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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Test (OECD 471): negative


HPRT Test (OECD 476): negative


MN Test (OECD 487): negative

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:
2005-06-20 to 2006-04-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2000
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: V3186/11
- Date of manufacture: December 7, 2004

Letter of Confirmation: CAS No 90530-15-7 and 93940-97-7 refer to the same substance and substance composition.
Target gene:
his; trp
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 mix of Aroclor induced rat liver
Test concentrations with justification for top dose:
both experiments:
0; 20; 100; 500; 2500 and 5000 μg/plate
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, DMSO was selected as the vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.
Untreated negative controls:
yes
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Untreated negative controls:
yes
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Untreated negative controls:
yes
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylendiamine
Untreated negative controls:
yes
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine
Untreated negative controls:
yes
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants; clearing or diminution of the background lawn (= reduced his- or trp- background growth); reduction in the titer

Rationale for test conditions:
according to guidelines
Evaluation criteria:
The test chemical is considered positive in this assay if the following criteria are met:
A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adding a metabolizing system.

A test substance is generally considered nonmutagenic in this test if:
The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
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
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Evaporation from medium: no
- Water solubility: not soluble in water
- Precipitation: no
- Other confounding effects: none

HISTORICAL CONTROL DATA
please refer to "any other informations on results"

 Experiment 1 Standard plate test

Dose (µg/plate)

Mean number of revertant colonies of 3 replicates (± S.D.) for different strains of S. typhimurium and E. coli

 

TA 100

TA 98

TA 1535

TA 1537

WP2 uvrA

 

 

 

Results without S9

DMSO

111 ± 8

31 ± 5

16 ± 3

8 ± 2

31 ± 4

20

108 ± 4

24 ± 6

17 ± 1

8 ± 1

27 ± 5

100

108 ± 6

28 ± 1

19 ± 3

8 ± 2

34 ± 3

500

102 ± 4

24 ± 4

15 ± 2

9 ± 2

29 ± 1

2500

102 ± 11

26 ± 1

16 ± 1

7 ± 1

31 ± 2

5000

107 ± 7

17 ± 4

16 ± 2

6 ± 1

32 ± 5

MNNG (5)

648 ± 36

-

634 ± 43

-

-

NOPD (10)

-

504 ± 26

-

-

-

AAC (100)

-

-

-

429 ± 20

-

4-NQO (5)

-

-

-

-

556 ± 24

 

 

 

Results with S9 (1:9)

DMSO

114 ± 9

35 ± 3

16 ± 3

13 ± 2

35 ± 5

20

117 ± 12

30 ± 3

16 ± 2

11 ± 3

32 ± 6

100

116 ± 11

35 ± 11

15 ± 2

10 ± 2

35 ± 3

500

105 ± 4

33 ± 3

18 ± 3

8 ± 1

38 ± 5

2500

101 ± 4

30 ± 5

13 ± 3

9 ± 3

40 ± 6

5000

114 ± 18

31 ± 3

14 ± 3

7 ± 1

33 ± 2

2- AA (2.5; 60)

828 ± 27

593 ± 39

130 ± 21

133 ± 8

231 ± 18

 

Experiment 2 Preincubation test

Dose (µg/plate)

Mean number of revertant colonies of 3 replicates (± S.D.) for different strains of S. typhimurium and E. coli

 

TA 100

TA 98

TA 1535

TA 1537

WP2 uvrA

 

 

 

Results without S9

DMSO

110 ± 4

30 ± 1

17 ± 2

8 ± 1

31 ± 5

20

108 ± 6

27 ± 3

15 ± 1

8 ± 3

29 ± 6

100

101 ± 10

28 ± 3

17 ± 2

8 ± 2

29 ± 3

500

103 ± 2

28 ± 4

16 ± 1

9 ± 5

27 ± 6

2500

101 ± 7

23 ± 4

15 ± 1

8 ± 2

25 ± 4

5000

99 ± 3

14 ± 2

15 ± 1

5 ± 3

21 ± 0

MMNG (5)

674 ± 50

-

553 ± 24

-

-

NOPD (10)

-

422 ± 29

-

-

-

AAC (100)

-

-

-

397 ± 25

-

4-NQO (5)

-

-

-

-

620 ± 19

 

 

 

Results with S9 (1:9)

DMSO

107 ± 6

31 ± 2

17 ± 3

9 ± 2

35 ± 6

20

104 ± 12

32 ± 3

15 ± 2

11 ± 3

30 ± 6

100

104 ± 1

31 ± 3

17 ± 2

11 ± 4

30 ± 6

500

101 ± 5

25 ± 3

15 ± 3

7 ± 2

27 ± 2

2500

99 ± 6

22 ± 3

14 ± 4

7 ± 1

27 ± 6

5000

96 ± 11

13 ± 2

12 ± 1

5 ± 3

24 ± 4

2- AA (2.5)

631 ± 21

598 ± 78

141 ± 19

120 ± 20

225 ± 22

 

Table 3 Historical control values for revertants/plate: Standard Plate Test

 

 

Bacterial strains

Historical control data of DMSO control

-S9

 

TA 98

TA 100

TA 1535

TA 1537

E. coli

Average

29

108

18

10

32

SD

4

8

2

2

5

Minimum

19

87

11

5

25

Maximum

48

140

25

16

53

+S9 (2 -AA)

 

TA 98

TA 100

TA 1535

TA 1537

E. coli

Average

36

110

17

11

35

SD

5

9

2

2

5

Minimum

21

91

10

5

25

Maximum

50

154

24

18

58

Historical control data of positive

control

-S9

 

TA 98

TA 100

TA 1535

TA 1537

E. coli

Average

750

953

853

440

671

SD

125

258

205

94

159

Minimum

332

506

502

300

502

Maximum

1200

1795

1582

1042

1478

+S9 (2-AA

 

TA 98

TA 100

TA 1535

TA 1537

E. coli

Average

786

957

139

136

323

SD

157

225

29

26

28

Minimum

504

573

85

200

153

Maximum

1745

1878

254

136

324

 

Table 4 Historical control values for revertants/plat: Preincubation test

 

 

Bacterial strains

Historical control data of DMSO control

-S9

 

TA 98

TA 100

TA 1535

TA 1537

E. coli

Average

28

109

17

10

32

SD

4

9

2

2

5

Minimum

17

84

12

5

25

Maximum

40

148

23

17

51

+S9 (2 -AA)

 

TA 98

TA 100

TA 1535

TA 1537

E. coli

Average

32

110

17

10

33

SD

5

9

2

2

5

Minimum

20

147

10

5

25

Maximum

49

91

24

16

50

Historical control data of positive

control

-S9

 

TA 98

TA 100

TA 1535

TA 1537

E. coli

Average

739

842

747

420

599

SD

123

170

145

72

77

Minimum

388

511

502

278

500

Maximum

1129

1583

1830

842

1150

+S9 (2-AA)

 

TA 98

TA 100

TA 1535

TA 1537

E. coli

Average

665

740

122

115

230

SD

103

143

21

20

27

Minimum

501

500

80

80

150

Maximum

1120

1380

258

200

307

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03/2018 - 07/2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
yes
Remarks:
see below
Principles of method if other than guideline:
A series of in-house non-GLP validation experiments was performed to get distinct responses of statistical significance when using the specified positive controls. To achieve such response the test design, specifically for the treatment, the recovery phase and harvest time, was slightly modified comparing the current proposal given in the OECD Guideline 487.
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
CAS number: 93940-97-7
Purity: 96.1% area-% (mixture of isomers)
Identity: Confirmed
Homogeneity: Given
Expiry Date: 01 May 2019
Storage Conditions: At room temperature
Physical state / Appearance: Yellowish clear, liquid
Molecular weight: 276.42 g/mol
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
Blood samples were drawn from healthy non-smoking donors not receiving medication. For this study, blood was collected from a male donor (27 years old) and from a male donor (34 years old). The lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation with PHA and to positive control substances. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes.
Human lymphocytes were stimulated for proliferation by the addition of the mitogen PHA to the culture medium for a period of 48 hours. The cell harvest time point was approximately 2 – 2.5 x AGT (average generation time). Any specific cell cycle time delay induced by the test item was not accounted for directly.
Cytokinesis block (if used):
cytokinesis blocker: cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
Due to the limited capacity for metabolic activation of potential mutagens in in vitro methods an exogenous metabolic activation system was used.
Phenobarbital/β-naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 was prepared and stored according to the currently valid version of the Envigo CRS GmbH SOP for rat liver S9 preparation. Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.
An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. S9 mix contained MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4).
The protein concentration of the S9 preparation used for this study was 34.3 mg/mL
Test concentrations with justification for top dose:
Dose selection was performed according to the current OECD Guideline for the in vitro micronucleus test. If no precipitate or limiting cytotoxicity is observed, the highest test concentration should correspond to 10 mM, 2 mg/mL or 2 μl/mL.

With regard to the the purity (96.1%) of the test item, 2081 μg/mL were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 13.5 to 2081 μg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, phase separation of the test item was observed at the end of treatment at 2081 μg/mL in the absence and presence of S9 mix. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.
No cytotoxic effects were observed in Experiment I after 4 hours treatment in the absence and presence of S9 mix.

Therefore, 2081 μg/mL were chosen as top treatment concentration.
Vehicle / solvent:
DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Demelcolcine
Details on test system and experimental conditions:
Test System (Reason for the choice of human lymphocytes)
Human lymphocytes are the most common cells in the micronucleus test and have been used successfully for a long time in in vitro experiments. They show stable spontaneous micronucleus frequencies at a low level.

Cell cultures
Blood samples were drawn from healthy non-smoking donors not receiving medication. For this study, blood was collected from a male donor (27 years old) for Experiment I and from a male donor (34 years old) for Experiment II. The lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation with PHA and to positive control substances. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes.
Human lymphocytes were stimulated for proliferation by the addition of the mitogen PHA to the culture medium for a period of 48 hours. The cell harvest time point was approximately 2 – 2.5 x AGT (average generation time). Any specific cell cycle time delay induced by the test item was not accounted for directly.

Culture conditions
Blood cultures were established by preparing an 11 % mixture of whole blood in medium within 30 hrs after blood collection. The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 μg/mL), the mitogen PHA (3 μg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL).
All incubations were done at 37 °C with 5.5 % CO2 in humidified air.

Test Item Preparation
Stock formulations of the test item and serial dilutions were made in DMSO. The final concentration of DMSO in the culture medium was 0.5 %. The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures. All formulations were prepared freshly before treatment and used within two hours of preparation.

Cytogenetic Experiment

Pulse exposure
About 48 hrs after seeding, 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration and each control. The culture medium was replaced with serum-free medium containing the test item. For the treatment with metabolic activation, the culture medium was supplemented with approx. 2.5 % S9 fraction (50 μL S9 mix/mL culture medium). After 4 hrs the cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were resuspended in and washed with "saline G" (pH 7.2, containing 8000 mg/L NaCl, 400 mg/L KCl, 1100 mg/L glucose •H2O, 192 mg/L Na2HPO4 • 2 H2O and 150 mg/L KH2PO4). The washing procedure was repeated once as described. The cells were resuspended in complete culture medium with 10 % FBS (v/v) and cultured for a 16-hour recovery period. After this period Cytochalasin B (4 μg/mL) was added and the cells were cultured another approximately 20 hours until preparation.

Continuous exposure (without S9 mix)
About 48 hrs after seeding, 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration and each control. The culture medium was replaced with complete medium (with 10 % FBS) containing the test item. After 20 hours the cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were re-suspended in and washed with "saline G". The washing procedure was repeated once as described. After washing the cells were re-suspended in complete culture medium containing 10 % FBS (v/v). Cytochalasin B (4 μg/mL) was added and the cells were cultured another approximately 20 hours until preparation.
Evaluation criteria:
A test item can be classified as non-clastogenic and non-aneugenic if:
− None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− There is no concentration-related increase
− The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data (95% control limit realized as 95% confidence interval).

A test item can be classified as clastogenic and aneugenic if:
− At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− The increase is concentration-related in at least one experimental condition
− The results are outside the range of the laboratory historical solvent control data (95% control limit realized as 95% confidence interval).
Statistics:
Statistical significance was confirmed by the Chi square test (α < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure period was 20 hours without S9 mix. The cells were prepared 40 hours after start of treatment with the test item.
In each experimental group, two parallel cultures were analyzed. 1000 binucleate cells per culture were evaluated for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is described as % cytostasis.
The highest treatment concentration in this study, 2081 μg/mL was chosen with regard to the purity (96.1%) of the test item and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test.
Conclusions:
In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration, which showed phase separation in both parts of Experiment I.
In the absence and presence of S9 mix, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test item. In the presence of S9 mix, however, one statistically significant increase (0.80 %) was observed after treatment with 1189 μg/mL. Since the value is clearly within the range of the laboratory historical control data (0.08 – 1.38% micronucleated cells), the finding can be regarded as biologically irrelevant.
Demecolcine (125 ng/mL), MMC (0.8 μg/mL) or CPA (17.5 μg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.
Executive summary:

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.

Therefore, 3-[[3-[[(2-cyanoethyl)amino]methyl]-3,5,5-trimethylcyclohexyl]amino]propiono-nitrile is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03/2018 - 08/2018
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)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial forward mutation assay
Specific details on test material used for the study:
BASF Test substance number: 13/0241-4
Batch: 16315004
CAS number: 93940-97-7
Purity: 96.1% area-% (mixture of isomers) (Final Report, Study code: 17L00091)
Identity:Confirmed
Homogeneity: Given
Expiry Date: 01 May 2019
Storage Conditions: At room temperature
Physical state / Appearance: Yellowish clear, liquid
Molecular weight: 276.42 g/mol
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany) are stored in liquid nitrogen in the cell bank of Envigo CRS GmbH allowing the repeated use of the same cell culture batch in experiments. Before free-zing, the level of spontaneous mutants may be reduced by treatment with HAT-medium. Each master cell stock is screened for mycoplasm contamination and checked for karyotype stability and spontaneous mutant frequency. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells.
Thawed stock cultures were propagated at 37 °C in 75 cm2 plastic flasks. About 2-3×10^6 cells were seeded into each flask with 15 mL of MEM (minimal essential medium) containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1%). The cells were sub-cultured once or twice weekly.
All incubations were done at 37°C with 1.5% carbon dioxide (CO2) in humidified air.

Culture Medium
For seeding of the cell cultures the complete culture medium was MEM (minimal essential medium) containing Hank’s salts, neomycin (5 μg/mL), 10% FBS, and amphotericin B (1 %). During 4 hours treatment no FBS was added to the medium. For the selection of mutant cells the complete medium was supplemented with 11 μg/mL 6-thioguanine. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
32.5, 65, 130.1, 260.1, 520.3, 1040.5 (PS), 2081 (PS) µg/mL (PS= Phase separation visible at the end of treatment)

The concentration range of the main experiment was chosen according to the data generated in the pre-experiment. The individual concentrations were spaced by a factor of 2.0.
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
Test Item Preparation
On the day of the experiment (immediately before use), the test substance was dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO in the culture medium was 0.5% (v/v). The solvent was chosen according to its solubilisation properties and its compatibility with cell cultures.
All formulations were prepared freshly before treatment and used within two hours of preparation.

Reasons for the Choice of the Cell Line V79
The V79 cell line has been used successfully in in vitro experiments for many years. Especially the high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50%) both necessary for the appropriate performance of the study, recommend the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22.

Cell Cultures
Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany) are stored in liquid nitrogen in the cell bank of Envigo CRS GmbH allowing the repeated use of the same cell culture batch in experiments. Before free-zing, the level of spontaneous mutants may be reduced by treatment with HAT-medium. Each master cell stock is screened for mycoplasm contamination and checked for karyotype stability and spontaneous mutant frequency. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells.
Thawed stock cultures were propagated at 37 °C in 75 cm2 plastic flasks. About 2-3×10^6 cells were seeded into each flask with 15 mL of MEM (minimal essential medium) containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1%). The cells were sub-cultured once or twice weekly.
All incubations were done at 37°C with 1.5% carbon dioxide (CO2) in humidified air.

Culture Medium
For seeding of the cell cultures the complete culture medium was MEM (minimal essential medium) containing Hank’s salts, neomycin (5 μg/mL), 10% FBS, and amphotericin B (1 %). During 4 hours treatment no FBS was added to the medium. For the selection of mutant cells the complete medium was supplemented with 11 μg/mL 6-thioguanine. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).

Experimental Performance
- Seeding
Two to four days after sub-cultivation stock cultures were trypsinized at 37 °C for approximately 5 to 10 minutes. Then the enzymatic digestion was stopped by adding complete culture medium with 10% FBS and a single cell suspension was prepared. The trypsin concentration for all sub-culturing steps was 0.2% in saline. Prior to the trypsin treatment the cells were rinsed with PBS. Approximately 0.7 to 1.2×10^7 cells were seeded in plastic flasks. The cells were grown for 24 hours prior to treatment.

- Treatment
After approx. 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 μl/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. 4 hours after treatment, this medium was replaced with complete medium following two washing steps with PBS. Immediately after the end of treatment the cells were trypsinised as described above and sub-cultivated. At least 2.0×10^6 cells per experimental point (concentration series plus controls) were subcultured in 175 cm² flasks containing 30 mL medium.
Two additional 25 cm² flasks were seeded per experimental point with approx. 500 cells each to determine the relative survival (cloning efficiency I) as measure of test item induced cytotoxicity. The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2.
The colonies used to determine the cloning efficiency I were fixed and stained 6 to 8 days after treatment as described below.
Three or four days after first sub-cultivation approximately 2.0×106 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of approximately 7 days five 75 cm² cell culture flasks were seeded with about 4 to 5×10^5 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability (cloning efficiency II).
The cultures were incubated at 37 °C in a humidified atmosphere with 1.5% CO2 for about 8 days. The colonies were stained with 10% methylene blue in 0.01% KOH solution.
The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.
Rationale for test conditions:
The gene mutation assay is considered acceptable if it meets the following criteria:
a) The mean values of the numbers of mutant colonies per 106 cells found in the solvent controls of both parallel cultures remain within the 95% confidence interval of the laboratory historical control data range.
b) Concurrent positive controls should induce responses that are compatible with those generated in the historical positive control data base and produce a statistical significant increase compared with the concurrent solvent control.
c) Two experimental conditions (i.e. with and without metabolic activation) were tested unless one resulted in positive results.
d) An adequate number of cells and concentrations (at least four test item concentrations) are analysable even for the cultures treated at concentrations that cause 90% cytotoxicity during treatment.
e) The criteria for the selection of the top concentration are fulfilled
Evaluation criteria:
A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).

A test item is classified as clearly non-mutagenic if, in all experimental conditions examined, all of the following criteria are met:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data (based 95% control limits).

In cases when the response is neither clearly negative nor clearly positive as described above, or in order to judge the biological relevance of a result, the data should be evaluated by expert judgement or further investigations.
Statistics:
A linear regression analysis (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.
A t-test was not performed at the test item concentrations since the 95% confidence interval was not exceeded at any experimental point.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
Phase separation was observed at 1040.5 μg/mL and above in the presence and absence of metabolic activation.
No relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50% in both cultures occurred up to the highest concentration with and without metabolic activation.
No biologically relevant increase in mutant colony numbers was observed in the main experiment up to the maximum concentration scored for gene mutations.
The 95% confidence interval was not exceeded at any of the test item concentrations.
The linear regression analysis (least squares) showed no significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05.
The mutant frequencies obtained in the main experiment for the solvent controls (mean values) with and without S9 mix were in the range from 8.5 to 19.3 mutants per 106 cells. The values were well within the 95% confidence interval of our laboratory’s historical solvent control data and, thus, fulfilled the requirements of the current OECD Guideline 476. The range of the mutant frequencies (mean values) of the groups treated with the test item was from 13.4 up to 20.2 mutants per 106 cells.
EMS (300 μg/mL) and DMBA (2.3 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.
The mean mutant frequency in the positive control DMBA did not quite reach the range of the historical positive control data (52.8 mutant colonies per 106 cells compared to a range of 55.6 – 739.9 colonies per 106 cells). This deviation was judged as irrelevant as it was minor and the t-test performed at these data showed a significant response (p =0.000).

The osmolarity and the pH-value were determined in culture medium of the solvent control and of the maximum concentration in the pre-experiment without metabolic activation:

 

Solvent control

Test item 2081 μg/mL

Osmolarity [mOsm]

385

379

pH-value

7.24

7.69

Conclusions:
In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, 3-[[3-[[(2-cyanoethyl)amino] methyl]-3,5,5-trimethylcyclohexyl]amino]propiononitrile is considered to be non-mutagenic in this HPRT assay.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. A bactieral reverse mutation assay, a micronucleus assay and a gene mutation assay (HPRT) with the test substance were negative. As a result the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the eighth time in Regulation (EU) No 2016/218.