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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames-Test (Harlan CCR, 2015): negative
HPRT-Test (Harlan CCR, 2015): negative
In vitro Micronucleus-Test (Harlan CCR, 2015): 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
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:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.: 0008924462
Target gene:
HIS/TRP
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix
Test concentrations with justification for top dose:
Pre-experimet/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Vehicle / solvent:
- Vehicle/solvent used: ethanol
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

Experimental Performance
For each strain and dose level, including the controls, three plates were used.
The following materials were mixed in a test tube and poured onto the selective agar plates:
Experiment I (Plate Incorporation)
100 μL Test solution at each dose level (solvent or reference mutagen solution (positive control)),
500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 μL Bacteria suspension (cf. test system, pre-culture of the strains),
2000 μL Overlay agar
Experiment II (Pre-Incubation)
In the pre-incubation assay 50 μL test solution or solvent or 100 μL reference mutagen solution (positive control)), 500 μL S9 mix / S9 mix substitution buffer and 100 μL bacterial suspension were mixed in a test tube and incubated at 37 °C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45 °C) was added to each tube. The mixture was poured on minimal agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.
In parallel to each test a sterile control of the test item was performed and documented in the raw data. Therefore, 100 μL of the stock solution, 500 μl S9 mix / S9 mix substitution buffer were mixed with 2.0 mL overlay agar and poured on minimal agar plates.

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 hours

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Dose Selection
In the pre-experiment the concentration range of the test item was 3 – 5000 μg/plate. The pre-experiment is reported as experiment I. Since minor toxic effects were observed seven concentrations were tested in experiment II. 5000 μg/plate was chosen as maximal concentration.
The concentration range included two logarithmic decades. The following concentrations were tested in experiment II adjusted to the prior purity of 97%: 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce a significant increase in mutant colony frequencies
- a minimum of five analysable dose levels should be present with at least three dose levels showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5.
- the sterility controls have to reveal no indication of bacterial contamination.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
333-5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
333-5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
333-5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
333-5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

The test item Triisooctylamine, dissolved in Ethanol, was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations adjusted to the prior purity of 97%:

Pre-experimet/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

The test item precipitated in the overlay agar in the test tubes at 5000 μg/plate in experiment I. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 1000 to 5000 μg/plate in experiment I. The undissolved particles had no influence on the data recording. No precipitation of the test item was observed in the overlay agar either in the test tube or on the incubated agar plates in experiment II.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Triisooctylamine at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix).

in induced revertant colonies.

CONCLUSION

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, Triisooctylamine is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Endpoint:
in vitro gene mutation study in mammalian cells
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 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
other: mammalian cell gene mutation test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.: 0008924462
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
The V79 cell line has been used successfully in in vitro experiments for many years. Especially the high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50 %) both necessary for the appropriate performance of the study, recommend the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix
Test concentrations with justification for top dose:
0.063 - 56.0 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 hours
- Exposure duration: The treatment period was 4 hours with and without metabolic activation.
- Expression/fixation time: Three or four days after treatment 1.5x10^6 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of 7 days five 80 cm² cell culture flasks were seeded with about 3 - 5x10^5 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability.
The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 for about 8 days. The colonies were stained with 10 % methylene blue in 0.01 % KOH solution.

NUMBER OF REPLICATIONS: The study was performed in two independent experiments, using identical experimental procedures.

NUMBER OF CELLS EVALUATED: The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity of the test item is indicated by a reduction of the cloning efficiency (CE).

Acceptability of the Assay
The gene mutation assay is considered acceptable if it meets the following criteria:
a) the numbers of mutant colonies per 106 cells found in the solvent controls fall within the laboratory historical control data range (see Appendix 1, Historical Data).
b) the positive control substances should produce a significant increase in mutant colony frequencies (see Appendix 1).
c) the cloning efficiency II (absolute value) of the solvent controls must exceed 50 %.
The data of this study comply with the above mentioned criteria (see tables of results, mutation rate and factor calculated referring to the cloning efficiency of the solvent controls) and Appendix 1 (Historical Data).

Dose selection
According to the current OECD Guideline for Cell Gene Mutation Tests at least four analysable concentrations should be used in two parallel cultures. For freely-soluble and non-cytotoxic test items the maximum concentration should be 5 mg/mL, 5 μL/mL or 10 mM, whichever is the lowest. For cytotoxic test items the maximum concentration should result in approximately 10 to 20 % relative survival or cell density at subcultivation and the analysed concentrations should cover a range from the maximum to little or no cytotoxicity. Relatively insoluble test items should be tested up to the highest concentration that can be formulated in an appropriate solvent as solution or homogenous suspension. These test items should be tested up or beyond their limit of solubility. Precipitation should be evaluated at the beginning and at the end of treatment by the unaided eye.
The pre-experiment was performed in the presence and absence of metabolic activation (4 hours treatment). Test item concentrations between 28.5 μg/mL and 3646 μg/mL (equal to a molar concentration of approximately 10 mM) were used. The highest concentration of the pre-experiment was chosen with regard to the purity (preliminary information at study start: Tertiary Amine: 97%) and the molecular weight (353.67 g/mol) of the test item.
Relevant toxic effects occurred after 4 hours treatment at 28.5 μg/mL and above without metabolic activation. In the presence of metabolic activation (4 hours treatment) the cell growth was completely inhibited at 57.0 μg/mL and above.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 hours) prior to removal to the test item. Phase separation occurred at 113.9 μg/mL and above after 4 hours treatment with and without metabolic activation.
There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.
The dose range of the first experiment was set according to the data generated in the pre-experiment. The experimental part without metabolic was repeated in experiment IA and IB due to severe cytotoxicity. The repeat experiments were performed using lower concentrations. The dose range of the second experiment was adjusted to the data on cytotoxicity observed in the first experiment.
The individual concentrations were generally spaced by a factor of 2.0. A narrower spacing was used at high concentrations to cover the cytotoxic range more closely.
To overcome problems with possible deviations in toxicity the main experiments were started with more than four concentrations.
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 within the laboratory´s historical control data range, 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. The number of mutant colonies obtained for the groups treated with the test item was 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 was considered together.
Key result
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:
valid
Positive controls validity:
valid
Additional information on results:
The test item Triisooctylamine was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.
The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The experimental part without metabolic activation was prematurely terminated as exceedingly severe cytotoxicity occurred already at low concentrations. This experimental part was repeated as experiment IA with an extended concentration range. Experiment IA was again terminated due to severe cytotoxicity and repeated as experiment IB with lower concentrations. The data of experiment IB are reported as first experiment without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and without metabolic activation.
Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% in both cultures occurred in experiment I at 1.0 μg/mL and above without metabolic activation. In the experimental part with metabolic activation no relevant cytotoxicity was noted up to 42.0 μg/mL. Exceedingly severe cytotoxic effects occurred at the next higher concentration of 56.0 μg/mL. In experiment II relevant cytotoxic effects as described above were noted at 1.0 μg/mL and above without metabolic activation and at 20.0 μg/mL and above with metabolic activation. The recommended cytotoxic range of approximately 10%-20% relative cloning efficiency or relative cell density was covered with and without metabolic activation. Precipitation was noted at 56.0 μg/mL in the first experiment with metabolic activation. The precipitate however, was judged to be based on denatured protein of the S9 as the test item is liquid.
No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiments up to the maximum concentration. The mutant frequency did not exceed the historical range of solvent controls.
The induction factor exceeded the threshold of three times the corresponding solvent control in the first culture of the first experiment at all concentrations with metabolic activation. In the second culture of experiment I with metabolic activation the threshold was reached or exceeded at 3.5, 7.0, and 42.0 μg/mL. These effects however, were based upon rather low solvent controls of 3.8 and 6.0 mutant colonies/106 cells respectively, and thus, biologically irrelevant.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in the first culture of the first experiment without metabolic activation and in the first culture of the second experiment with metabolic activation. These trends however, were inverse going down versus increasing concentrations and consequently, irrelevant. A significant dose dependent trend of the mutation frequency occurred in the first culture of experiment I with metabolic activation. Since the trend was not reproduced in the parallel culture and all of the values of the mutation frequency remained within the historical solvent control range, the isolated trend was judged as irrelevant fluctuation.
In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 3.8 up to 24.4 mutants per 106 cells; the range of the groups treated with the test item was from 3.9 up to 41.3 mutants per 106 cells.
EMS (150 μg/mL) and DMBA (2.2 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Table 1 Summary of Results of Experiment I and II

 

 

conc. µg/mL

P

S9 mix

Relative cloning efficiency I

%

Relative cell density

%

Relative cloning efficiency II

%

Mutant colonies/ 106cells

Induction factor

Relative cloning efficiency I

%

Relative cell density

%

Relative cloning efficiency II

%

Mutant colonies/ 106cells

Induction factor

Column

1

2

3

4

5

6

7

8

9

10

11

12

13

Experiment I / 4h treatment

 

 

 

culture I

culture II

Solvent control with ethanol

 

 

-

100.0

100.0

100.0

16.7

1.0

100.0

100.0

100.0

24.4

1.0

Positive control (EMS)

150.0

 

-

97.8

93.3

94.6

117.7

7.0

96.3

91.3

93.0

108.3

4.4

Test item

0.063

 

-

89.5

98.8

96.5

17.4

1.0

90.4

87.9

91.8

20.6

0.8

Test item

0.13

 

-

86.5

108.7

94.2

12.6

0.8

86.1

88.6

93.3

8.2

0.3

Test item

0.25

 

-

70.3

82.4

94.7

10.0

0.6

69.4

93.8

92.4

18.7

0.8

Test item

0.50

 

-

53.3

92.9

95.8

5.6

0.3

54.5

33.2

95.3

14.7

0.6

Test item

1.0

 

-

49.7

30.3

94.5

3.9

0.2

42.1

25.6

96.0

7.0

0.3

Test item

2.0

 

-

37.9

culture was not continued #

38.3

culture was not continued #

Test item

3.0

 

-

11.9

culture was not continued #

16.7

culture was not continued #

Test item

4.0

 

-

9.8

culture was not continued #

7.5

culture was not continued #

Solvent control with ethanol

 

 

+

100.0

100.0

100.0

3.8

1.0

100.0

100.0

100.0

6.0

1.0

Positive control (DMBA)

2.2

 

+

108.4

78.9

133.1

90.5

23.7

86.3

71.9

75.9

152.6

25.5

Test item

1.8

 

+

101.3

culture was not continued ##

99.2

culture was not continued ##

Test item

3.5

 

+

102.8

87.8

94.5

23.7

6.2

89.2

73.9

74.9

20.1

3.3

Test item

7.0

 

+

109.4

83.8

100.8

19.0

5.0

96.0

83.7

76.5

23.9

4.0

Test item

14.0

 

+

100.8

98.8

117.4

14.1

3.7

91.0

69.5

85.7

17.6

2.9

Test item

28.0

 

+

103.1

66.9

102.5

24.5

6.4

83.1

59.8

94.5

11.9

2.0

Test item

42.0

 

+

101.1

81.5

93.1

41.3

10.8

85.2

55.2

91.0

17.7

3.0

Test item

56.0

P

+

21.7

culture was not continued #

14.9

culture was not continued #

Experiment II / 4h treatment

 

 

 

culture I

culture II

Solvent control with ethanol

 

 

-

100.0

100.0

100.0

17.9

1.0

100.0

100.0

100.0

12.7

1.0

Positive control (EMS)

150.0

 

-

94.2

90.3

100.2

100.8

5.6

96.2

100.5

103.6

136.2

10.7

Test item

0.09

 

-

99.9

92.0

123.3

23.6

1.3

96.9

119.3

116.0

10.1

0.8

Test item

0.19

 

-

95.4

97.6

106.6

10.5

0.6

96.2

111.4

128.1

18.0

1.4

Test item

0.38

 

-

73.1

99.4

113.8

12.5

0.7

71.8

101.9

120.1

21.3

1.7

Test item

0.75

 

-

63.7

58.4

99.1

18.5

1.0

62.5

72.1

126.0

13.7

1.1

Test item

1.0

 

-

27.3

26.1

125.7

17.6

1.0

33.9

22.4

103.8

33.2

2.6

Test item

1.5

 

-

7.5

culture was not continued #

13.1

culture was not continued #

Test item

2.0

 

-

0.0

culture was not continued #

0.0

culture was not continued #

Test item

2.5

 

-

0.0

culture was not continued #

0.0

culture was not continued #

Solvent control with ethanol

 

 

+

100.0

100.0

100.0

20.3

1.0

100.0

100.0

100.0

19.5

1.0

Positive control (DMBA)

2.2

 

+

92.1

64.2

116.4

220.5

10.8

92.3

114.5

100.7

141.1

7.3

Test item

2.5

 

+

98.5

93.6

111.1

13.7

0.7

99.7

119.3

105.8

14.7

0.8

Test item

5.0

 

+

98.5

73.2

78.4

15.6

0.8

98.6

76.2

93.1

20.0

1.0

Test item

10.0

 

+

96.7

87.8

106.2

16.9

0.8

96.4

96.2

96.5

8.7

0.4

Test item

20.0

 

+

65.0

23.3

100.5

6.9

0.3

64.9

41.6

94.4

19.3

1.0

Test item

30.0

 

+

45.4

12.0

80.3

5.9

0.3

36.8

14.8

109.5

9.7

0.5

Test item

40.0

 

+

24.3

culture was not continued #

18.9

culture was not continued #

Test item

50.0

 

+

0.8

culture was not continued #

1.0

culture was not continued #

 

P =   Precipitation visible at the end of treatment

#       culture was not continued due to exceedingly severe cytotoxic effects

##     culture was not continued as a minimum of only 4 analysable concentrations is required

 

 

Endpoint:
in vitro cytogenicity / micronucleus study
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 487 (In vitro Mammalian Cell Micronucleus Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.: 0008924462
Target gene:
N/A
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
The V79 cell line has been used successfully for many years in in vitro experiments. The high proliferation rate (doubling time of V79 cells in stock cultures: approximately 13 hours, determined on December 17, 2010) and a reasonable plating efficiency of untreated cells (as a rule more than 70 %) both necessary for the appropriate performance of the study, support the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22 ± 1.
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix
Test concentrations with justification for top dose:
without S9-mix:
Exp. IB: 0.2 - 1.3 μg/mL
Exp. II: 0.02 - 0.64 μg/mL
with S9-mix:
Exp. IA: 16.1 - 64.4 μg/mL
Exp. II: 5.0 - 20.0 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: [ethanol]
- Justification for choice of solvent/vehicle: Ethanol was a suitable vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Griseofulvin
Details on test system and experimental conditions:
Chinese Hamster V79 cells
The V79 cell line has been used successfully for many years in in vitro experiments. The high proliferation rate (doubling time of V79 cells in stock cultures: approximately 13 hours, determined on December 17, 2010) and a reasonable plating efficiency of untreated cells (as a rule more than 70 %) both necessary for the appropriate performance of the study, support the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22 ± 1.
Large stocks of the V79 cell line (obtained from Labor für Mutagenitätsprüfungen (LMP), Technical University Darmstadt, 64287 Darmstadt, Germany) are stored in liquid nitrogen in the cell bank of Harlan CCR. This allows the repeated use of the same cell culture batch in experiments. Before freezing each batch is screened for mycoplasm contamination and checked for karyotype stability. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells.

Culture conditions
Thawed stock cultures were propagated at 37 °C in 80 cm2 plastic flasks. About 5 x 105 cells per flask were seeded in 15 mL of MEM (minimal essential medium) containing Hank’s salts, glutamine and Hepes (25 mM). Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 μg/mL) and 10 % (v/v) fetal bovine serum (FBS). The cells were sub-cultured twice a week.
Exponentially growing stock cultures more than 50 % confluent were rinsed with Ca-Mg-free salt solution containing 8000 mg/L NaCl, 200 mg/L KCl, 200 mg/L KH2PO4 and 150 mg/L Na2HPO4. Afterwards the cells were treated with trypsin-EDTA-solution at 37 °C for approx. 5 minutes. Then, by adding complete culture medium including 10 % (v/v) FBS the enzymatic treatment was stopped and a single cell suspension was prepared. The trypsin concentration for all subculturing steps was 0.25% (w/v) in Ca-Mg-free salt solution. The cells were seeded into Quadriperm dishes containing microscopic slides. Into each chamber 1.0 x 105 – 1.5 x 105 cells were seeded.
All incubations were done at 37 °C in a humidified atmosphere with 1.5 % carbon dioxide (98.5 % air).

Test Item Preparation
All formulations were prepared freshly before treatment and used within two hours of preparation. The formulation was assumed to be stable for this period unless specified otherwise by the Sponsor.
The osmolarity and pH-value were determined in the solvent control and the maximum concentration without metabolic activation.

Dose Selection
Dose selection was performed according to the current OECD Guideline for the in vitro micronucleus test. The highest test item concentration should be 10 mM, 2 mg/mL or 2 μL/mL, whichever is the lowest. At least three test item concentrations should be evaluated for cytogenetic damage. In case of test item induced cytotoxicity, measured by the relative increased cell count (RICC), or precipitation (observed at the end of test item exposure by the unaided eye) the dose selection should reflect these properties of the test item. Where cytotoxicity will occur the applied concentrations should cover a range from no to approximately 55 ± 5 % cytostasis. Where precipitation will occur the dose range should cover at least one concentration in the non-precipitating range. If cytotoxicity will occur in concentrations higher than precipitating concentrations, evaluation at the border of precipitation is considered sufficient. Furthermore, test item induced changes in osmolarity will influence the dose selection.
2062.0 μg/mL of Triisooctylamine were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 4.0 to 2062.0 μg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, no precipitation of the test item was observed. Since the cultures fulfilled the requirements for cytogenetic evaluation in the presence of S9 mix, this preliminary test was designated Experiment IA. The experimental part without S9 mix was repeated with a top dose of 50.0 μg/mL (Exp. IB) due to strong cytotoxicity.
In Experiment IA in the presence of S9 mix and in Experiment IB in the absence of S9 mix, concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage. However, in Experiment IB in the absence of S9 mix moderate cytotoxicity of 68 %, indicated as proliferation index, was observed at the highest evaluated concentration of 1.3 μg/mL.
Therefore, 10.0 μg/mL (without S9 mix) and 120.0 μg/mL (with S9 mix) were chosen as top concentrations in Experiment II.
The cytogenetic evaluation of concentrations in Experiment IB (without S9 mix) higher than indicated in Table 2 was impossible due to strong test item-induced toxic effects (low cell numbers, partially paralleled by poor cell quality).

Pre-experiment
A preliminary cell growth inhibition test (determination of proliferation index) was performed to determine the concentrations to be used in the main experiment. The experimental conditions in this pre-experimental phase were identical to those required and described below for the mutagenicity assay.
The pre-test was performed with 10 concentrations of the test item separated by no more than a factor of √10 and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 4 hrs (with and without S9 mix). The preparation interval was 24 hrs after start of the exposure.

Cytogenetic Experiment
Pulse exposure
The culture medium of exponentially growing cell cultures was replaced with serum-free medium containing the test item. For the treatment with metabolic activation 50 μL S9 mix per mL culture medium was added. After 4 hours the cultures were washed twice with "Saline G" (pH 7.2) containing 8000 mg/L NaCl, 400 mg/L KCl, 1100 mg/L glucose • H2O, 192 mg/L Na2HPO4 • 2 H2O and 150 mg/L KH2PO4. The cells were then cultured in complete medium containing 10 % (v/v) FBS for the remaining culture time of 20 hours.
Continuous exposure (without S9 mix)
The culture medium of exponentially growing cell cultures was replaced with complete medium containing 10 % (v/v) FBS including the test item. The medium was not changed until preparation of the cells.

Preparation of micronuclei
The cells were treated on the slides in the chambers with deionised water for 1 – 1.5 min at 37 °C. Afterwards the cells were fixed twice with a mixture of methanol and glacial acetic acid (3+1 parts, respectively) containing 1.25 % formaldehyde. The slides were stained with Giemsa, mounted after drying and covered with a cover slip. All slides were labelled with a computer-generated random code to prevent scorer bias.

Evaluation of cytotoxicity and cytogenetic damage
Evaluation of the slides was performed using microscopes with 40 x objectives. The micronuclei were counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle. The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus. Per culture at least 1000 cells from clones with 2 - 8 cells were scored for cytogenetic damage on coded slides. The frequency of micronucleated cells was reported as % micronucleated cells.
Cytotoxicity was assessed by the determination of the relative increase in cell counts (RICC).

Cytotoxicity [%] = 100 – RICC
In addition, cytotoxicity was assessed via counting the number of clones consisting of 1 cell (c1), 2 cells (c2), 3 - 4 cells (c4), and 5 - 8 cells (c8) among the cells that were scored for the presence of micronuclei. These clusters represent the cells that have divided 1, 2, or 3 times within the experiment. From these data, a proliferation index (PI) was calculated (see formula below). Only those cultures were evaluated which showed a PI > 1.3, in order to guarantee a sufficient cell proliferation during treatment and recovery.
Evaluation criteria:
Interpretation of Results
Many experiments with Chinese Hamster V79 cells have established a range of micronucleus frequencies acceptable for control cultures. The current historical data range together with the statistical significance, confirmed by the Chi square test (α < 0.05), should be considered for classification of the test item.
The micronucleus assay is considered acceptable if it meets the following criteria:
a) The rate of micronuclei in the solvent controls falls within the historical laboratory control data range.
b) The rate of micronuclei in the positive controls is statistically significant increased.
c) The quality of the slides must allow the evaluation of a sufficient number of analyzable cells.
A test item can be classified as non-mutagenic if:
− the number of micronucleated cells in all evaluated dose groups is in the range of the historical laboratory control data and
− no statistically significant or concentration-related increase of the number of micronucleated cells is observed in comparison to the respective solvent control.
A test item can be classified as mutagenic if:
− The number of micronucleated cells exceeds both the value of the concurrent negative control and the range of the historical negative control data.
− A significant, dose-related and reproducible increase in the number of cells containing micronuclei is observed
If the above mentioned criteria for the test item are not clearly met, the test item is classified as equivocal or a confirmatory experiment may be performed. However, results may remain questionable regardless of the number of times the experiment is repeated.
Statistics:
Chi square test (α < 0.05)
Key result
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:
valid
Positive controls validity:
valid
Additional information on results:
The test item Triisooctylamine, dissolved in ethanol, was assessed for its potential to induce micronuclei in Chinese hamster V79 cells in vitro in the absence and presence of metabolic activation by S9 mix.
Three independent experiments were performed. In Experiment IA, the exposure period was 4 hours with S9 mix. In Experiment IB, the exposure period was 4 hours without S9 mix. In Experiment II, the exposure periods were 4 hours with S9 mix and 24 hours without S9 mix. The cells were prepared 24 hours after start of treatment with the test item.
In each experimental group two parallel cultures were analysed. At least 1000 cells per culture were scored for cytogenetic damage on coded slides. To determine a cytotoxic effect the RICC and the PI were determined.
The highest treatment concentration in this study, 2062.0 μg/mL was chosen with regard to the purity (97 %, preliminary information at study start) of the test item and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test.
In Experiment IB in the absence of S9 mix, precipitation of the test item in the culture medium was observed at 50.0 μg/mL. In all other experimental parts no precipitation occurred.
No relevant influence on osmolarity or pH value was observed.
In Experiment IA in the presence of S9 mix and in Experiment IB in the absence of S9 mix, concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage. However, in Experiment IB in the absence of S9 mix moderate cytotoxicity of 68 %, indicated as proliferation index, was observed at the highest evaluated concentration. In Experiment II in the absence and presence of S9 mix, cytotoxicity, indicated as relative increased cell count, was observed at the highest evaluated concentrations.
In the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying micronuclei was observed. The micronucleus rates of the cells after treatment with the test item (0.30 – 1.45 % micronucleated cells) exceeded the range of the solvent control values (0.55 – 1.05 % micronucleated cells), but were within the range of the laboratory historical control data (see Appendix 1).
However, in Experiment II in the presence of S9 mix one single statistically significant increase in micronucleated cells (1.35 %) was observed after treatment with 10.0 μg/mL. Since the value is within the range of the laboratory historical solvent control data (0.15 – 2.00% micronucleated cells), the finding has to be regarded as biologically irrelevant.
Either Griseofulvin (8.0 μg/mL), MMC (0.1 μg/mL) or CPA (10.0 μg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.

Table 1     Summary of results

 

 

Exp.

Preparation interval

Test item concentration

in µg/mL

Proliferation index

RICC

in %

Cytotoxicity in %

Micronucleated cells*

in %

Exposure period 4 hrs without S9 mix

I B

24 hrs

Solvent control1

2.88

100

0

0.90

Positive control2

2.58

340

0#

6.00S

0.2

2.88

189

0#

1.45

0.5

2.68

202

0#

1.25

1.3

1.97

235

0#

1.05

Exposure period 24 hrs without S9 mix

II

24 hrs

Solvent control1

3.06

100

0

0.60

Positive control3

2.81

54

46

9.05S

0.02

3.03

78

22

0.65

0.04

3.06

32

68

0.45

0.10

3.00

61

39

0.30

0.26

3.06

79

21

0.40

0.64

3.05

37

63

0.70

Exposure period 4 hrs with S9 mix

I A

24 hrs

Solvent control1

2.22

100

0

1.05

Positive control4

1.67

-71

n.a.

13.65S

16.1**

2.31

83

17

1.05

32.2

1.75

-112

n.a.

0.80

64.4

1.66

-234

n.a.

1.10

II

24 hrs

Solvent control1

2.46

100

0

0.55

Positive control4

1.67

-17

n.a.

15.70S

5.0

2.24

77

23

0.70

10.0

2.45

71

29

1.35S

20.0

1.76

42

58

1.05

 

*       The number of micronucleated cells was determined in a sample of 2000 cells

**      The number of micronucleated cells was determined in a sample of 4000 cells

n.a.   Not analysable, because the cell number of the solvent control or treated cultures was lower after treatment

#       Not cytotoxic since the RICC is higher than the solvent control value

S       Number of micronucleated cells statistically significantly higher than corresponding control values

1       Ethanol            0.5 % (v/v)

2       Mitomycin C    0.1 μg/mL

3       Griseofulvin      8.0 μg/mL

4       CPA                 10.0 μg/mL

 

 

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames-Test

This study was performed to investigate the potential of Triisooctylamine dissolved in Ethanol to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.

The study was conducted according to OECD 471 guideline and GLP (Harlan CCR, 2015). The assay was performed with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations adjusted to the prior purity of 97%:

Pre-experimet/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

The test item precipitated in the overlay agar in the test tubes at 5000 μg/plate in experiment I. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 1000 to 5000 μg/plate in experiment I. The undissolved particles had no influence on the data recording. No precipitation of the test item was observed in the overlay agar either in the test tube or on the incubated agar plates in experiment II.

The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used in experiment I and in the presence of S9 mix in experiment II. Reduced background growth was observed in all strains in experiment II without S9 mix.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in strains TA 1537, TA 98 and TA100.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Triisooctylamine at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, Triisooctylamine is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

HPRT-Test

The study was performed to investigate the potential of Triisooctylamine to induce gene mu-tations at the HPRT locus in V79 cells of the Chinese hamster. The study was conducted according to OECD 476 guideline and GLP (Harlan CCR, 2015).

The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The experimental part without metabolic activation was prematurely terminated as exceedingly severe cytotoxicity occurred already at low concentrations. This experimental part was repeated as experiment IA with an extended concentration range. Experiment IA was again terminated due to severe cytotoxicity and repeated as experiment IB with lower concentrations. The data of experiment IB are reported as first experiment without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and without metabolic activation.

The highest concentration of 3646 μg/mL in the pre-experiment was equal to approximately 10 mM. The concentration range of the main experiments was limited by cytotoxicity of the test item. The test item was dissolved in ethanol.

No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation.

Appropriate reference mutagens (EMS and DMBA), used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

Conclusion

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, Triisooctylamine is considered to be non-mutagenic in this HPRT assay.

In vitro Micronucleus-Test

The test item Triisooctylamine, dissolved in ethanol, was assessed for its potential to induce micronuclei in Chinese hamster V79 cells in vitro in three independent experiments according to OECD 487 guideline and GLP (Harland CCR, 2015).

In each experimental group two parallel cultures were analysed. Per culture at least 1000 cells were evaluated for cytogenetic damage.

The highest applied concentration in this study (2062.0 μg/mL of the test item) was chosen with regard to the purity (97 %, preliminary information at study start) of the test item and with respect to the current OECD Guideline 487.

Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 487.

In Experiment IA in the presence of S9 mix and in Experiment IB in the absence of S9 mix, concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage. However, in Experiment IB in the absence of S9 mix moderate cytotoxicity of 68 %, indicated as proliferation index, was observed at the highest evaluated concentration. In Experiment II in the absence and presence of S9 mix, cytotoxicity, indicated as relative increased cell count, was observed at the highest evaluated concentrations.

No relevant increases in micronucleated cells were observed with and without S9 mix. However, in Experiment II in the presence of S9 mix one single statistically significant increase in micronucleated cells (1.35 %) was observed after treatment with 10.0 μg/mL. Since the value is within the range of the laboratory historical solvent control data (0.15 – 2.00 % micronucleated cells), the finding has to be regarded as biologically irrelevant.

Appropriate mutagens (MMC, Griseofulvin and CPA) were used as positive controls. They induced statistically significant increases in cells with micronuclei.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in Chinese hamster V79 cells.

Therefore, Triisooctylamine is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to cytotoxic or the highest evaluable concentrations.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified as genotoxic under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.