Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro studies:
- Ames assay: negative (comparable to OECD TG 471, with and without metabolic activation)
- HPRT assay: negative (OECD TG 476; V79 cells of the Chinese hamster, with and without metabolic activation)
- Chromosome aberration assay: negative (OECD TG 473; V79 cells of the Chinese hamster, with and without metabolic activation)

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
only four strains used in study, only one positive control used for the assay with metabolic activation
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
no strain used for the detection of oxidising mutagens and cross-linking agents; 2-aminoanthracene used as single positive control for the assays with metabolic activation
Principles of method if other than guideline:
The study was conducted according to the testing protocol published by Ames (1973, 1975) and in accordance with OECD 471 from 26 May 1983, which was replaced.
Ames BN et al. (1973). Proc. Nat. Acad. Sci. USA 70: 2281 -2285
Ames BN et al. (1975). Mut. Res. 31: 347 - 364
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Analytical purity: approx. 98 %
- Impurities (identity and concentrations): approx. 1.5 % Di-2-ethylhexylamine
Target gene:
Histidine operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
MEDIA USED
- Type and composition of media: nutrient broth solution (8 g Difco bacto nutrient broth + 5 g NaCl/liter)
- temperature: 37°C
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : 5 male Sprague-Dawley rats, injected with Aroclor 1254, 5 days before sacrifice
- method of preparation of S9 mix: livers washed, homogenized in three volumes of KCl solution (150 mM), centrifugation at 9,000 g for 10 min, use of supernatent
Test concentrations with justification for top dose:
20, 100, 500, 2500 and 5000 µg/plate
Vehicle / solvent:
- Ssolvent used: acetone
Untreated negative controls:
yes
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
Remarks:
water control
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
other: with S-9 mix: all strains 2-aminoanthracene; without S-9 mix: strains TA100, TA1535: N-methyl-N'-nitro-N-nitrosoguanidine; TA98: 4-nitro-o-phenylendiamine
Details on test system and experimental conditions:
METHOD OF APPLICATION: standard plate test

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 test plates per test concentration or control; two experiments were carried out independently.
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 non-mutagenic 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.
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Complete solubility of the test substance in acetone up to the highest dose of 5000 µg/plate.

Table 1: Standard plate test 1 (Mean ± SD)

Dose (µg/plate) TA 1535 TA 100 TA 1537 TA 98
-S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
0 17±4 14±5 130±6 113±6 6±1 6±1 27±3 37±5
20 14±3 11±1 135±26 142±9 10±2 9±2 21±4 34±7
100 16±5 14±1 127 136±9 10±2 12±5 18±6 34±4
500 17±5 13±1 152±17 139±10 9±2 12±5 22±3 33±6
2500 16±2 16±3 118±20 132±5 11±2 10±1 25±1 28±6
5000 17±4 13±6 124±6 152±8 5±3 10±1 23±3 19±1
2-AA - 453±76 - 2500±278 - 141±42 - 1423±45
MNNG 2367±29 - 2467±333 - - - - -
AAC - - - - 60±16 - - -
NPD - - - - - - 563±140 -

Table 2: Standard plate test 2 (Mean ± SD)

Dose (µg/plate) TA 1535 TA 100 TA 1537 TA 98
-S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
0 15±3 14±4 101±6 108±4 7±2 7±1 27±4 41±6
20 14±4 12±2 94±12 128±10 7±1 6±2 28±6 31±3
100 14±4 13±1 107±7 113±10 5±1 8±2 21±9 31±3
500 10±1 16±5 96±11 107±3 4±1 8±2 25±5 31±3
2500 11±1 16±2 87±8 110±15 5±1 4±1 27±5 30±4
5000 11±1 13±5 83±4 115±3 4±2 3±1 29±5 37±9
2-AA - 319±20 - 2017±206 - 211±15 - 1533±166
MNNG 1640±284 - 1910±62 - - - - -
AAC - - - - 632±35 - - -
NPD - - - - - - 873±44 -

2-AA: 2-aminoanthracene;

MNNG; N-methyl-N-nitro-N-nitrosoguanidine

NPD: 4-nitro-o-phenylendiamine

AAC: 9-aminoacridine chloride monohydrate

Conclusions:
Under the conditions tested the test substance is non mutagenic in the bacterial AMES-test.
The positive control gave the expected values.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental: 24 Sep 2009 - 10 Dec 2009
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)
Version / remarks:
21 July 1997
Deviations:
no
Principles of method if other than guideline:
The assay was performed 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.
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and batch No.of test material: BASF and batch 407
- Production date: 16 July 2009
- Expiration date of the batch: 16 July 2011
- Purity test date: 17-18 August 2010

- Analytical purity: 99.7%
- Lot/batch No.: Behälter 407 v. 16.07.2009

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Solubility the vehicle: solubility in the vehicle determined

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: vortexing for formulation used

FORM AS APPLIED IN THE TEST: solution of different substance concentrations within the vehicle
Target gene:
HPRT locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: Laboratory for Mutagenicity Testing; Technical University; 64287 Darmstadt, Germany
- Suitability of cells: V79 cell line is a trusted model in this assay (high proliferation rate, good cloning efficiancy)

For cell lines:
- Absence of Mycoplasma contamination: approved by screening of each batch
- Methods for maintenance in cell culture: subcultured twice weekly at 80 Cm2 plastic flasks with 15 mL media
- Cell cycle doubling time: 12-16 h
- Modal number of chromosomes: 22
- Periodically checked for karyotype stability: yes, approved by screening of each batch
- Periodically ‘cleansed’ of spontaneous mutants: yes , approved by screening of each batch (and depressed before freezing by treatment of cells with HAT-medium)

MEDIA USED
- Type and composition of media: minimal essential medium (SEROMED, 12247 Berlin, Germany), supplemented with 10% FCS and 1% neomycin
- CO2 concentration: 1.5%
- temperature: 37°C
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-naphthoflavone induced male Wistar HsdCpb:WU rat liver S9
Test concentrations with justification for top dose:
- Experiment 1: with and without S-9 mix: 112.5, 225, 450, 900, and 3600 µg/mL
- Experiment 2: with and without S-9 mix: 225, 450, 900, 1800, and 3600 µg/mL
Vehicle / solvent:
- Vehicle used: THF (tetrahydrofurane)
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its relative nontoxicity to the cells. The final concentration of THF in the culture medium was 0.5 % (v/v).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: two

METHOD OF APPLICATION: in suspension

TREATMENT AND HARVEST SCHEDULE:
- Seeding: experiment I: three days old exp. growing stock cultures, experiment II: five days old exp. growing stock cultures

DURATION
- Preincubation period: 24 h
- Exposure duration: 4 h (1st experiment with and without S-9 mix, 2nd experiment with S-9 mix) and 24 h (2nd experiment without S-9 mix), respectively
- Expression time (cells in growth medium): 7 days
- Selection time (incubation with a selection agent): 8 days

SELECTION AGENT (mutation assays): 6-thioguanine
STAIN: 10 % methylene blue in 0.01 % KOH solution (for cloning efficiency)

NUMBER OF REPLICATIONS: 5 flasks/concentration, 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency I and II
- Definition:
cloning efficiency I: survival; cloning efficiency determined immediately after treatment to measure toxicity.
cloning efficiency II: viability; cloning efficiency determined after the expression period to measure viability of the cells without selective agent.








Evaluation criteria:
A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at 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 non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations 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 corresponding solvent control data. If there is by chance a low spontaneous mutation rate in the range normally found (0.6 – 31.7 mutants per E+06 cells) a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT11 (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 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.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
The maximal concentration of the test item equals a molar concentration of 10 mM. No relevant cytotoxic effects occurred up to the maximal concentration of 3600 µg/mL.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Other confounding effects: Phase separation of the test item was observed as an oily film covering the surface of the medium at 900, 1800, and 3600 µg/mL in the first experiment with and without metabolic activation.



Table 1: Summary of the results from the two hprt experiments

 

 

 

relative

relative

mutant

 

relative

relative

mutant

 

 

conc. µg

S9

cloning

cloning

colonies/

induction

cloning

cloning

colonies/

induction

 

per m L

mix

efficiency I

efficiency II

106cells

factor

efficiency I

efficiency II

106cells

factor

 

 

 

%

%

 

 

%

%

 

 

Colum n

1

2

3

4

5

6

7

8

9

10

Experiment I / 4 h treatment

 

 

culture I

culture II

Solvent control with THF

 

-

100.0

100.0

14.7

1.0

100.0

100.0

12.3

1.0

Positive control with EMS

150.0

-

91.5

70.9

147.4

10.0

96.7

98.2

101.8

8.3

Test item

112.5

-

64.1

77.4

16.9

1.2

73.4

87.0

22.1

1.8

Test item

225.0

-

85.0

77.4

23.0

1.6

68.5

70.0

29.2

2.4

Test item

450.0

-

80.1

81.8

19.5

1.3

74.0

104.9

18.4

1.5

Test item

900 (p)

-

81.9

73.6

9.0

0.6

91.7

92.5

14.3

1.2

Test item

1800 (p)

-

76.8

culture was not continued#

83.2

culture was not continued#

Test item

3600 (p)

-

93.4

73.3

11.4

0.8

83.9

104.6

14.4

1.2

Solvent control with THF

 

+

100.0

100.0

18.1

1.0

100.0

100.0

5.5

1.0

Positive control with DMBA

1.1

+

24.4

71.5

1015.1

56.1

21.8

53.6

973.2

176.0

Test item

112.5

+

83.2

105.8

9.9

0.5

102.7

99.6

17.1

3.1

Test item

225.0

+

85.3

107.4

11.5

0.6

97.7

99.7

14.1

2.5

Test item

450.0

+

86.3

112.3

12.3

0.7

104.0

90.7

12.3

2.2

Test item

900 (p)

+

87.2

107.3

13.2

0.7

105.2

99.0

16.1

2.9

Test item

1800 (p)

+

86.4

culture was not continued#

92.4

culture was not continued#

Test item

3600 (p)

+

90.7

94.5

14.3

0.8

94.3

106.5

10.9

2.0

Experiment II / 24 h treatment

 

 

culture I

culture II

Solvent control with THF

 

-

100.0

100.0

4.8

1.0

100.0

100.0

22.3

1.0

Positive control with EMS

75.0

-

66.8

82.4

132.0

27.7

101.9

102.1

188.8

8.4

Test item

112.5

-

99.7

culture was not continued##

105.6

culture was not continued##

Test item

225.0

-

97.8

98.3

12.3

2.6

98.3

112.6

18.7

0.8

Test item

450.0

-

97.0

99.5

15.2

3.2

88.4

109.8

14.2

0.6

Test item

900.0

-

99.0

96.1

18.4

3.9

88.8

126.6

8.3

0.4

Test item

1800.0

-

99.2

101.3

12.5

2.6

94.0

103.0

17.2

0.8

Test item

3600.0

-

95.6

99.7

4.8

1.0

98.7

126.0

14.9

0.7

Experiment II / 4 h treatment

 

 

 

 

Solvent control with THF

 

+

100.0

100.0

11.9

1.0

100.0

100.0

4.9

1.0

Positive control with DMBA

1.1

+

97.3

93.8

403.9

33.8

97.5

79.4

143.0

29.5

Test item

112.5

+

98.1

culture was not continued##

97.7

culture was not continued##

Test item

225.0

+

91.8

107.9

17.1

1.4

97.5

78.6

6.1

1.3

Test item

450.0

+

106.7

98.9

18.9

1.6

89.5

90.4

8.0

1.6

Test item

900.0

+

103.6

98.1

24.3

2.0

97.7

85.3

10.9

2.3

Test item

1800.0

+

99.7

95.0

12.9

1.1

91.1

82.5

12.1

2.5

Test item

3600.0

+

103.6

90.5

13.1

1.1

92.6

87.0

11.2

2.3

#: Culture was not continued to avoid analysis of too many insoluble concentrations.

##: Culture was not continued since a minimum of only four analysable concentrations is required.

P: Phase separation visible to the unaided eye.

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, the test substance is considered to be non-mutagenic in the HPRT assay.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental: 30 Sep - 10 Dec 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
21 July 1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and batch No.of test material: BASF and batch 407
- Production date: 16 July 2009
- Expiration date of the batch: 16 July 2011
- Purity test date: 17-18 August 2010

- Analytical purity: 99.7%
- Lot/batch No.: Behälter 407 v. 16.07.2009

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Solubility the vehicle: solubility in the vehicle determined

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: vortexing for formulation used

FORM AS APPLIED IN THE TEST: solution of different substance concentrations within the vehicle
Target gene:
not applicable
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: Laboratory for Mutagenicity Testing; Technical University; 64287 Darmstadt, Germany
- Suitability of cells: V79 cell line is a trusted model in this assay (high proliferation rate, good cloning efficiancy)

For cell lines:
- Absence of Mycoplasma contamination: approved by screening of each batch
- Methods for maintenance in cell culture: subcultured twice weekly at 80 Cm2 plastic flasks with 15 mL media
- Cell cycle doubling time: 14 h
- Modal number of chromosomes: 22
- Periodically checked for karyotype stability: yes, approved by screening of each batch
- Periodically ‘cleansed’ of spontaneous mutants: yes, approved by screening of each batch (and depressed before freezing by treatment of cells with HAT-medium)

MEDIA USED
- Type and composition of media: minimal essential medium (SEROMED, 12247 Berlin, Germany), supplemented with 10% FCS, Neomycin (5 Pg/mL) and Amphotericin B (2.5 Pg/mL)
- CO2 concentration: 1.5%
- temperature: 37°C
Cytokinesis block (if used):
Colcemid based block, (0.2 µg/mL) 15.5 hours and 25.5 hours after the start of the treatment
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-naphthoflavone induced male Wistar HsdCpb:WU rat liver S9
Test concentrations with justification for top dose:
Experiment I, preparation interval 18 h, exposure period 4 h, without S9 mix: 0.02, 0.04, 0.08, 0.2, 0.3, 0.6, 1.3, 2.5, 5.0 µL/mL
Experiment I, preparation interval 18 h, exposure period 4 h, with S9 mix: 0.02, 0.04, 0.08, 0.2, 0.3, 0.6, 1.3, 2.5, 5.0 µL/mL

Experiment II, preparation interval 18 h, exposure period 18 h, without S9 mix: 0.02, 0.04, 0.08, 0.2, 0.3, 0.6, 1.3, 2.5, 5.0 µL/mL
Experiment II, preparation interval 28 h, exposure period 18 h, without S9 mix: 0.02, 0.04, 0.08, 0.2, 0.3, 0.6, 1.3, 2.5, 5.0 µL/mL
Experiment II, preparation interval 28 h, exposure period 18 h, with S9 mix: 0.02, 0.04, 0.08, 0.2, 0.3, 0.6, 1.3, 2.5, 5.0 µL/mL
Vehicle / solvent:
- Vehicle used: THF (tetrahydrofurane)
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its relative nontoxicity to the cells. The final concentration of THF in the culture medium was 0.5 % (v/v).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Tetrahydrofurane (THF)
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: two

METHOD OF APPLICATION: in Quadriperm dishes which contained microscopic slides

DURATION
- Exposure duration: 4h Experiment I with and without S9 mix; Experiment II with S9 mix), 18 and 28 h (Experiment II without S9 mix), respectively
- Recovery time: 4 h (Experiment I with and without S9 mix), 24 h (Experiment II with S9 mix)
- Preparation time (incubation with colcemid): Colcemid was added to the culture medium 15.5 and 25.5 h after the start of treatment. The cells were treated, 2.5 h later, on the slides in the chambers with hypotonic solution for 20 min at 37°C. After incubation in hypotonic solution the cells were fixed with a mixture of methanol and glacial acetic acid (3:1).
- Fixation time (start of exposure up to fixation or harvest of cells): 18 and 28 h, respectively

STAIN (cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Two parallel cultures were set up in each experimental group. Two independent experiments were performed.

NUMBER OF CELLS EVALUATED: At least 100 well spread metaphases per culture were evaluated for cytogenetic damage, except for the positive control in Exp. I without S9 mix.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: The number of polyploid cells in 500 metaphases per culture was determined.
Evaluation criteria:
A test item is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of the laboratory's historical control data range.
and/or
- no significant increase of the number of structural chromosome aberrations is observed.

A test item is classified as clastogenic if:
- the number of induced structural chromosome aberrations is not in the range of the laboratory's historical control data range.
and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of the laboratory's historical control data range.
Statistics:
Statistical significance was confirmed by means of the Fisher's exact test (p < 0.05). However, both biological and statistical significance were considered together.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
5 µL/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no effect.
- Effects of osmolarity: no effect.
- Precipitation: no test item precipitation observed.
- Other confounding effects: Phase separation occurred without S9 mix at 0.2 µL/mL and above and in the presence of S9 mix at 0.04 µL/mL and above in Experiment I. Presumably due to a longer preparation interval of 28 h, no phase separation occurred in Experiment II.

RANGE-FINDING/SCREENING STUDIES:
In the pre-experiment 5.0 µL/mL of Tris-(2-ethylhexyl)amin O 2446 was applied as top concentration for treatment of the cultures. At the selected dose no influence on pH value or osmolarity was detected. Phase separation occurred at concentrations of 0.2 µL/mL and above in the absence of S9 mix and at 0.04 µL/mL and above in the presence of S9 mix. Neither precipitation nor test item induced cytotoxicity occurred. Therefore, precipitation and cytotoxicity did not interfere with the evaluation and the experiment was designated main experiment I. However, the cultures did not fulfil the acceptance criteria for cytogenetic evaluation as the number of aberrations found in the solvent controls were not within the range of the historical control data range. Therefore, Experiment I was repeated with the same maximum test item concentration.



Table 1: Summary of results of the chromosome aberration study without metabolic activation

Exp. Preparation

interval

Test item concentration in µL/mL

Cell numbers in %

of control

Mitotic indices in %

of control

 

incl. gaps*

Aberrant cells in %

excl. gaps*

 

with exchanges

Exposure period 4 hrs without S9 mix

I

18 hrs

Solvent control1

100.0

100.0

3.0

2.5

1.5

 

 

Positive control2#

n.t.

62.9

49.0

45.0S

33.0

 

 

0.08

102.9

94.9

3.5

3.5

2.5

 

 

1.3PS

96.1

109.9

2.0

2.0

0.0

 

 

2.5PS

102.2

109.3

3.5

3.0

1.5

 

 

5.0PS

91.3

106.5

2.5

2.0

1.0

Exposure period 18 hrs without S9 mix

II

18 hrs

Solvent control1

100.0

100.0

1.0

0.5

0.0

 

 

Positive control3

n.t.

59.0

19.5

18.5S

6.0

 

 

1.3

109.4

102.2

1.5

1.0

0.0

 

 

2.5

109.6

126.7

2.5

2.5

0.0

 

 

5.0

103.1

121.6

1.5

1.0

0.0

Exposure period 28 hrs without S9 mix

II

28 hrs

Solvent control1

100.0

100.0

2.0

2.0

0.0

 

 

Positive control3

n.t.

54.3

28.5

28.0S

15.0

 

 

1.3

88.9

104.5

1.0

0.0

0.0

 

 

2.5

83.4

106.2

0.0

0.0

0.0

 

 

5.0

76.6

108.9

1.0

1.0

0.0

*: Inclusive cells carrying exchanges

#: Evaluation of 50 metaphases per culture

n.t.: Not tested

S: Aberration frequency statistically significant higher than corresponding control values

PS: Phase separation occurred

1: THF 0.5 % (v/v)

2: EMS 1000.0 Pg/mL

3: EMS 500.0 Pg/mL

Table 2: Summary of results of the chromosome aberration study with metabolic activation

Exp. Preparation

interval

Test item concentration in µL/mL

Cell numbers in %

of control

Mitotic indices in %

of control

 

incl. gaps*

Aberrant cells in %

excl. gaps*

 

with exchanges

Exposure period 4 hrs with S9 mix

I

18 hrs

Solvent control1

100.0

100.0

0.5

0.5

0.5

 

 

Positive control2

n.t.

72.5

17.0

14.5S

4.5

 

 

0.02

75.0

126.9

4.0

3.5S

1.0

 

 

1.3PS

90.5

101.8

2.5

2.5

0.5

 

 

2.5PS

68.7

106.0

2.5

2.0

0.5

 

 

5.0PS

94.7

119.3

2.5

2.0

0.5

II

28 hrs

Solvent control1

100.0

100.0

1.0

0.5

0.5

 

 

Positive control2

n.t.

103.8

10.0

8.0S

1.0

 

 

1.3

96.4

106.1

0.5

0.5

0.5

 

 

2.5

98.3

99.7

3.0

3.0S

1.0

 

 

5.0

93.8

97.7

1.0

0.5

0.0

*: Inclusive cells carrying exchanges

n.t.: Not tested

S: Aberration frequency statistically significant higher than corresponding control values

PS: Phase separation occurred

1: THF 0.5 % (v/v)

2: CPA 1.4 Pg/mL

Conclusions:
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line), when tested up to the highest by the OECD guideline required concentration.

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Genetic toxicity in bacteria:

The test substance was tested for mutagenicity in the Ames assay according to a protocol similar to OECD TG 471 (adopted 1981) and following the testing protocol published by Ames (1973, 1975). Different concentrations of the substance (dose range: 20 µg- 5000 µg/plate) were tested in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 in the standard plate test with and without metabolic activation. External metabolic activation was provided by adding S9-mix from Aroclor 1254 treated male Sprague-Dawley rats. No bacteriotoxic effect (reduced his-background growth) and no increase in the number of his+ revertants was observed either without S-9 mix or after addition of a metabolizing system. According to the results of the present study, the test substance Tri-2-ethylhexylamine is not mutagenic in the Ames-test under the experimental conditions.

 

Genetic toxicity in mammalian cells:

The potential of the test substance to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster was investigated in a study according to OECD TG 476 and GLP regulations.

The assay was performed 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 concentration (3600 µg/mL) was equal to a molar concentration of approximately 10 mM.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test item and the activity of the metabolic activation system.

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, the test substance is considered to be non-mutagenic in this HPRT assay.

 

In addition, the test substance, diluted in tetrahydrofuran (THF), was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments. The assays were conducted in accordance with OECD TG 473 and GLP regulations.

In the first experiment exposure periods of 4 hours with and without metabolic activation were employed. The preparation interval was 18 hours. In the second experiment exposure periods were 18 and 28 hours without metabolic activation, and 4 hours with metabolic activation, respectively. Accordingly, the preparation intervals were 18 and 28 hours without, and 28 hours with metabolic activation. Metabolic activation was provided by adding S-9 mix from Phenobarbital /ß-naphthoflavone induced male Wistar HsdCpb:WU rat livers.

In each experimental group two parallel cultures were set up. At least 100 metaphases per culture were evaluated for structural chromosome aberrations, except for the positive control in Experiment I without metabolic activation, where only 50 metaphases were evaluated. The highest applied concentration (5µL/mL) was chosen with respect to the current OECD Guideline 473. Dose selection for the cytogenetic experiments was performed considering the toxicity data.

 

In this study in the absence and presence of S9 mix neither test item precipitation nor cytotoxicity was observed up to the highest applied concentration. Phase separation occurred in Experiment I in the presence and absence of metabolic activation.

In both independent experiments no clastogenicity was observed at the concentrations evaluated for cytogenetic damage either with or without metabolic activation. However, two statistically significant increases in the number of aberrant cells excluding gaps were observed after treatment with 0.02 µL/mL (3.5 % aberrant cells excluding gaps) in Experiment I in the presence of S9 mix and with 2.5 µL/mL (3.0 % aberrant cells excluding gaps) in Experiment II in the presence of S9 mix. These observations have to be regarded as biologically irrelevant, since the values were in the range of the laboratory’s historical solvent control data (Exp. I, with S9 mix: 0.0 – 4.0 % aberrant cells excluding gaps; Exp. II, with S9 mix: 0.5 – 3.0 % aberrant cells excluding gaps) and no dose-related increases were observed. No relevant increase in polyploid metaphases was found after treatment with the test item as compared to the frequencies of the control cultures.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line) in vitro.

Therefore, the test substance is considered to be non-clastogenic in this chromosome aberration test in the absence and presence of metabolic activation, when tested up to the highest required concentration.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008, as amended for the thirteenth time in Regulation (EU) No 2018/1480.. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008.