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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An Ames test (OECD 471, GLP) was performed (Stepan, 2000). TA 1535, TA 1537, TA 98, TA 100 and E.coli WP2 uvr A were treated with concentrations ranging from 5 to 5000 µg/plate (+/- S9). Results for genotoxicity were reported to be negative.

A mammalian chromosome aberration test (OECD 473, GLP) was conducted (Stepan, 2000). Primary lymphocytes were treated with concentrations ranging from 9.77 to 312.5 µg/ml (+/- S9). Results for genotoxicity were reported to be negative.

A read across approach was conducted by using a mammalian gene mutation assay (OECD 476, GLP) performed for a structurally similar substance (amides, C8-C18 (even numbered), N-[3-(dimethylamino)propyl], N-oxides) in 2010. Mouse lymphoma L5178Y cells were treated with concentrations ranging from 1.25 to 70 µg/ml (+/- S9). Genotoxicity was 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:
From February 28, 2000 to March 25, 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
OECD guideline 471 and EU guideline B14. GLP study.
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)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: Stepan Company
- Batch No.of test material: 876 TK
- Date received: 10 May 1999
- Description: off-white paste

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test material was accurately weighed and approximately half-log dilutions prepared in dimethyl formamide. An allowance for test material purity (77%) was made prior to each days formulation.
Target gene:
his and trp
Species / strain / cell type:
E. coli WP2 uvr A
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction
Test concentrations with justification for top dose:
Experiment 1:
5, 15, 50, 150, 500 and 1500 µg/plate for the strains S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
15, 50, 150, 500, 1500 and 5000 µg/plate for E. coli WP2 uvr A

Experiment 2:
5, 15, 50, 150, 500 and 1500 µg/plate for the strains S. typhimurium TA 1535, TA 98 and TA 100
50, 150, 500, 1500 and 5000 µg/plate for E. coli WP2 uvr A
15, 50, 100, 150, 200 and 500 µg/plate for the strain S. typhimurium TA 1537

Experiment 3:
15, 50, 100, 150, 200 and 500 µg/plate for the strain S. typhimurium TA 1537

Justification:
In order to select an appropriate dose level for use in the main study, a preliminary test was carried out to determine the toxicity of the test material.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl formamide
- Justification for choice of solvent/vehicle: Dimethyl formamide is an acceptable vehicle for use in the Ames assay.
Negative solvent / vehicle controls:
yes
Remarks:
(Dimethyl formamide)
Positive controls:
yes
Remarks:
(without metabolic activation)
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
3 µg/plate for TA100, 5 µg/plate for TA1535 and 2 µg/plate for WP2uvrA
Positive controls:
yes
Remarks:
(without metabolic activation)
Positive control substance:
9-aminoacridine
Remarks:
80 µg/plate for TA1537
Positive controls:
yes
Remarks:
(without metabolic activation)
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
0.2 µg/plate for TA98
Positive controls:
yes
Remarks:
(with metabolic activation)
Positive control substance:
other: 2-aminoanthracene
Remarks:
1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537, 10 µg/plate for WP uvrA
Positive controls:
yes
Remarks:
(with metabolic activation)
Positive control substance:
benzo(a)pyrene
Remarks:
5 µg/plate for TA98
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth.

Evaluation criteria:
The test material was considered positive in this test system if there is a reproducible, dose-related and statistically significant increase in the revertant count in at least one strain of bacteria.
Statistics:
Dunnett's method of linear regression
Species / strain:
other: E. coli WP2 uvr A, TA1535, TA1537, TA98, TA100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(at and above 200 µg/plate)
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: E. coli WP2 uvr A, TA1535, TA1537, TA98, TA100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(at and above 500 µg/plate)
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation of the test substance was noted at the dose levels tested.

RANGE-FINDING/SCREENING STUDIES:
Preliminary toxicity study was performed with strains TA100 and WPuvrA with and without metabolic activation. Ten concentrations of the test material and a vehicle control (dimethyl formamide) were tested. The dose range of the test material was: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500, and 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
All tester strain cultures exhibit a characteristic number, within the range of historical data, of spontaneous revertants per plate in the vehicle and untreated controls.

Experiment 1

 

Table 1: without metabolic activation

With or without S9-Mix

Test substance concentration µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

  

TA100             TA1535        WP2uvrA

Frameshift type

   

    TA98         TA1537

-

0

146

10.1 #

22

2.9

19

3.1

20

6.1

9

0.6

-

5

144

12.5

20

3.1

 

N/T

20

3.8

10

1.5

-

15

145

11.4

14

4.2

23

3.8

25

2.3

14

4.4

-

50

143

7.6

12

2.1

16

7.0

28

8.4

16*

3.8

-

150

112

4.0

13

2.3

24

3.1

25

4.0

18$$$

2.3

-

500

23

3.2

3

2.0

22

9.3

6

2.0

3V

1.7

-

1500

0V

0.0

0V

0.0

20

3.5

0V

0.0

0T

0.0

-

5000

N/T

N/T

0V

0.0

N/T

N/T

Positive controls

Name

ENNG

ENNG

ENNG

4NQO

9AA

S9-Mix

Concentration

(µg/plate)

3

5

2

0.2

80

 

Nº Colonies per plate

425

56.4

372

1.5

615

77.6

111

12.3

807

226.2

 

ENNG  N-ethyl-N'-nitro-N-nilrosoguanidine N/T  Not tested at this dose level

4NQO  4-Nitroquinoline-l-oxide                                   S  Sparse bacterial background lawn

9AA    9-Aminoacridine                                           T  Toxic, no bacterial background lawn

V        Very weak bacterial background lawn            $$$ p≤0.005

#         Standard deviation                                         *    p≤ 0,05         

 

 Table 2: with metabolic activation 

With or without S9-Mix

Test substance concentration (µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

 

TA100           TA1535      WP2uvrA

Frameshift type

 

  TA98               TA1537

+

0

151

18.6#

13

2.1

22

0.0

24

3.6

7

1.0

+

5

174

51.0

14

1.5

N/T

21

3.2

12

2.0

+

15

132

13

11

5.1

22

1.2

29

2.5

9

3.1

+

50

144

11

12

2.1

19

4.5

30

8.3

13

5.3

+

150

154

14.6

14

1.7

23

1.0

27

3.6

13

4.2

+

500

38

5.3

5

4.0

27

6.1

17

7.2

4

1.7

+

1500

0V

0.0

0V

0.0

20

5.5

0V

0.0

0V

0.0

+

5000

N/T

N/T

0V

0.0

N/T

N/T

Positive controls

S9-Mix

+

 

 

 

Name

Concentration µg/plate)

Nº colonies per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

2122

505.5

212

41.0

479

7.6

231

25.4

546

60.5

 BP        Benzo(a)pyrene                                        N/T   Not testedat this dose level

2AA     2-Aminoanthracene                                      S      Sparse bacterial background lawn

V          Very weak bacterial background lawn           #      Standard deviation

*           p≤ 0,05

 

 Experiment 2

 

Table 3: without metabolic activation

With or without S9-Mix

Test substance concentration (µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

 

 TA100           TA1535     WP2uvrA

Frameshift type

    

  TA98           TA1537

-

0

81

12.3#

19

1.7

18

6.1

15

2.3

9

5.3

-

5

89

1.0

16

3.2

N/T

14

2.1

N/T

-

15

79

15.0

15

6.2

N/T

14

1.5

9

1.2

-

50

85

3.2

22

7.0

20

9.8

16

2.3

10

3.8

-

100

N/T

N/T

N/T

N/T

13

3.2

-

150

76

9.6

15

1.2

17

3.8

15

3.1

9

4.4

-

200

N/T

N/T

N/T

N/T

6

1.5

-

500

10S

6.0

3S

1.0

16

3.8

6S

3.1

0V

0.0

-

1500

0V

0.0

0V

0.0

14

3.6

0V

0.0

N/T

-

5000

N/T

N/T

2

1.7

N/T

N/T

Positive controls

S9-Mix

-

 

 

Name

 

ENNG

ENNG

ENNG

4NQO

9AA

Concentration

(µg/plate)

3

5

2

0.2

80

No. colonies per plate

 

359

12.2

269

35.5

817

40.6

89

8.4

940

164.2

 

ENNG   N-ethyl-N'-nitro-N-nitrosoguanidine                  N/T   Not tested at this dose level

4NQO   4-Nttroquinoline-l-oxide                                   S      Sparse bacterial background lawn

9AA      9-Aminoacridine                                            V      Very weak bacterial background lawn

#          Standard deviation

 

Table 4: with metabolic activation

With or without

S9-Mix

 

Test substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

  TA100        TA1535       WP2uvrA

Frameshift type

TA98        TA1537

+

0

93

4.0#

12

4.5

21

2.6

22

3.1

10

4.2

+

5

97

8.2

13

2.3

N/T

23

4.6

N/T

+

15

83

11.8

12

1.7

N/T

17

3.5

11

3.1

+

50

91

12.1

12

1.0

20

2.5

29

9.2

10

2.0

 

100

N/T

N/T

N/T

N/T

13

1.7

+

150

82

12.7

13

2.1

20

3.5

31

12.4

17

6.6

+

200

N/T

N/T

N/T

N/T

11

4.6 

+

500

30

10.8

4S

1.7

19

5.3

11S

4.2

5S

2.5

+

1500

0V

0.0

0V

0.0

13

3.1

0V

0.0

N/T

+

5000

N/T

N/T

2S

2.1

N/T

N/T

Positive controls

S9-Mix

+

 

 

Name

 

2AA

2AA

2AA

BP

2AA

Concentration (µg/plate)

1

2

10

5

2

No. colonies per plate

929

68.6

128

36.0

708

24.3

174

36.3

435

56.5

 

BP       Benzo(a)pyrene                      N/T   Not tested at this dose level

2AA     2-Aminoanthracene                  S      Sparse bacterial background lawn

#         Standard deviation                    V      Very weak bacterial background lawn

 

 

Experiment 3

Table 5: with and without metabolic activation

Test substance concentration µg/plate)

Number of revertants (mean number of colonies per plate)

Frameshift type

(Without metabolic activation)

TA1537

Frameshift type

(With metabolic activation)

TA1537

0

8

3.2#

12

3.1

15

9

2.0

11

4.2

50

10

1.5

13

2.6

100

9

0.7

13

4.5

150

11

3.2

13

3.5

200

2S

1.7

14

4.0

500

0V

0.0

10S

7.8

Name

9AA

2AA

Concentration (µg/plate)

80

2

No. colonies per plate

940

87.0

399

137.2

9AA     9-Aminoacridine

2AA     2-Arninoanthracene

S         Sparse bacterial background lawn

V         Very weak bacterial background lawn

#          Standard deviation

Conclusions:
Interpretation of results: negative

The test material was considered to be non-mutagenic under the conditions of this test.
Executive summary:

The Bacterial Reverse mutation Assay (Ames test) for the test substance was performed with five histidine dependents bacteria, four strains of Salmonella typhimurium T1535, TA98, TA100, and TA1537 and E.Coli WP2 uvrA. Plate Incorporation Method was carried out at the concentrations of 5000, 1500, 500, 150, 50 and 15 µg/plate for the strains of Salmonella typhimurium in the presence and absence of metabolic activation and at the concentrations of 1500, 500, 150, 50, 15 and 5 µg/plate for the E.Coli WP2 uvrA in the presence and absence of metabolic activation.The dose range for TA1537 (with/without S9) was modified slightly, based on results observed in the Experiment 1and was15, 50, 100, 150, 200 and 500 µg/plate. Intermediate doses were included to allow for small but statistically significant increases observed in the first experiment. The test material caused a visible reduction in the growth of the bacterial lawn to all of the bacterial tester strains both with and without metabolic activation. The toxicity of the test material to the bacterial tester strains varied both between strains and between exposures with or without S9-mix. The test material was, therefore, tested up to its toxic limit. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. Very small, statistically significant increases were observed to TA1537 in Experiment 1 only both with and without S9. However, these increases were not considered to be biologically significant as they were within the strains historical range and were non-reproducible over three separate experiments. According to the obtained results, the test substance was, therefore, considered to be non-mutagenic under the conditions of this test.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 26, 2000 to September 7, 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
OECD guideline 473 and EU guideline B.10. GLP study.
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability.
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Preliminary Toxicity test: 0, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/ml
Experiment 1: with and without S9: 0, 9.77, 19.53, 39.06, 78.13, 156.25, 234.38 and 312.5 µg/ml
Experiment 2: without S9: 0, 4.89, 9.77, 19.53, 39.06, 78.13, 117.19 µg/ml
Experiment 2: with S9: 0, 19.53, 39.06, 78.13, 156.25, 234.38 and 312.5 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Eagle's minimal essential medium (MEM)
Negative solvent / vehicle controls:
yes
Remarks:
Eagle's minimal essential medium (MEM)
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Without metabolic activation system
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation system
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium. 1.0 ml of the appropriate solution of vehicle, positive control or test material was added to 9.0 ml 0f each culture. 1 ml of 10% S9 (ie 1 % final concentration of S9) was used for the Preliminary Toxicity Test and Experiment 1, and 1 ml of 20% S9 (ie 2% final concentration of S9) for Experiment 2.
- Exposure duration and Expression time (cells in growth medium):
Experiment 1: with S9-mix was exposed 4 hours with a cell harvest after a 20-hour expression period.
Experiment 1: without S9-mix was exposed 4 hours with a cell harvest after a 20-hour expression period.
Experiment 2: with S9-mix was exposed 4 hours with a cell harvest after a 20-hour expression period.
Experiment 2: without S9-mix the exposure time was 24 hours.
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours, the mitosis was arrested two hours before the required harvest time

SELECTION AGENT (mutation assays): Colcemid
STAIN (for cytogenetic assays): the slides were stained in 5% Gurrs Giemsa for 5 minutes.

NUMBER OF REPLICATIONS: all cultures were set up in duplicate.

NUMBER OF CELLS EVALUATED: where possible the first 100 consecutive well-spread metaphases from each culture were counted

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index. A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
Evaluation criteria:
If the metaphase cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing. Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides. A statistically significant increase in the frequency of cells with chromosome aberrations (excluding gaps) is considered as a positive result.
Statistics:
The frequency of cells with aberrations (both including and excluding gaps) and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 156.25 µg/ml and above
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: there was no observable change in pH when the test substance was dosed into the media.
- Effects of osmolality: the osmolality did not increase (50 mOSM)
- Precipitation: there was no precipitate of the test material seen in the cultures.

COMPARISON WITH HISTORICAL CONTROL DATA: The vehicle control cultures had frequencies of cell with chromosome aberrations within the expected range and the positive control treatments gave statistically significant increases in the frequency of cells with aberrations.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Metaphase cells were present at dose levels up to 2500 µg/ml in the 4(20) hour treatment in the absence of metabolic activation (S9) and up to 156.25 µg/ml in the 4(20) hour treatment in the presence of S9. The maximum dose with metaphases present in the 24-hour continuous exposure was 78.13 µg/ml. In the 4 hour exposure group without S9, approximately 50% mitotic inhibition was achieved at 156.25 µg/ml and complete mitotic inhibition at 5000 µg/ml.

Table 1:Mitotic Index Experiment 1

MITOTIC INDEX - EXPERIMENT 1 (4-HOUR TREATMENT, 24-HOUR HARVEST)

DOSE LEVEL

4 HOURS TREATMENT WITHOUT S9

4 HOURS TREATMENT WITH S9

µg/ml

A

B

Mean

% of

Control

A

B

Mean

% of

Control

0

4.20

6.20

5.20

100

6.05

6.15

6.10

100

9.77

-

-

-

-

-

-

-

-

-

19.53

-

-

-

-

-

-

-

-

-

39.06

3.80

5.70

4.75

91

7.05

6.95

6.95

7.00

-

78.1 3

4.65

4.55

4.60

88

6.40

5.60

5.60

6.00

98

156.25

2.65

1.50

2.08

40

J.40

2.25

2.25

2.83

46

234.38

NM

NM

0.00

0

NM

NM

NM

0.00

0

312.5

NM

NM

0.00

0

NM

NM

NM

0.00

0

MMC 0.4

2.75

2.20

2.48

48

NA

NA

NA

NA

NA

CP 25

NA

NA

NA

NA

1.95

1.05

1.95

1.50

25

MMC: mitomycin C              CP: cyclophosphamide        - : not assessed for mitotic index            NA: not applicable

NM: no scorable metaphases

 

Table 2: Mitotic Index Experiment 2 

MITOTIC INDEX - EXPERIMENT 2 (24-HOUR HARVEST)

DOSE LEVEL

24 HOURS TREATMENT WITHOUT S9

4 HOURS TREATMENT WITH S9

µg/ml

A

B

Mean

% of

Control

A

B

Mean

% of

Control

0

4.35

3.65

4.00

100

3.90

4.50

4.20

100

4.89

-

-

-

-

NA

NA

NA

NA

9.77

-

-

-

-

NA

NA

NA

NA

19.53

-

-

-

-

-

-

-

-

39.06

4.35

2.90

3.63

91

5.00

4.55

4.78

1 14

78.13

2.55

2.60

2.58

65

6.45

4.00

5.23

125

11 7.19

1.10

1.40

1.25

31

NA

NA

NA

NA

156.25

NA

NA

NA

NA

1.95

1.30

1.63

39

234.38

NA

NA

NA

NA

NM

NM

0.00

0

312.5

NA

NA

NA

NA

NM

NM

0.00

0

MMC 0.2

1.50

0.95

1.23

31

NA

NA

NA

NA

CP 25

NA

NA

NA

NA

0.60

0.35

0.48

11

 MMC: mitomycin C              CP: cyclophosphamide        - : not assessed for mitotic index            NA: not applicable

NM: no scorable metaphases

Conclusions:
Interpretation of results: negative

The test substance was shown to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

The In Vitro Mammalian Chromosome Aberration Test for the test substance was performed with human lymphocytes. A total of two experiments with four treatment conditions were used for the study, ie. 4 hours exposure with the addition of metabolic activation system (S9) with cell harvest after a 20-hour expression period and a 4-hour exposure in the absence of activation with a 20-hour expression period, this was Experiment 1. In the Experiment 2 the 4-hour exposure with addition of S9 was repeated (using a 2% final concentration of S9), whilst in the absence of activation the exposure time was increased to 24 hours. For the metaphase analysis the dose levels selected were: 39.06, 78.13, and 156.25 µg/ml in the Experiment 1; 39.06, 78.13 and 117.19 µg/ml in the Experiment 2, treatment time 24 hours, and 39.06, 78.13 and 156.25 µg/ml in the Experiment 2, treatment time 4 hours. The test substance showed some evidence of toxicity in all cases, a complete mitotic inhibition was observed in the 4 hour exposure group without S9, at 5000 µg/ml (pre-test). The test substance did not induce a statistically significant increase in the frequency of cells with chromosome aberrations (excluding gaps) in either the presence or absence of a metabolic activation system in either of two separate experiments. The test substance was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
Read-across approach from experimental data on an analogue.
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: PA19400382
- Concentration: approximately 30% solution of substance in water
- Physical appearance: pale amber coloured liquid

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: To achieve the maximum recommended dose level of 50 mg/ml (5000 µg/ml) for the preliminary test, the test substance was measured out and diluted 1:5 with R0 medium.
- Final dilution of a stock liquid: 1:5
Target gene:
thymidine kinase locus, TK +/-
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
S9 obtained from rat liver induced phenobarbital/beta-naphthoflavone
Test concentrations with justification for top dose:
Experiment 1: 5 to 55 µg/ml without metabolic activation; 20 to 70 µg/ml with metabolic activation
Experiment 2: 1.25 to 25 µg/ml without metabolic activation; 10 to 65 µg/ml with metabolic activation

A preliminary test has been conducted in order to establish the highest dose which could be used for further testing. The maximum dose applied was 5000 µg/ml resulting in very high levels of toxicity and thus, leading to a second experiment with the top dose being 80 µg/ml.
Results from the pre-test were used to evaluate the top dose according to the follwing criteria:
1) maximum recommended dose level, 5000 µg/ml or 10 mM
2) the presence of excessive precipitate where no test material-induced toxicity was observed
3) test-material induced toxicity, where the maximum dose level used should produce 10 to 20% survival
Vehicle / solvent:
RPMI 1640 without serum
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Solvent control is the negative control
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Remarks:
EMS (positive control without S9), CP (positive control with S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension (Exp. 1); in medium (Exp. 2)
- Cell density at seeding: 1 x 10^6 (Exp. 1); 0.3 x 10^6 (Exp. 2)

DURATION
- Exposure duration: 4 h (Exp. 1), 24 h (Exp. 2)
- Method: Incubation at 37°C with continuous shaking

SELECTION AGENT: trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2

METHODS OF STAINING TECHNIQUE USED: MTT

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
- Any supplementary information relevant to cytotoxicity: to assist the scoring of the TFT mutant colonies 0.025 ml MTT solution was added


Rationale for test conditions:
Dose selection for the mutagenicity experiments was made using data from the preliminary toxicity test in an attempt to obtain the desired levels of toxicity. This optimum toxicity is approximately 20% survival (80% toxicity), but no less than 10% survival (90% toxicity). The test material inducing a mutation frequency value greater than the previously set value of 126 x 10^-6 (Global Evaluation Factor, GEF) is considered to be positive.
Evaluation criteria:
Relative suspension growth (%RSG), calculation of day 2 viability (%V), calculation of relative total growth (RTG), calculation of mutation frequency (MF).
Statistics:
Test for linear trend
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment 1: at 55 μg/ml in the absence of metabolic activation, and at and above 60 μg/ml in the presence of metabolic activation; Experiment 2: at 20 μg/ml in the absence of metabolic activation and at 55 μg/ml in the presence of metabolic activation
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no

RANGE-FINDING/SCREENING STUDIES: In all three of the exposure groups there were marked dose related reductions in the %RSG of cells treated with the test material when compared to the concurrent vehicle controls. No precipitate of the test material was observed at any of the dose levels.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: MTT

HISTORICAL CONTROL DATA
- Positive historical control data: MF of 530.76 to 2223.55 (without S9); MF of 466.26 to 1248.34 (with S9)
- Negative historical control data: MF of 65.97 to 184.74 (without S9); Mf of 91.96 to 164.97 (with S9)
Conclusions:
Interpretation of results: negative

The substance did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic.
Executive summary:

A mammalian gene mutation assay using mouse lymphoma L5178Y cells was conducted according to OECD guideline 476 in compliance with GLP. Mouse lymphoma L5178Y cells were treated in duplicate with concentrations ranging from 1.25 to 70 µg/ml either with or without metabolic activation. Exposure times for experiment 1 and experiment 2 were 4 h and 24 h, respecticely. Cell viability was measured using the MTT assay. Trifluorothymidine was used as selection agent. Relative suspension growth (%RSG), calculation of day 2 viability (%V), calculation of relative total growth (RTG), and calculation of mutation frequency (MF) were parameter evaluated. Results for genotoxicity were reported to be negative. Cytotoxicity occurred at 20 µg/ml (with metabolic activation) and at 55 µg/ml (with metabolic activation) and above.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Jul 1998
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:
May 1983
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
- Batch No.of test material: 8058790
- Expiration date of the batch: April 1999
- Purity: 30% active substance. The 30% active substance was taken into consideration. The concentration in both experiments are based on 100% active substance.
- Physical appearance/color: liquid/yellowish

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability of the test substance pure in the solvent: 12 months

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was dissolved in bidistilled water and diluted with bidistilled water to the desired concentration just before the start of the test.
Target gene:
his
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
S9 mix obtained by phenobarbital/beta-naphthoflavone induced rat liver
Test concentrations with justification for top dose:
1st experiment: 8, 40, 50, 200, 1000 and 5000 µg/plate
2nd experiment: 12.5, 25, 50, 100 and 200 g/plate

The 30% active substance was taken into consideration. The concentrations in both experiments are based on 100% active substance. The solution medium was chosen according to the properties tested preliminary before start of the study.
Vehicle / solvent:
- Solvent used: Bidistilled water
- Justification for choice of solvent: The solution medium was chosen according to the solubility properties tested preliminary before the start of the study.
Untreated negative controls:
yes
Remarks:
untreated fresh cells
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 4-nitro-o-phenylendiamine, 2-aminoanthracene (with S9 mix, all strains)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
- Any supplementary information relevant to cytotoxicity: The colonies were counted with an Cardinal automatic colony counter.
Evaluation criteria:
A combination of the following criteria was considered as a positive result:
- the plate background of non-converted bacteria did not show any growth reduction versus the respective negative controls
- the spontaneous mutation rates of each tester strain per plate were within the characteristic spontaneous mutation range
- as a rule, the positive controls showed mutation rates exceeding the control values of TA 100 at least by the factor 2.0 and those of the other tester strains at least by the factor 3.0
- at more than one dose tested, the test substance caused at least a 2.0-fold increase in comparison with the negative controls in the tester strain TA 100. For the other tester strains, an increase in the mutation rate of more than 3.0 above the corresponding negative controls was considered positive
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1,000 g/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1,000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 200 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1,000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 200 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1,000 µg/plate
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:
cytotoxicity
Remarks:
at 1,000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Test results in the table above presents the results from the 1st experiment only. The results of the 2nd experiment are comparable to those obtained from the 1st experiment. Cytotoxicity was observed at 200 µg/plate only in strain TA 1538 without metabolic activation. All other strains, in the absence and presence, experienced no cytotoxicity at the highest concentration chosen (200 µg/plate). In addition, no genotoxicity was observed and the controls were valid.
No precipitations were noted.
Conclusions:
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce point mutations by base-repair changes or frameshifts in the genoe of the strains used. Therefore, the test substance is not considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.
Executive summary:

The sample of the test substance was tested in the Salmonella typhimurium Reverse Mutation Assay for the induction of reverse mutations in a bacterial test system.

The assay was performed with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 in two independent experiments, both with and without metabolic activation by S9-mix.

Solutions of the test substance were prepared in bidist. water and diluted with the same solvent just before use. The following concentrations were tested:

1st test: 8, 40, 200, 1000 and 5000 pg/plate

2nd test: 12.5, 25, 50, 100 and 200 pg/plate

The direct plate incorporation assay was utilized.

The following results were obtained:

Toxic effects were noted at the concentration of 200 pg/plate or higher. Precipitations were not noted.

No enhanced revertant rates compared to concurrent negative controls were observed in all tested strains, neither in the presence nor in the absence of metabolic activation.

Therefore the test substance did not induce reverse mutations in the tested strains of Salmonella typhimurium in this bacterial mutagenicity test.

In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test substance did not induce gene mutations in Salmonella typhimurium strains.

Subsequently, the test substance is considered not to be mutagenic in this bacterial mutagenicity test in vitro.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In vitro gene mutation study in bacteria

The Bacterial Reverse mutation Assay (Ames test) for the test substance was performed with five histidine dependents bacteria, four strains ofSalmonella typhimuriumT1535, TA98, TA100, and TA1537 and E.Coli WP2 uvrA. Plate Incorporation Method was carried out at the concentrations of 5000, 1500, 500, 150, 50 and 15 µg/plate for the strains ofSalmonella typhimurium in the presence and absence of metabolic activation and at the concentrations of 1500, 500, 150, 50, 15 and 5 µg/plate for the E.Coli WP2 uvrA in the presence and absence of metabolic activation.The dose range for TA1537 (with/without S9) was modified slightly, based on results observed inthe Experiment 1and was15, 50, 100, 150, 200 and500 µg/plate. Intermediate doses were included to allow for small but statistically significant increases observed in the first experiment.The test material caused a visible reduction in the growth of the bacterial lawn to all of the bacterial tester strains both with and without metabolic activation.The toxicity of the test material to the bacterial tester strains varied both between strains and between exposures with or without S9-mix. The test material was, therefore, tested up to its toxic limit. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. Very small, statistically significant increases were observed to TA1537 in Experiment 1 only both with and without S9. However, these increases were not considered to be biologically significant as they were within the strains historical range and were non-reproducible over three separate experiments.According to the obtained results, the testsubstance was, therefore, considered to benon-mutagenic under the conditions of this test.

The sample of the test substance was tested in the Salmonella typhimurium Reverse Mutation Assay for the induction of reverse mutations in a bacterial test system by Henkel KGaA according to OECD guideline 471 in compliance with GLP in 1998.

The assay was performed with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 in two independent experiments, both with and without metabolic activation by S9-mix.

Solutions of the test substance were prepared in bidist. water and diluted with the same solvent just before use. The following concentrations were tested:

1st test: 8, 40, 200, 1000 and 5000 pg/plate

2nd test: 12.5, 25, 50, 100 and 200 pg/plate

The direct plate incorporation assay was utilized.

The following results were obtained:

Toxic effects were noted at the concentration of 200 pg/plate or higher. Precipitations were not noted.

No enhanced revertant rates compared to concurrent negative controls were observed in all tested strains, neither in the presence nor in the absence of metabolic activation.

Therefore the test substance did not induce reverse mutations in the tested strains of Salmonella typhimurium in this bacterial mutagenicity test.

In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test substance did not induce gene mutations in Salmonella typhimurium strains. Subsequently, the test substance is considered not to be mutagenic in this bacterial mutagenicity test in vitro.

In vitro cytogenicity study in mammalain cells

The In VitroMammalian Chromosome Aberration Test for the test substance was performed with human lymphocytes. A total of two experiments with four treatment conditions were used for the study, ie.4 hours exposure with the addition of metabolic activation system (S9) with cell harvest after a 20-hour expression period and a 4-hour exposure in the absence of activation with a 20-hour expression period, this was Experiment 1. In the Experiment 2 the 4-hour exposure with addition of S9 was repeated (using a 2% final concentration of S9), whilst in the absence of activation the exposure time was increased to 24 hours.For the metaphase analysis the dose levels selected were: 39.06, 78.13, and 156.25 µg/ml in the Experiment 1; 39.06, 78.13 and 117.19 µg/ml in the Experiment 2, treatment time 24 hours, and39.06, 78.13 and 156.25 µg/ml in the Experiment 2, treatment time 4 hours.The test substance showed some evidence of toxicity in all cases, a complete mitotic inhibition was observed in the 4 hour exposure group without S9, at 5000 µg/ml (pre-test).The testsubstancedid not induce a statistically significant increase in the frequency of cells with chromosome aberrations (excluding gaps) in either the presence or absence of ametabolic activation systemin either of two separate experiments.The test substance was therefore considered to be non-clastogenic to human lymphocytes in vitro.

In vitro gene mutation in mammalian cells

For the mammalian gene mutation assay, a read-across approach was used. The analogue Amides, C12-C18 (even numbered), N-[3-(dimethylamino) propyl], N´-oxides which shares the same functional groups with the substance Amides, C12-C14 (even numbered), N-[3-(dimethylamino) propyl], N´-oxides, also has comparable values for the relevant molecular properties. The analogue has been tested in a mammalian gene mutation assay using mouse lymphoma L5178Y cells conducted according to OECD guideline 476 in compliance with GLP in 2010. Mouse lymphoma L5178Y cells were treated in duplicate with concentrations ranging from 1.25 to 70 µg/ml either with or without metabolic activation. Exposure times for experiment 1 and experiment 2 were 4 h and 24 h, respecticely. Cell viability was measured using the MTT assay. Trifluorothymidine was used as selection agent.Relative suspension growth (%RSG), calculation of day 2 viability (%V), calculation of relative total growth (RTG), and calculation of mutation frequency (MF) were parameter evaluated. Results for genotoxicity were reported to be negative. Cytotoxicity occurred at 20 µg/ml (with metabolic activation) and at 55 µg/ml (with metabolic activation) and above. Based on the results obtained in the mammalian gene mutation assay using the analogue it is concluded that the test substance is also negative for genotoxicity in the gene mutation assay.

Conclusion

Further testing was considered not be necessary, because the required studies for this endpoint, as part of the integrated testing strategy set forth in the Regulation (EC) 1906/2006, were all reported to be negative.

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 the purpose of classification under Regulation 1272/2008. Based on the criteria laid down in Regulation (EC) No. 1272/2008, as amended for the second time in Directive EC 286/2011, classification as a mutagen is not warranted.