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Genetic toxicity in vitro

Description of key information
GENETIC TOXICITY IN VITRO Gene mutation in bacteria: S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538, with and without metabolic activation, OECD 471: negative with and without metabolic activation (BASF SE, 1999, 9901542) Gene mutation in mammalian cells: HPRT, V79, with and without metabolic activation, OECD 476, up to 70.0 μg/mL: non mutagenic with and without metabolic activation (BASF SE, 2013, 50M0674/12M311) Cytogenetic in mammalian cells: Micronucleus test in vitro, V79, with and without metabolic activation, three experiments , OECD 487: non mutagenic with and without metabolic activation (BASF SE, 2013, 33M0674/12M331)
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
OECD guideline study conducted in compliance with GLP regulations, with acceptable restrictions (neither E. coli strain WP2 uvrA nor S. typhimurium strain TA102 were employed in this study for the detection of oxidising mutagens and cross-linking agents)
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(adopted 1983)
Deviations:
yes
Remarks:
(neither E. coli strain WP2 uvrA pKM101a nor S. typhimurium strain TA102 were employed in this study for the detection of oxidising mutagens and cross-linking agents; 2-aminoanthracene used as sole indicator of the efficacy of the S9 mix)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
Species / strain / cell type:
S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537, TA 1538
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/B-Naphthoflavone induced rat liver S-9 mix
Test concentrations with justification for top dose:
1st and 2nd assay: 8, 40, 200, 1000 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Tween 80/bidest. water
- Justification for choice of solvent/vehicle: The suspension medium was chosen according to the solubility properties tested preliminary before start of the study.
Untreated negative controls:
yes
Remarks:
untreated cell suspension
Negative solvent / vehicle controls:
yes
Remarks:
Tween 80/bidest. water
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 4-nitro-o-phenylenediamine, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 12 hours

NUMBER OF REPLICATIONS: triplicate plates

DETERMINATION OF CYTOTOXICITY
- Method: background lawn

DETAILS ON CONTROLS:
- Negative controls: suspension medium Tween 80/bidist. water and the untreated fresh cell suspensions
- Positive controls:
- Without metabolic activation:
sodium azide (2 µg/plate) for TA 100 and TA 1535;
9-aminoacridine (80 µg/plate) for TA 1537
4-nitro-o-phenylendiamine (40 µg/plate) for TA 98 and TA 1538
- With metabolic activation:
2-aminoanthracene in all strains (2.5 µg/plate for TA 1535, TA 1537; 5.0 µg/plate for TA 100, TA 98, TA 1538)
- Sterility controls:
The test batches containing S9-mix were each controlled for sterility by adding 0.5 mL of S9-mix fraction to untreated agar plates.
Species / strain:
S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537, TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

RESULTS:

 

Experiment 1

Treatment groups

[µg/plate]

Strains

 

 

TA 100

TA 1535

TA 1537

TA 1538

TA 98

 

 

-

SD

+

SD

-

SD

+

SD

-

SD

+

SD

-

SD

+

SD

-

SD

+

SD

medium

-

108

6

125

15

9

3

18

12

16

5

10

0

10

4

11

6

19

8

32

2

solvent

-

129

15

117

14

10

1

13

1

17

5

11

3

8

1

12

7

22

6

25

4

test substance

8

121

18

137

17

10

2

11

3

14

3

20

2

10

3

9

6

29

8

22

3

40

121

15

134

16

14

5

14

4

9

2

13

4

15

5

11

2

24

3

27

2

200

115

12

113

17

7

2

11

3

14

1

4

3

13

3

15

3

23

8

31

5

1000

113

11

98

9

9

1

11

6

13

3

12

3

8

3

11

3

20

4

29

5

5000

106

8

87

16

11

4

7

4

5

1

5

2

11

5

14

1

14

3

22

7

positive

controls

A

736

40

302

6

542

8

222

13

906

59

463

56

2421

54

1784

75

1718

100

1381

166

B

148

13

2990

18

11

1

194

35

15

6

181

15

17

3

1992

122

19

3

2125

110

Experiment 2

Treatment groups

[µg/plate]

Strains

 

 

TA 100

TA 1535

TA 1537

TA 1538

TA 98

 

 

-

SD

+

SD

-

SD

+

SD

-

SD

+

SD

-

SD

+

SD

-

SD

+

SD

medium

-

103

12

116

15

8

5

10

2

14

5

20

7

13

7

18

8

23

3

20

6

solvent

-

111

15

96

16

24

2

9

3

10

2

12

1

16

7

12

2

26

4

24

2

test substance

8

115

6

117

15

14

7

16

1

10

1

15

5

10

3

17

6

23

0

23

6

40

111

3

125

24

12

4

15

4

14

3

10

4

13

1

14

3

23

2

28

2

200

106

11

119

23

15

2

13

5

7

1

8

3

13

4

13

3

21

2

30

5

1000

81

11

82

3

12

5

10

1

7

2

9

5

12

4

12

3

13

2

23

2

5000

82

11

86

9

152

2

12

6

8

3

10

5

8

2

11

1

13

1

25

2

positive

controls

A

764

19

165

22

615

21

76

9

409

120

112

28

2387

235

1227

297

1587

21

872

96

B

118

9

943

91

27

9

115

25

18

4

78

8

16

1

951

123

27

6

727

75

-: without metabolic activation

+: with metabolic activation

SD: standard deviation

Positive controls:         A:        TA 1535, TA 100:                     sodium azide

                                           TA 1537:                                9-aminoacridine

                                           TA 1538, TA 98:            4-nitro-o-phenylenediamine

                                B :       TA 1535, TA 1537:                    2-aminoanthracene

                                           TA 100, TA 1538, TA 98:            2-aminoanthracene

Conclusions:
Interpretation of results (migrated information):
negative

Amines, tri-C8-10-alkyl is considered not to be mutagenic in this bacterial mutagenicity test in vitro.
Executive summary:

The test item with the batch number 5J167 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 according to OECD 471 (BASF SE, 1999, 9901542).

Solutions of the test substance were prepared in Tween 80/bidist. water and diluted with Tween 80/bidist. water just before use. The following concentrations were tested:

1st and 2nd test: 8, 40, 200, 1000 and 5000 µg/plate

The direct plate incorporation assay was utilized. The following results were obtained:

Toxic effects 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, Amines, tri-C8-10-alkyl is considered not to be mutagenic in this

bacterial mutagenicity test in vitro.

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

Additional information

Additional information from genetic toxicity in vitro:

Valid experimental data are available to assess the genetic toxicity of Amines, tri-C8-10-alkyl. 

GENETIC TOXICITY IN VITRO

 

Gene mutation in bacteria:

OECD conform studies:

The test item with the batch number 5J167 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 according to OECD 471 (BASF SE, 1999, 9901542).

Solutions of the test substance were prepared in Tween 80/bidist. water and diluted with Tween 80/bidist. water just before use. The following concentrations were tested:

1st and 2nd test: 8, 40, 200, 1000 and 5000 µg/plate

The direct plate incorporation assay was utilized. The following results were obtained:

Toxic effects 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, Amines, tri-C8-10-alkyl is considered not to be mutagenic in this

bacterial mutagenicity test in vitro.

Gene mutation in mammalian cells:

 

OECD conform studies:

The substance Amines, tri-C8-10-alkyl was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro (BASF SE, 2013, 50M0674/12M311). Two independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation). According to an initial range-finding cytotoxicity test for the determination of the experimental doses and taking into account the cytotoxicity actually found in the main experiments, the following doses were tested.

1st Experiment

without S9 mix

0; 6.3; 12.5; 25.0; 50.0 μg/mL

with S9 mix

0; 2.2; 4.4; 8.8; 17.5; 35.0; 70.0 μg/mL

2nd Experiment

without S9 mix

0; 5.0; 10.0; 20.0; 40.0; 80.0; μg/mL

with S9 mix

0; 1.6; 3.1; 6.3; 12.5; 25.0 μg/mL

Following attachment of the cells for 20-24 hours, cells were treated with the test substance for 4 in the absence of metabolic activation and for 4 hours in the presence of metabolic activation. Subsequently, cells were cultured for 6-8 days and then selected in 6-thioguaninecontaining medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line.

Both positive control substances, EMS and DMBA, led to the expected increase in the frequencies of forward mutations.

In this study, in the 1st and 2nd Experiment, at least the highest concentrations evaluated for gene mutations were clearly cytotoxic in the absence and the presence of metabolic activation. Based on the results of the present study, the test substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other. Thus, under the experimental conditions of this study, the test substance Amines, tri-C8-10- alkyl is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

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, Amines, tri-C8-10- alkyl is considered to be non-mutagenic in this HPRT assay.

 

Cytogenetic in mammalian cells:

OECD conform studies:

The substance Amines, tri-C8-10-alkyl was assessed for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity) according to OECD guideline 487 (BASF SE, 2013, 33M0674/12M331). Three independent experiments were carried out with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation).

The test substance was poorly soluble in culture medium. Due to strong test substance precipitation in an initial range-finding test the following concentrations were tested in this study.

1st Experiment

4 hours exposure; 24 hours harvest time; without S9 mix:

0; 31.3; 62.5; 125.0 µg/mL

4 hours exposure, 24 hours harvest time, with S9 mix:

0; 31.3; 62.5; 125.0 µg/mL

2nd Experiment

24 hours exposure, 24 hours harvest time, without S9 mix

0; 50.0; 100.0; 200.0 µg/mL

4 hours exposure, 24 hours harvest time, with S9 mix

0; 50.0; 100.0; 200.0 µg/mL

3rd Experiment

4 hours exposure, 24 hours harvest time, with S9 mix

0; 100.0; 150.0; 200.0; 250.0 and 300.0 μg/mL

A sample of at least 1 000 cells for each culture were analyzed for micronuclei, i.e.

2 000 cells for each test group. In several cases the sample was increased up to 4 000 cells. The vehicle controls gave frequencies of micronucleated cells within our historical negative control data range for V79 cells. Both positive control substances, EMS and cyclophosphamide, led to the expected increase in the number of cells containing micronuclei. In the main experiments, no cytotoxicity indicated by reduced relative increase in cell count or proliferation index was observed up to the highest applied test substance concentrations. On the basis of the results of the present study, the slight increase in the number of cells containing micronuclei in single test groups as well as the statistical significance both observed only in the presence of a metabolizing system, have to be regarded as biologically irrelevant due to missing reproducibility.

Thus, under the experimental conditions described, Amines, tri-C8-10-alkyl is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.

GENETIC TOXICITY IN VIVO

In vivo cytogenicity:

According to integrated testing strategy, based on available information no further in vivo testing is suggested.

Key study assignment:

For each toxicological endpoint one reliable and suitable study is available. No further reliable and relevant information was available; therefore these studies are integrated as key studies.

Assessment of genetic toxicity:

According to the ITS (guidance on information requirements, R.7.7.6 Integrated Testing Strategy (ITS) for mutagenicity), first in vitro tests were conducted to assess the mutagenic potential. All performed invitro tests assessing the mutagenicity in bactria (BASF SE, 1999, 9901542), the gene mutation in mammalian cell (BASF SE, 2013, 50M0674/12M311) and the cytogenetic in mammalian cells (BASF SE, 2013, 33M0674/12M331)are negative.

As all available in vitro test did not show any positive result and no further hints for mutagenicity can be draw from other information (like structure elements etc.) no further testing is suggested and summarizing no mutagenic effects of the test substance is expected.


Justification for selection of genetic toxicity endpoint
one test choose as example, all tests according to integrated test strategy negativ

Justification for classification or non-classification

In summary no hints for mutagenicity are obtained from experiments in vitro, therefore the test material does not fulfil the requirement for germ cell mutagens cat. 2 according to GHS (Regulation (EU) 1272/2008) or mutagenic category 3 DPD (67/548/EEC).

 

Labelling mutagenic:

GHS: no labelling

DSD: no labelling

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