Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames : negative (BASF 1989, BASF 2018)

HPRT: negative (BASF 2010)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT (hypoxanthine-guanine phosphoribosyl transferase)
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: Thawed stock cultures are propagated at 37 °C in 80 cm2 plastic flasks (GREINER, 72632 Frickenhausen, Germany). About 5×105 cells are seeded into each flask with 15 mL of MEM (minimal essential medium; SEROMED, 12247 Berlin, Germany) supplemented with 10 % foetal calf serum (FCS, PAA Laboratories GmbH, 35091 Cölbe, Germany) and 1 % neomycin. The cells are subcultured twice weekly. The cell cultures are incubated at 37 °C in a 4.5 % carbon dioxide atmosphere (95.5 % air).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes


Metabolic activation:
with and without
Metabolic activation system:
Mammalian Microsomal Fraction S9 Mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO; ethanol; Nutrient medium
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Evaluation criteria:
A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible positive response for one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces reproducibly with one of the concen-trations a mutation frequency that is three times higher than the spontaneous mutation fre-quency in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corre-sponding solvent control data. If there is by chance a low spontaneous mutation rate in the range normally found (0.6 – 31.7 mutants per 106 cells), a concentration-related in-crease of the mutations within this range has to be discussed. The variability of the muta-tion rates of solvent controls within all experiments of this study is also taken into consideration.
Statistics:
A linear regression (least squares) will be performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT®11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for the groups treated with the test item will be 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 will be considered to-gether.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Excessive cytotoxicity at 2 or 4µg/mL without S9 after treatment for 4h or 24h, respectively, and at 14µg/mL with S9

      relative relative mutant   relative relative mutant  
  conc. µg S9 cloning cloning colonies/ induction cloning cloning colonies/ induction
  per mL mix efficiency I efficiency II 10^6 cells factor efficiency I efficiency II 10^6 cells factor
      % %     % %    
Column 1 2 3 4 5 6 7 8 9 10
Experiment I / 4 h treatment     culture I culture II
Solvent control with ethanol   - 100.0 100.0 12.4 1.0 100.0 100.0 9.4 1.0
Positive control with EMS 150.0 - 99.8 53.6 178.2 14.3 80.8 88.7 96.5 10.3
Test item 0.13 - 101.3 78.0 19.6 1.6 98.7 98.5 5.3 0.6
Test item 0.25 - 105.6 94.2 13.2 1.1 95.8 97.6 16.6 1.8
Test item 0.5 - 94.2 34.5 39.5 3.2 100.3 44.5 12.8 1.4
Test item 1.0 - 77.1 37.7 32.2 2.6 77.4 41.1 35.6 3.8
Test item 2.0 - 6.5 55.9 16.1 1.3 7.3 66.0 5.6 0.6
Test item 3.0 - culture was not continued## culture was not continued##
Test item 4.0 - culture was not continued## culture was not continued##
Solvent control with ethanol     100.0 100.0 15.6 1.0 100.0 100.0 31.5 1.0
Positive control with DMBA 1.1 + 84.3 97.5 1033.2 66.3 73.1 88.3 1083.7 34.4
Test item 0.5 + 102.8 culture was not continued# 130.6 culture was not continued#
Test item 1.0 + 36.8 79.6 19.7 1.3 81.1 104.7 37.5 1.2
Test item 2.0 + 38.0 85.8 33.1 2.1 38.2 98.9 10.8 0.3
Test item 4.0 + 31.9 105.3 36.2 2.3 35.4 91.5 18.7 0.6
Test item 8.0 + 23.4 86.4 30.2 1.9 34.7 91.3 17.0 0.5
Test item 12.0 + 16.5 67.2 20.4 1.3 4.8 88.9 12.6 0.4
Test item 16.0 + 0.0 culture was not continued## 0.0 culture was not continued##
Experiment II / 24 h treatment     culture I culture II
Solvent control with ethanol   - 100.0 100.0 23.8 1.0 100.0 100.0 14.1 1.0
Positive control with EMS 150.0 - 108.5 99.5 384.0 16.2 106.6 108.7 350.6 24.9
Test item 0.13 - 107.4 culture was not continued# 105.1 culture was not continued#
Test item 0.25 - 92.2 103.9 22.2 0.9 104.2 98.3 17.4 1.2
Test item 0.5 - 102.5 85.8 25.2 1.1 115.1 70.0 19.8 1.4
Test item 1.0 - 99.3 103.6 16.2 0.7 96.1 92.9 29.8 2.1
Test item 2.0 - 92.9 97.2 28.0 1.2 66.6 96.8 22.0 1.6
Test item 3.0 - 26.6 89.4 29.3 1.2 12.7 101.0 14.5 1.0
Test item 4.0 - 0.0 culture was not continued## 0.0 culture was not continued##
Experiment II / 4 h treatment        
Solvent control with ethanol   + 100.0 100.0 12.4 1.0 100.0 100.0 12.0 1.0
Positive control with DMBA 1.1 + 52.1 90.7 880.1 70.8 39.7 73.3 731.9 60.8
Test item 1.0 + culture was not continued#   culture was not continued#  
Test item 2.0 + 84.3 100.7 16.8 1.4 41.6 93.3 33.3 2.8
Test item 4.0 + 86.3 106.5 17.4 1.4 42.9 71.4 40.7 3.4
Test item 8.0 + 71.6 96.6 20.8 1.7 39.1 100.8 15.7 1.3
Test item 10.0 + 56.2 93.8 38.3 3.1 19.4 72.5 17.2 1.4
Test item 12.0 + 42.8 91.4 14.7 1.2 17.8 86.3 36.4 3.0
Test item 14.0 + 1.6 culture was not continued## 0.8 culture was not continued##

# culture was not continued since a minimum of only four analysable concentrations is required

## culture was not continued due to strong toxic effects

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from male Wistar rats livers
Test concentrations with justification for top dose:
1st Exp: 0; 33; 100; 333; 1000; 2600 and 5200 μg/plate (all strains, with and without S9 mix) SPT
2nd Exp: 0; 0.33; 1; 3.3; 10; 33 and 100 μg/plate (all strains, with and without S9 mix) SPT
3rd Exp: 0; 0.1; 0.33; 1; 3.3; 10 and 33 μg/plate (TA strains without S9 mix), 0; 0.33; 1; 3.3; 10; 33 and 100 μg/plate (TA strains with S9 mix and E.coli with and without S9 mix) PIT
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
other: 2-aminoanthracene; N-methyl-N-nitro-N-nitrosoguanidine; 4-nitro-o-phenylenediamine
Details on test system and experimental conditions:
Standard plate test
The experimental procedure of the standard plate test (plate incorporation method) was based on the method of Ames et al..
• Salmonella typhimurium
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation) or 0.5 mL phosphate buffer (without metabolic activation).
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.
Escherichia coli
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation) or 0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (trp+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.
Preincubation Test
The experimental procedure was based on the method described by Yahagi et al. and Matsushima et al.
0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S9 mix (with
metabolic activation) or phosphate buffer (without metabolic activation) were incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar was added and, after mixing, the samples were poured onto the agar plates within approx. 30 seconds. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis
System.
Evaluation criteria:
The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
Species / strain:
other: TA 1535, TA 100, TA 1537, TA 98 and E.coli WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid

SOLUBILITY: No precipitation of the test substance was found with and without S9 mix.

TOXICITY: A strong bacteriotoxic effect was observed depending on the strain and test conditions from about 0.33 μg/plate onward.

MUTAGENICITY: A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system.

Conclusions:
Under the experimental conditions of this study, the test substance Tridecylamine, branched and linear (PSN 18/0023-1) is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
E. coli WP2 or S. typhimurium TA102 strain missing
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S-9 mix
Test concentrations with justification for top dose:
Standard plate test: 6 - 5000 µg/plate; Preincubation test: 1.25 - 50 µg/plate
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: Copmplete solubility of test substance in DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
without S-9: 5 µg N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for strains TA 100 and TA 1535, 10 µg 4-nitro-o-phenylendiamine for the strain TA 98 and 100 µg 9-aminoacridine chloride monohydrate for the strain TA 1537; with S-9: 10 µg 2-aminoanthracene fo
Details on test system and experimental conditions:
METHOD OF APPLICATION: standard plate test (in agar, plate incorporation) and preincubation test


DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours


NUMBER OF REPLICATIONS:
- Standard plate test: 2 individual experiments in triplicates
- Preincubation test: one experiment in triplicates


DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (his- background growths)
Evaluation criteria:
A substance is regarded as positive if the following is fulfilled:
- doubling of the spontaneous mutation rate
- dose-response relationship
- reproducibility of results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from 25-50 µg/plate (standard test); from 20 µg/plate (preincubation test)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY:
A bacteriotoxic effect was observed in the standard plate test from about 25 µg - 50 µg/plate onward (without S-9 mix) or from about 50 µg - 100 µg onward (with S-9 mix). In the preincubation test bacteriotoxicity was found using all tester strains at 20 µg/plate (without S-9 mix) or using TA 1535 at 50 µg/plate (with S-9 mix).

Standard plate test:

Dose/plate

(µg)

Without S9 mix

With S9 mix

Mean

SD

Mean

SD

Salmonella typhimurium TA 1535

0 (DMSO)

18

8

18

3

6

18

6

18

2

12

21

4

27

4

20

17

1

25

3

25

18

3

24

10

50

14 B

3

24

2

100

6 B

4

21

2

500

0

0

3 B

1

2500

0

0

0

0

5000

0

0

0

0

MNNG

5.0 µg

1130

140

2-AA

2.5 µg

221

20

Salmonella typhimurium TA 100

0 (DMSO)

100

15

102

13

6

112

9

117

5

12

111

9

117

10

20

88

8

111

9

25

93

6

116

11

50

20 B

8

105

16

100

1

2

82 B

29

500

0

0

2

0

2500

0

0

0

1

5000

0

0

0

0

MNNG

5.0 µg

1123

148

2-AA

2.5 µg

1480

197

Salmonella typhimurium TA 1537

0 (DMSO)

15

2

13

2

6

13

1

12

1

12

11

1

13

7

20

16

4

16

2

25

13 B

7

17

3

50

4 B

1

13

3

100

2 B

2

9 B

3

500

0

0

6

1

2500

0

0

0

0

5000

0

0

0

0

AAC

100.0 µg

327

29

2-AA

2.5 µg

108

15

Salmonella typhimurium TA 98

0 (DMSO)

25

5

43

8

6

23

7

46

7

12

29

6

48

8

20

29

3

40

3

25

30 B

3

36

4

50

B

43

8

100

3 B

4

35 B

10

500

0

0

0

0

2500

0

0

0

0

5000

0

0

0

0

NOPD

10.0 µg

782

76

2-AA

2.5 µg

834

61

B = reduced his- background

.

Preincubation Test:

Dose/plate

(µg)

Without S9 mix

With S9 mix

Mean

SD

Mean

SD

Salmonella typhimurium TA 1535

0 (DMSO)

18

3

17

2

1.25

21

1

2.5

24

2

3

11

1

5

18

6

6

12

1

10

14

3

12

15

4

20

8 B

2

25

15

3

50

8 B

2

MNNG 5.0 µg

1383

90

2-AA 2.5 µg

134

15

Salmonella typhimurium TA 100

0 (DMSO)

100

10

105

5

1.25

134

21

2.5

123

5

3

122

7

5

119

14

6

118

5

10

115

23

12

138

18

20

78 B

8

25

110

9

50

98

22

MNNG 5.0 µg

1140

53

2-AA 2.5 µg

513

86

Salmonella typhimurium TA 1537

0 (DMSO)

9

1

7

2

1.25

12

3

2.5

10

1

3

6

1

5

7

3

6

8

2

10

9

1

12

13

5

20

B

25

8

2

50

5

1

AAC 100.0 µg

416

49

2-AA 2.5 µg

95

5

Salmonella typhimurium TA 98

0 (DMSO)

20

2

29

1

1.25

20

3

2.5

19

1

3

32

3

5

19

4

6

29

2

10

16

1

12

33

6

20

6 B

3

25

28

8

50

33

6

NOPD 10.0 µg

796

105

2-AA 2.5 µg

1093

155

B = reduced his- background

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

Genetic toxicity in vivo

Description of key information

MNT negative (BASF 2002)

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
other: Crl:NMRI
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland GmbH
- Age at study initiation: 5 - 8 weeks
- Weight at study initiation: mean ca. 28 g
- Housing: single housing in Makrolon cages, type Ml
- Diet: Kliba Haltungsdiät (Provimi Kliba SA, Kaiseraugst, Switzerland) ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
oral: gavage
Vehicle:
- Vehicle: olive oil Ph.Eur./DAB
- Justification for choice of vehicle: Due to the insolubility of the test substance in water, olive oil was selected as the vehicle, which had been demonstrated to be suitable in the in vivo micronucleus test and for which historical data are available.
- Concentration of test material in vehicle: 2, 4 or 6 g/100 ml
Details on exposure:
APPLIED VOLUME:
10 ml/kg bw
Duration of treatment / exposure:
- Sacrifice intervals: 24 hours (vehicle control, 200, 400 and 600 mg/kg TS and positive controls) and 48 hours (vehicle control and 600 mg/kg TS)
Frequency of treatment:
single oral administration
Remarks:
Doses / Concentrations:
200, 400, 600 mg/kg
Basis:

No. of animals per sex per dose:
5 males
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (CPP) and Vincristine Sulphate (VCR)
- Justification for choice of positive control: The stability of CPP and VCR is well-defined under the selected conditions, since both positive control articles are well-defined clastogens and aneugens, respectively.
- Route of administration: oral by gavage
- Doses / concentrations: CPP: 20 mg/kg bw, VCR: 0.15 mg/kg bw
Tissues and cell types examined:
Erythrocytes were prepared from femur bone marrow and stained for microscopic evaluation. 2000 polychromatic erythrocytes per animal were evaluated and the following parameters were recorded: number of polychromatic and normochromatic erythrocytes, each with and without micronuclei; number of small and large micronuclei. The ratio of polychromatic to normochromatic erythrocytes was calculated from the recorded data.
Evaluation criteria:
The test is considered valid and acceptable if the slides allow to evaluate sufficient cells, i.e. >/= 2000 polychromatic cells; and if the proportion of micronuclei in negative controls was within the range of historical control data; and if the two positive control chemicals induced an increase within the range of historical positive control data. The test chemical is considered positive if a dose-related and significant increase in the number of micronucleated polychromatic erythrocytes is noted at any of the intervals; and if the proportion of micronuclei bearing cells exceeded both the concurrent and the historical negative control range. If these criteria are not met the test chemical is considered negative in this test.
Statistics:
The Wilcoxon test for the hypothesis of equal medians was used to compare dose groups with vehicle groups.
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
CLINICAL SIGNS
In the pretest, mortalities were noted down to 1000 mg/kg body weight. 800 mg/kg were survived by both male and female mice, but caused tremor, squatting posture, and poor general state. In the main test, 5/10 animals died at 800 mg/kg within 48 h. The highest dose was therefore lowered to 600 mg/kg. No clinical signs were noted at 200 mg/kg. Transient squatting posture was seen in animals receiving 400 mg/kg 1 h after dosing. At 600 mg/kg, squatting posture was seen in all animals on the first and second day after treatment as was tremor. Poor general state was seen 1 h after treatment in all high dose animals.


ERYTHROCYTES
In the negative control groups, polychromatic erythrocytes containing micronuclei amounted to 1.0 ‰ after 24 h and 0.7 ‰ after 48 h. Cyclophosphamide led to exclusively small micronuclei, incidence was 14.8 ‰. Proportion following vincristine treatment was 99.6 ‰, with an expected amount of large micronuclei, i.e. 27.8 ‰. Effects of both positive control chemicals were statistically significant on the P<0.01 level.
Single doses of Tridecylamine led to 0.8 ‰ (200 mg/kg), 0.9 ‰ (400 mg/kg). The high dose led to 2.2 ‰ (24 h) and 1.1 ‰ (48 h). This was within the range of historical control data.
The number of normochromatic erythrocytes containing micronuclei was unchanged in the various dose groups. The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) or large micronuclei (d > D/4) did not deviate from the vehicle control value at any of the sacrifice intervals and was within the historical control range. A slight inhibition of erythropoiesis was seen at 600 mg/kg, as determined from the ratio of polychromatic to normochromatic erythrocytes.

Ratio polychromatic (PCE) and normochromatic (NCE) erythrocytes:

Dose (mg/kg bw)

Interval (h)

Ratio PCE/NCE

0

24

3.11

0

48

2.82

200

24

3.36

400

24

3.03

600

24

2.16

600

48

2.13

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

Additional information

In vitro:

TDA was tested negative in the Ames test with and without metabolic activation, both in the standard plate test and in the preincubation test (BASF AG, 1989). The study was conducted according to OECD TG 471. The substance was tested in the range 6 – 5000 μg/plate in the standard test and 1.25 – 50 μg/plate in the Preincubation test using the S. typhimurium test strains TA1535, TA100, TA1537, and TA98. Bacteriotoxic effects were seen in the standard plate test from about 25 – 50 μg/plate without metabolic activation and from 50 – 100 μg/plate with metabolic activation. In the preincubation test cytotoxicity was seen in all test strains at 20 μg/plate without metabolic, or with metabolic activation at 50 μg/plate only in TA 1535.

 

In addition, an Ames-Tests with Tridecylamine, branched and linear was conducted in 2018 to evaluate the toxicological properties of a new batch of TDA and to include all 5 required strains.

The test substance Tridecylamine, branched and linear (PSN 18/0023 -1) was tested for ist mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay. Strains tested TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA at a dose range of 0.33 μg - 5200 μg/plate (SPT) and 0.1 μg - 100 μg/plate (PIT). Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats). No precipitation of the test substance was found with and without S9 mix. A strong bacteriotoxic effect was observed depending on the strain and test conditions from about 0.33 μg/plate onward. A relevant increase in the number of his+or trp+revertants (factor2: TA 100, TA 98 and E.coli WP2 uvrA or factor3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system. Under the experimental conditions of this study, the test substance Tridecylamine, branched and linear (PSN 18/0023-1) is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation (BASF SE, 2018).

 

Gene mutation in mammalian cells was examined in an HPRT assay (BASF 2010). Chinese hamster V79 cells were exposed to the registered substance in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a

treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The highest applied concentrations of 4.0 μg/mL (4 hours treatment without S9 mix) in experiment I, 16.0 μg/mL (4 hours treatment with S9 mix) in experiment I, 4.0 μg/mL (24 hours treatment without S9 mix) in experiment II and 14 μg/mL (4 hours treatment with S9 mix) in experiment II were limited by test item induced cytotoxicity. No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. Vehicle and appropriate positive controls yielded the expected results.

In vivo:

TDA was tested negative in a Mouse Micronucleus test that was performed in accordance with OECD TG 474 (BASF AG, 2002). The mice received single doses of 200, 400, and 600 mg TDA dissolved in olive oil/kg bw by oral gavage which caused clinical signs of toxicity at the mid- and the high-dose level. Transient squatting posture was seen in animals receiving 400 mg/kg bw at 1 h after dosing. At 600 mg/kg bw, squatting posture and tremor were seen in all animals on the first and second day after treatment. Poor general state was seen at 1 h after treatment in all high dose animals. In the main test, 5/10 animals died at 800 mg/kg within 48 h. The highest dose was therefore lowered to 600 mg/kg bw in the course of the study.

 

The evaluation of 2000 polychromatic erythrocytes 24 and 48 hours after dosing revealed that the proportion of polychromatic erythrocytes containing micronuclei was 0.8, 0.9, and 2.2 ‰ in the groups at 200, 400, and 600 mg/kg bw, respectively, after 24 hours, and 1.1 ‰ after 48 hours in animals receiving the high dose. This was within the range of the historical control data for this vehicle (mean ± SD: 2.0±0.6 ‰; n=56). At the high dose, TDA slightly inhibited erythropoiesis, showing that the test material had reached the target. The positive control substances, cyclophosphamide and vincristine, gave significantly positive effects on the p< 0.01 level as expected. Overall, there was no indication of a clastogenic or aneugenic activity.

Justification for classification or non-classification

TDA was not mutagenic bacteria and mammalian cells in vitro. The substance did not induce micronuclei in vivo. Consequently, it does not have to be classified for mutagenicity according to Regulation (EC) No 1272/2008 (CLP).