.53

11.64

166.8

125.1

8.34

8.70

4.57

3.96

125.1

83.42

 

501 µg/pl.

Mean

107

100

6

7

139

133

206

220

6

9

sd

7

25

3

4

9

23

4

9

4

2

f(I)

0.80

1.16

1.00

0.88

1.25

1.16

0.94

0.87

0.75

0.75

 

151 µg/pl.

Mean

124

99

8

7

92

105

292

252

11

10

sd

17

19

3

2

23

41

21

71

1

2

f(I)

0.93

1.15

1.33

0.88

0.83

0.91

1.33

1.00

1.38

0.83

 

50 µg/pl.

Mean

80

112

7

5

111

102

250

257

9

5

sd

28

36

1

1

13

11

99

53

2

2

f(I)

0.60

1.30

1.17

0.63

1.00

0.89

1.14

1.02

1.13

0.42

 

15 µg/pl.

Mean

112

109

9

9

101

94

213

200

13

10

sd

32

34

1

2

14

22

16

35

3

4

f(I)

0.84

1.27

1.50

1.13

0.91

0.82

0.97

0.79

1.63

0.83

 

5 µg/pl.

Mean

114

112

8

9

89

98

201

189

7

11

sd

19

37

2

2

24

13

27

26

4

2

f(I)

0.86

1.30

1.33

1.13

0.80

0.85

0.92

0.75

0.88

0.92

 

1.5 µg/pl.

Mean

97

103

8

6

119

92

244

243

9

12

sd

29

41

2

1

12

5

62

81

5

4

f(I)

0.73

1.20

1.33

0.75

1.07

0.80

1.11

0.96

1.13

1.00

In this table, "> 1OOO" is represented by "1001". Induction factors for the respective treatments are stated as lower limit.

Table 2: Mean revertants Experiment 2

Strain

TA97a

TA98

TA100

TA102

TA1535

Induction

 

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

 

H20

 

Mean

99

108

10

9

122

130

220

232

11

13

sd

9.8

10.7

3.1

3.5

13.4

15.2

8.3

8.2

1.7

2.9

DMSO

Mean

103

100

8

10

117

117

237

265

13

14

sd

11.5

10.5

1.5

1.3

12.6

12.5

25.7

14.7

3.2

2.6

 

Pos.Contr.

Mean

1001

1001

1001

1001

1001

1001

1001

1001

1001

1001

sd

0

0

 

0

0

0

0

0

0

0

0

f(I)

9.72

10.01

125.1

100.1

8.20

8.56

4.22

3.78

91.00

71.50

 

498 µg/pl.

Mean

 

15

12

7

5

20

51

16

24

3

10

sd

15

7

3

4

10

5

4

5

1

2

f(I)

0.15

0.12

0.88

0.50

0.17

0.44

0.07

0.09

0.23

0.71

 

149 µg/pl.

Mean

129

112

9

5

119

121

256

228

10

11

sd

26

9

1

1

24

49

40

27

4

1

f(I)

1.25

1.12

1.13

0.50

1.02

1.03

1.08

0.86

0.77

0.79

 

50 µg/pl.

 

Mean

90

109

7

7

103

69

236

150

11

10

sd

15

6

4

5

12

6

27

17

2

2

f(I)

0.87

1.09

0.88

0.70

0.88

0.59

1.00

0.57

0.85

0.71

 

15 µg/pl.

Mean

137

113

9

5

93

98

151

186

8

8

sd

7

23

1

2

21

15

13

28

2

2

f(I)

1.33

1.13

1.13

0.50

0.79

0.84

0.64

0.70

0.62

0.57

 

5 µg/pl.

Mean

126

135

10

9

89

94

147

187

7

9

sd

23

14

3

3

9

14

14

52

2

5

f(I)

1.22

1.35

1.25

0.90

0.76

0.80

0.62

0.71

0.54

0.64

In this table, "> 1OOO" is represented by "1001". Induction factors for the respective treatments are stated as lower limit.

Table 3: Historical control data

Strain

 

TA97a

TA98

TA100

TA102

TA1535

Induction

 

 

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

 

 

H20

Mean

105

105

7

8

119

112

215

225

11

13

Min

92

99

6

7

107

100

 

165

205

8

10

Max

124

109

9

10

130

122

261

237

14

16

Exo 1

92

108

6

10

120

113

220

237

8

10

Exp 2

124

109

9

7

107

100

165

205

14

14

 

 

DMSO

Mean

123

95

7

8

121

123

247

223

10

11

Min

106

86

6

5

111

115

219

162

8

9

Max

133

108

8

10

138

131

269

253

13

13

Exo 1

133

86

6

8

111

115

219

253

8

12

Exo 2

129

90

7

10

115

131

269

162

9

9

 

Mean

1001

1001

1001

1001

1001

1001

1001

1001

1001

1001

 

Pos. Contr.

Min

1001

1001

1001

1001

1001

1001

1001

1001

1001

1001

Max

1001

1001

1001

1001

1001

1001

1001

1001

1001

1001

Exp 1

1001

1001

1001

1001

1001

1001

1001

1001

1001

1001

Exo 2

1001

1001

1001

1001

1001

1001

1001

1001

1001

1001

In this table, "> 1OOO" is represented by "1001".

Conclusions:
Interpretation of results: negative
Executive summary:

The test item did not show mutagenic effects in both experiments. The number of revertant colonies was not increased in comparison with the spontaneous revertants (solvent only). Cytotoxicity of the test item was detected in the second experiment in the highest concentration 498 µg/plate. The background lawn was visible but the number of revertants was decreased. In the lower concentrations, normal growth occurred.

The test item is considered not mutagenic for the reasons given above.

In the cytotoxicity experiment, the test item showed cytotoxicity towards the bacteria in the follow concentrations: 5000 µg/plate and 1500 µg/plate; on the base of these results the highest concentration in the first experiment was chosen with 500 µg/plate. The test item showed any cytotoxicity towards the bacteria in the second experiment (using the pre-incubation method) in the highest concentration (500 µg/plate). The lower concentrations showed normal growth.

The confirmation tests of the genotype did not show any irregularities. The control of the titre was above the demanded value. The number of revertant colonies of the positive controls was in the range of the historical data of the laboratory and the revertants were increased in comparison with the negative controls, as well as showing mutagenous potential of the diagnostic mutagenes. Some of the spontaneous revertants are lower than the values given by Prof. Ames; in comparison with the historical data of the LAUS GmbH, they were within the normal range. For these reasons, the result of the test is considered valid.

Therefore it can be stated, that under the test conditions, the test item is not mutagenic in the Bacterial Reverse Mutation Test using Salmonella typhimurium, strains TA 97a, TA 98, TA 100, TA 102 and TA 1535.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 Dec 2019 - 08 Apr 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
Version / remarks:
adopted in 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes: cultured peripheral human lymphocytes
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: Collected from healthy adults, non-smoking and without any recent exposure to drugs or radiation.
- Suitability of cells: Cell type selected is listed as one of the recommended cell types in OECD guideline 473.
- Normal cell cycle time (negative control): Average generation time 14.4 - 14.6 h.

For lymphocytes:
- Sex, age and number of blood donors: Female, 27/28, two (main assay 1, collection date 18 December 2019) and male, 30, two (main assay 2, collection date: 22 January 2020)
- Whether whole blood or separated lymphocytes were used: Whole blood was used.
- Whether blood from different donors were pooled or not: Blood from two donors was pooled for each main assay.
- Mitogen used for lymphocytes: Phytohaemagglutinin (PHA), 10 mL in 500 mL medium.

MEDIA USED
- Type and composition of media, CO2 concentration: RPMI 1640 medium (Dutch modification) supplemented with 20% (v/v) heat-inactivated foetal calf serum, L-glutamine (200 mM) and antibiiotic solution (not further specified); 5% CO2
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- Supplier: Trinova Biochem GmbH, Germany
- Source of S9 : Prepared from the livers of male Sprague Dawley rats (age: 5 - 6 weeks) treated with phenobarbital and 5,6-benzoflavone.
- Concentration or volume of S9 mix and S9 in the final culture medium: S9 tissue fraction 0.1 mL (= 10% v/v), NADP (100 mM) 0.04 mL, G-6-P (100 mM) 0.05 mL, MgCl2 (100 mM) 0.02 mL, Phosphate buffer (pH 7.4, 200 mM) 0.5 mL, distilled water 0.29 mL
- Quality controls of S9: Enzymatic activity, sterility and metabolic capability checked and certified.
Test concentrations with justification for top dose:
Main assay 1:
3 h treatment with and without metabolic activation: 195, 293, 439, 658, 988, 1480, 2220, 3330 and 5000 μg/mL
24 h treatment without metabolic activation: 130, 195, 293, 439, 658, 988, 1480, 2220, 3330 and 5000 μg/mL

Due to the steep increase of cytotoxicity it was not possible to select a maximum dose level for scoring presenting moderate cytotoxicity. The whole experiment was therefore repeated in main assay 2.

Main assay 2:
3 h treatment with and without metabolic activation: 273, 313, 360, 415, 477, 548, 630 and 725 µg/mL
24 h treatment without metabolic activation: 118, 136, 156, 179, 206, 237, 273, 313, 360 and 415 µg/mL
Vehicle / solvent:
- Vehicle/solvent used: DMSO (batch no. H079S; supplier: HoneyWell)

- Justification for choice of solvent/vehicle: Based on the limited solubility of the test substance in water, DMSO was selected as the vehicle since it is compatible with the survival of the cells and the S9 metabolic activity.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Remarks:
3 h treatment: tests with and without metabolic activation were done concurrently, positive control cultures were treated only with CP at 18.0 and 23.0 μg/mL. 24 h treatment: positive control cultures were treated with MMC at 0.300 and 0.450 μg/mL.
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate
- Number of independent experiments : Two main assays were performed but only main assay 2 was used for evaluation of chromosome aberrations.

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 48 h
- Exposure duration/duration of treatment: 3 and 24 h
- Harvest time after the end of treatment: 21 h for 3 h treatment and none for 24 h treatment.

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): Colcemid (0.2 μg/mL final concentration) added for the last 3 h until harvesting.
- Methods of slide preparation and staining technique used including the stain used: The fixed cell suspension was dropped onto clean, wet, grease-free, glass slides and air-dried. The slides were stained in 3% Giemsa in tap water and rinsed twice in tap and distilled water.
- Number of cells spread and analysed per concentration: 300 (150 per replicate culture)
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: mitotic index (MI)
- Any supplementary information relevant to cytotoxicity: Due to the steep increase of cytotoxicity observed in main assay 1, it was not possible to select the maximum dose level for scoring which presented a moderate cytotoxicity (as recommended by OECD guideline 473). The whole experiment was repeated in main assay 2 using a lower dilution factor of 1.15 and a narrower dose range. Although dose levels in main assay 2 were very narrowly spaced, the test substance still produced a very steep increase of cytotoxicity in the short-term (3 h) treatment experiment with and without metabolic activation. Since a steep increase of cytotoxicity was seen at the high dose levels and increases of MI over the concurrent negative control were seen at the low dose levels, it was considered appropriate to examine additional 1000 cells for each culture on slides not previously examined. Hence, determination of the MI was based on 2000 instead of 1000 cells for the 3 h treatment (with and without metabolic activation).
Rationale for test conditions:
Due to a steep increase of cytotoxicity observed in main assay 1, it was not possible to select the maximum dose level for scoring which presented a moderate cytotoxicity as recommended by OECD guideline 473. The whole experiment was, therefore, repeated in main assay 2 using a lower dilution factor (1.15) and a narrower dose range.
Evaluation criteria:
A test item is considered clearly positive if the following criteria are met:
– At least one dose level shows a statistically significant increase in aberration-bearing cells (excluding gaps), compared with the concurrent negative control.
– Any of the results is outside the distribution of the historical negative control data.
– The increase is dose-related when evaluated with an appropriate trend test (Cochran-Armitage).

A test item is considered clearly negative if the following criteria are met:
– None of the dose levels shows a statistically significant increase in aberration-bearing cells (excluding gaps).
– There is no concentration-related increase, when evaluated with an appropriate trend test.
– All the results are inside the distribution of the historical control data.
Statistics:
Fisher’s Exact Test was used to compare the number of cells bearing aberrations (assumed to be Poisson distributed) in control and treated cultures. Bonferroni’s corrections were applied for multiple comparisons. The analysis was performed using sets of data either including or excluding gaps. Cochran-Armitage Trend Test (one-sided) was performed to aid determination of concentration response relationship.
Key result
Species / strain:
lymphocytes: cultured peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Steep increase in 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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No relevant change of pH observed at any dose level, in the absence or presence of metabolic activation.
- Data on osmolality: No relevant change of osmolality observed at any dose level, in the absence or presence of metabolic activation.
- Possibility of evaporation from medium: Negligible as vapour pressure is 1.84E-03 Pa at 20 °C and 2.85E-03 Pa at 25 °C.
- Water solubility: Poorly soluble in aqueous solutions, therefore, DMSO was used as vehicle/solvent.
- Precipitation and time of the determination: No precipitation observed at the beginning and the end of the treatment period, in the presence and absence of metabolic activation.

SOLUBILITY STUDIES: The highest concentration used in main assay 1 was selected based on the solubility of the test substance in the vehicle DMSO.

STUDY RESULTS
- Concurrent vehicle negative and positive control data provided as pdf file under 'Attached background material'.

Chromosome aberration test (CA) in mammalian cells: Detailed results provided under 'Any other information on results' and 'Attached background material'.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data) : Provided under 'Attached background material'.

Additional information in tabular form can be found under 'Attached background material'. 

 

Table 2: Cytotoxicity results main assay 2

S9

Treatment time

(hours)

Harvest time

(hours)

Dose level

(µg/mL)

Relative mitotic index

(%)

Presence of precipitation/opacity

3

24

0.00

548

477

415

100

79

124

148

-

No

No

No

+

3

24

0.00

548

477

415

100

68

91

115

-

No

No

No

24

24

0.00

237

179

136

100

40

66

91

-

No

No

No

 

Table 3: Summary of genotoxicity results main assay 2

S9

Treatment time

(hours)

Harvest time

(hours)

Dose level

(µg/mL)

Incidence of cells bearing aberrations

(excluding gaps) (%)

Statistical significance

Incidence of cells bearing numerical changes (polyploid and endoreduplicated cells) (%)

3

24

0.00

548

477

415

0.0

0.7

0.0

0.0

-

NS

NS

NS

0.0

2.6

1.0

2.3

Linear trend

Historical control data (95% confidence limits)

 

NS

 

0.00-0.83

-

+

3

24

0.00

548

477

415

0.00 0.3

0.00

0.3

-

NS

NS

NS

0.0

2.3

1.3

2.0

Linear trend

Historical control data (95% confidence limits)

 

NS

 

0.00-0.59

-

24

24

0.00

237

0.0

0.3

-

NS

0.3

0.0

179

0.0

NS

0.3

136

0.3

NS

0.0

Linear trend

Historical control data (95% confidence limits)

 

NS

 

0.00-0.66

-

 

Table 4: Positive control results

S9

Treatment time

(hours)

Harvest time

(hours)

 

Dose level

(µg/mL)

Incidence of cells bearing aberrations

(%)

+/-

3

24

Cyclophosphamide

18.0

25.0

-

24

24

Mitomycin-C

0.300

22.7

 

Conclusions:
Interpretation of results: negative with and without metabolic activation
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 Jan - 09 Mar 2020
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 using the Hprt and xprt genes)
Version / remarks:
adopted in 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
HGPRT locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Dr. J. Thacker, MRC Radiobiology Unit, Harwell, UK.
- Suitability of cells: Cell type selected is listed as one of the recommended cell types in OECD guideline 476
- Methods for maintenance in cell culture: Freshly thawed cells from stock cultures were maintained in 175 cm2 culture flasks in minimal essential medium (MEM) and cultured at a humidified atmosphere of 5% CO2 and at 37 °C incubation temperature.

MEDIA USED
- Type and identity of media:
Minimal medium: 10 x Eagles Minimal Essential (MEM, 58.7 mL) was diluted in sterile bidistilled water (500 mL) and supplemented with L-glutamine (5.9 mL 200mM), sodium bicarbonate (15.7 mL 7.5%), non-essential amino acids (5.9 mL 100x), streptomycin sulfate (1.2 mL 50,000IU/mL) and penicillin G (1.2 mL 50,000 IU/mL).
Complete medium: EMEM minimal medium was supplemented with 10% foetal calf serum
- 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:
Type and composition of metabolic activation system: Cofactor supplemented post-mitochondrrial fraction (S9 mix).
- source of S9 : MOLTOX, Molecular Toxicology, Inc. (Batch no. 3971)
- method of preparation of S9 mix: S9 tissue mix was prepared from the livers of Sprague-Dawley rats treated with phenobarbital and 5,6 benzoflavone.
- amount of S9 in the final culture medium: 10%
- quality controls of S9: S9 mix was checked for alkoxyresufurin-0-dealkylase activities and for pro-mutagen activation (ability of the sample to activate ethidium bromide and cyclophosphamide to intermediates mutagenic to S. typhimurium strains TA98 and TA1535).
Test concentrations with justification for top dose:
Without metabolic activation: 36.2*, 72.3*, 145*, 174*, 208*, 250 and 300 µg/mL (3 h)
With metabolic activation: 72.3*, 145*, 289*, 347*, 417*, 500* and 600 µg/mL (3 h)

*Dose levels analysed for mutant frequency.
The selection of the concentrations used in the main experiments was based on data from the preliminary cytotoxicity test, in which cytotoxicity (no survival of cells) became evident at ≥ 313 µg/mL without S9 mix and at ≥ 625 µg/mL with S9 mix.
Vehicle / solvent:
- Vehicle/solvent used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS: duplicate cultures in a single experiment

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: Two days before the experiment, sufficient numbers of 175 cm2 flasks were inoculated with 5 million freshly trypsinised V79 cells from a common pool, in order to have at least 20 million of cells for treatment.
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration: 3 h exposure with and without S9 mix
- Expression time (cells in growth medium): At the end of the treatment period, the cells were trypsinised and replated in fresh medium for determination of the cloning efficiency and the mutation frequency and incubated for 8 days. The cells were sub-cultured on Days 2 and 5 of the expression period.
- Selection time: After the expression period the cells were plated for determination of the mutant frequency and incubated for 8 days.
- Fixation time (start of exposure up to fixation or harvest of cells): 16 days

SELECTION AGENT: 7.5 µg/mL 6-Thioguanine (6-TG)

STAIN: Giemsa

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative survival and cloning efficiency
Evaluation criteria:
A test item was considered to be clearly positive if:
– At least one of the test concentrations exhibited a statistically significant increase, compared with the concurrent solvent/vehicle control.
– The increase was concentration-related.
– Any of the results were outside the distribution of the historical negative control data (95% confidence limits).

A test item was considered to be clearly negative if:
– None of the test concentrations exhibited a statistically significant increase, compared with the concurrent solvent/vehicle control.
– There was no concentration-related increase.
– All results were inside the distribution of the historical negative control data (95% confidence limits).
Statistics:
The individual mutation frequency values at each test point were transformed to induce homogeneous variance and normal distribution. The appropriate transformation was estimated using the procedure of Snee and Irr (1981), and was found to be y = {x + a)b where a = 0 and b = 0.275. The mutant frequency in the solvent control and treated cultures was compared using the Dunnett’s test (one-tailed).
The results of the experiment were subjected to an Analysis of Variance in which the effect of replicate culture and dose level in explaining the observed Variation were examined. For each experiment, a two way analysis of variance was performed (without interaction) fitting to two factors:
- Replicate culture: to identify differences between the replicate cultures treated.
- Dose level: to identify dose-related increases (or decreases) in response, after allowing for the effects of replicate cultures and expression time.
The analysis was performed separately with the sets of data obtained in the absence and presence of S9 metabolism.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at ≥ 208 µg/mL without S9 mix and at ≥ 500 µg/mL with S9 mix
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: A preliminary cytotoxicity test was performed in the presence and absence of metabolic activation using test item concentrations in the range of 19.5 - 5000 µg/mL. Cytotoxicity (no survival of cells) was observed at ≥ 313 µg/mL without S9 mix and at ≥ 625 µg/mL with S9 mix. No reduction in relative survival was noted at the remaining concentrations. Precipitation of the test item in medium was noted following incubation in the absence of S9 mix at ≥ 1250 µg/mL.

STUDY RESULTS
No statistically significant or biologically relevant increase in mutant frequency was observed following treatment with the test item at any dose level, in the absence or presence of S9 metabolism, in any experiment. Solvent and positive controls showed the expected results and matched the acceptability criteria. For details please refer to Tables No. 1 and 2 under "Any other information on results incl. tables".

HISTORICAL CONTROL DATA:
- Positive historical control data:
The mean number of mutants with metabolic activation is 254 ± 58.4 (range: 178 - 425, n=16) for 10 µg/mL DMBA. The mean number of mutants without metabolic activation is 1422 ± 596 (range: 603 - 2681, n=15) for 10 mM EMS. The results of the test fell within the historical control data range and matched the acceptability criteria for the positive control.
- Vehicle historical control data:
The mean number of mutants with metabolic activation is 9.46 ± 5.73 (range: 2.31 - 20.0, n=16), upper confidence limit (5%) = 20.9. The mean number of mutants without metabolic acativation is 5.96 ± 5.47 (range: 1.88 - 21.4, n=15), upper confidence limit (5%) = 16.9. The results of the test fell within the historical control data range and matched the acceptability criteria for the solvent control.

Table 1: Result of the HGPRT test without S9 mix

      Survival after treatment Viability after expression Genotoxicity
Treatment   Dose Level 106cells Plate counts Mean CE Adjusted Mean RS Plate counts Mean %CE Total plate
counts
Mean ± SD MF MF pooled
 cultures
mg/mL post treatment CE
Solvent A 0 20 118 120 138 125 0.63 0.55 0.53 100 151 156 158 173 86 20 1.0 ± 0.86 12.9 9.28
control B 21 108 110 105 108 0.54 0.5 179 190 201 12 0.6 ± 0.94 6.32
Test Item A 36.2 23 134 132 145 137 0.69 0.7 0.71 134 180 182 182 191 96 5 0.3 ± 0.72 2.76 3.66
  B 22.8 140 140 146 142 0.71 0.72 204 199 201 9 0.5 ± 0.6 4.47
Test Item A 72.3 21.6 134 133 129 132 0.66 0.63 0.61 116 162 179 185 184 92 11 0.55 ± 0.83 6.27 7.33
  B 20.6 131 132 126 130 0.65 0.59 186 187 206 16 0.8 ± 0.7 8.29
Test Item A 145 17.6 150 147 149 149 0.74 0.58 0.6 113 171 184 176 168 84 24 1.2 ± 1.01 13.56 19.31
  B 18.8 145 147 150 147 0.74 0.61 160 160 159 41 2.1 ± 0.89 25.68
Test Item A 174 17.9 109 110 115 111 0.56 0.44 0.41 78 140 150 148 145 73 20 1 ± 0.79 13.7 16.55
  B 17 90 107 107 101 0.51 0.38 135 153 144 28 1.4 ± 1.23 19.44
Test Item A 208 9.8 70 70 71 70 0.35 0.15 0.15 28 150 160 143 151 75 11 0.6 ± 0.76 7.28 8.97
  B 11 54 56 60 57 0.28 0.14 150 152 148 16 0.8 ± 0.83 10.67
EMS   10.0 mM 25 115 118 123 119 0.59 0.66 0.66 124 148 138 154 147 73 1850** 92 .5** 1261.36**  

EMS = Ethylmethanesulfonate
CE = Cloning efficiency
RS = Relative survival
MF = Mutant frequency
SD = Standard deviation

 

Table 2: Results of the HGPRT test with S9 mix

      Survival after treatment Viability after expression Genotoxicity
Treatment   Dose Level 106cells Plate counts Mean CE Adjusted Mean RS Plate counts Mean %CE Total plate
counts
Mean ± SD MF MF pooled
 cultures
mg/mL post treatment CE
Sovent A 0 21.8 158 163 166 162 0.81 0.77 0.81 100 196 189 205 166 83 10 0.5 ± 0.83 5.08 7.85
control B 23.4 168 165 170 168 0.84 0.85 137 124 143 16 0.8 ± 0.7 11.88
Test Item A 72.3 21 150 167 167 161 0.81 0.73 0.74 91 169 157 158 166 83 8 0.4 ± 0.82 4.96 3.92
  B 20.8 157 170 164 164 0.82 0.74 169 171 171 5 0.3 ± 0.44 2.94
Test Item A 145 20.2 170 150 151 157 0.79 0.69 0.69 85 150 145 160 139 70 8 0.4 ± 0.5 5.27 9.71
  B 20 166 157 155 159 0.8 0.69 125 130 124 19 0.95 ± 0.94 15.04
Test Item A 289 23.8 128 112 103 114 0.57 0.59 0.67 83 137 129 133 128 64 11 0.55 ± 0.6 8.27 7.45
  B 24 133 145 152 143 0.72 0.74 127 120 119 8 0.4 ± 0.68 6.56
Test Item A 347 22.6 114 118 131 121 0.61 0.59 0.61 75 129 138 129 143 72 8 0.4 ± 0.75 6.06 8.37
  B 20.4 135 147 138 140 0.7 0.62 146 159 159 16 0.8 ± 0.83 10.34
Test Item A 417 21.6 144 156 132 144 0.72 0.67 0.65 80 140 138 131 140 70 11 0.6 ± 0.6 8.07 5.38
  B 20.4 136 132 155 141 0.71 0.62 145 142 141 4 0.2 ± 0.41 2.8
Test Item A 500 13.6 134 130 148 137 0.69 0.4 0.37 46 145 140 153 159 80 9 0.5 ± 0.69 6.16 4.71
  B 13.2 116 118 114 116 0.58 0.33 173 170 175 6 0.3 ± 0.57 3.47
DMBA   10 12.1 153 167 169 163 0.82 0.43 0.43 53 118 102 107 109 55 483** 24.2** 443.12**  

DMBA = Dimethyl benzanthracene
CE = Cloning efficiency
RS = Relative survival
MF = Mutant frequency
SD = Standard deviation

Conclusions:
Interpretation of results: negative with and without metabolic activation
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Gene mutation in bacteria

Fatty acids, C10-12, esters with polylactic acid, sodium salts (Dermosoft decalact, CAS No. 1312021-45-6, EC No. 700-937-1) was tested for its mutagenic potential in bacteria (Ames test) according to OECD guideline 471 and in compliance with GLP (Paulus , 2011). Salmonella typhimurium strains TA 97a, TA 98, TA 100, TA 102 and TA 1535 were exposed to the test item in the presence and absence of metabolic activation (S9 mix). Two independent experiments were performed with concentration ranges identified in a preliminary cytotoxicity test. In the first experiment of the Ames test, the bacterial strains were exposed to test item concentrations from 1.5 – 502 µg/plate using the plate-incorporation method. In the second experiment, 5 – 498 µg/plate were tested using the pre-incubation method. Solvent (DMSO) and positive controls were included in each experiment. Precipitation of the test item was not reported. Cytotoxicity, evident as a reduction in the number of revertant colonies when compared to the solvent controls, was observed in the second experiment at the highest concentration of 498 µg/mL only. The test item did not induce a biologically relevant increase in the number of revertant colonies in any of the tested strains either with or without metabolic up to the highest tested concentration in the presence and absence of S9 mix. Positive and vehicle controls were in the range of historical control data and confirmed the validity of the test system. Thus, based on the results of the present study and under the experimental conditions chosen, the test substance is not mutagenic in bacteria with and without metabolic activation.

Chromosome aberration in mammalian cells

The potential of Dermosoft decalact to induce chromosomal damage in mammalian cells was assessed in an in vitro chromosome aberration study performed according to OECD guideline 473 observing GLP provisions in the absence and presence of S9 metabolic activation (Ciliutti,  2021). The study was performed using cultured peripheral human lymphocytes as test system. Three treatment series were included in Main Assay 1 of the study. A short-term treatment was performed where the cells were treated for 3 h in the presence and absence of S9 mix. The harvest time of 24 h, corresponding to approx. 1.5 cell cycle lengths, was used. A long-term (continuous) treatment was also performed in the absence of S9 metabolism until harvest at 24 h. The test item was assayed at the highest dose level of 5000 μg/mL, the maximum concentration as indicated in the OECD guideline for UVCB substances and at the following lower dose levels: 195, 293, 439, 658, 988, 1480, 2220 and 3330 μg/mL. For the long-term treatment the additional dose level of 130 μg/mL was employed. Due to the steep increase of cytotoxicity it was not possible to select the maximum dose level for scoring which presented a moderate cytotoxicity as recommended by the guideline. The whole experiment was therefore repeated in Main Assay 2 using a lower dilution factor (1.15) and a narrower dose range. Treatment concentrations in Main Assay 2 were as follows: 273, 313, 360, 415, 477, 548, 630 and 725 µg/mL (3 h treatment, with and without metabolic activation) and 118, 136, 156, 179, 206, 237, 273, 313, 360 and 415 µg/mL (24 h treatment, without metabolic activation). Each experiment included appropriate negative and positive controls. Two replicate cell cultures were prepared at each test point. Dose levels were selected for the scoring of chromosomal aberrations on the basis of the cytotoxicity of the test item as determined by the reduction in mitotic index. For the 3 h exposure in the absence and presence of S9 mix, despite a very narrow space interval between dose levels, once again a steep increase of cytotoxicity was seen. In addition, increases of the mitotic index over the concurrent negative control were seen at lower dose levels. In order to better evaluate these results it was considered appropriate to perform an additional scoring of mitotic index on slides not previously examined. Cytotoxicity results were confirmed. No further treatments were performed and it was decided to analyse the concentration inducing 79% and 68% cytotoxicity as top dose. For each culture, 150 well spread metaphases were scored to assess the frequency of aberrant cells. Adequate cell proliferation was observed in negative control cultures and the appropriate number of doses and cells (300 metaphases per test point) was analysed. Statistically significant increases in the incidence of cells bearing aberrations were observed following treatments with the positive controls cyclophosphamide and mitomycin C and the responses were compatible with those generated in the historical control database of the test facility, indicating the correct functioning of the test system. Following treatment with the test item, no statistically significant increase in the incidence of cells bearing structural aberrations, including or excluding gaps, was observed at any dose level and treatment condition. All the results obtained were inside the distribution of historical control data and no dose effect relationship was indicated by a linear trend test. Increases in the number of cells bearing numerical aberrations (polyploid and endoreduplicated cells) which reached statistical significance at the high and low dose levels selected for scoring were observed after the short exposure both in the absence and presence of metabolic activation. Polyploidy (including endoreduplication) can arise in chromosome aberration assays in vitro. While aneugens can induce polyploidy, polyploidy alone does not indicate aneugenic potential and can simply indicate cell cycle perturbation or cytotoxicity. This chromosome aberration test is not designed to measure aneuploidy. It is concluded that the test item did not induce structural aberrations in human lymphocytes after in vitro treatment, under the reported experimental conditions.

Gene mutation in mammalian cells

Dermosoft decalact was evaluated for point mutation effects at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster V79 cells (Bisini,  2020). Duplicate cultures were exposed to the test item, solvent (DMSO) and positive controls (10 mM ethylmethanesulfonate (EMS) without S9 mix and 10 µg/mL 7,12-dimethylbenz(a)anthracene (DMBA) with S9 mix) in the presence of metabolic activation (phenobarbital, 5,6-benzoflavone-induced rat liver S9 fraction). Concentrations were selected based on the results of a preliminary cytotoxicity test, in which cytotoxicity was observed at ≥ 313 µg/mL without S9 mix and at ≥ 625 µg/mL with S9 mix. In the main mutation assay, the cells were exposed for 3 hours to 36.2 – 300 µg/mL in the absence of S9 mix and to 72.3 – 600 µg/mL in the presence of S9 mix. Following exposure, the cells were incubated for 8 days to allow expression of the mutant phenotype. The expression period was followed by a selection period, in which the cells were cultivated in 6-thioguanidine-enriched medium for 8 days. There was no precipitation of the test material in medium at the tested concentrations, neither in the presence nor absence of S9 mix. Cytotoxicity was observed at ≥ 208 µg/mL without S9 mix and at ≥ 500 µg/mL with S9 mix. There was no statistically significant increase over the spontaneous mutant frequency observed for any concentration in the absence or presence of S9 mix. Results of the mutant frequency at all concentrations tested were within the distribution of the historical solvent control range. In addition, mutant frequencies of the solvent control cultures remained within the range of the laboratories historical control data. The positive controls EMS and DMBA induced clear mutagenic and statistically significant effects, demonstrating the functionality of the S9 mix and the sensitivity of the test. Under the conditions of the test, the test item did not induce gene mutation in mammalian cells in vitro, neither in the presence nor in the absence of metabolic activation.

Justification for classification or non-classification

The available data on genetic toxicity in vitro obtained with fatty acids, C10-12, esters with polylactic acid, sodium salts (Dermosoft decalact, CAS No. 1312021-45-6, EC No. 700-937-1) do not meet the criteria for classification according to Regulation (EC) No. 1272/2008 (CLP), and are therefore conclusive but not sufficient for classification.