Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

TIPA did not cause gene mutations in Salmonella typhimurium (Ames test) or in Chinese hamster ovary cells (CHO/HGPRT; OECD TG 476), nor were chromosomal aberrations induced in rat lymphocytes (OECD TG 473). All in vitro studies were performed in the absence and presence of 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
Study period:
1987
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:
No E.coli strain or TA102 tested
Principles of method if other than guideline:
Haworth S, Lawlor T, Mortelmans K, Speck W, Zeiger E (1983): Salmonella mutagenicity results for 250 chemicals. Environ Mutagen S[Suppl 1]:3-142.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Purity > 94%
Target gene:
His locus
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced male Sprague-Dawley rats and Syrian hamsters S9-mix
Test concentrations with justification for top dose:
0, 100, 333, 1000, 3333, 10000 µg/plate
Vehicle / solvent:
Water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 4-nitro-o-phenylene diamine: TA 98, without S9-mix; 2-aminoanthracene: all strains, with S9-mix
Details on test system and experimental conditions:
The test chemical (0.05 mL), Salmonella culture (0.10 mL), and S-9 mix or buffer (0.50 mL) were incubated at 37°C, without shaking, for 20 min. The top agar was added and the contents of the tubes were mixed and poured onto the surface of petri dishes containing Vogel-Bonner medium. The histidine-independent (his+) colonies arising on these plates were counted following two days incubation at 37°C. Plates were machine counted unless precipitate was present which interfered with the count, or the color of the test chemical on the plate reduced the contrast between the colonies and the background agar.
Evaluation criteria:
Evaluations were made at both the individual trial and overall chemical levels. Individual trials were judged mutagenic (+), weakly mutagenic (+ W), questionable (?), or nonmutagenic (-), depending on the magnitude of the increase of his+ revertants, and the shape of the dose-response. A trial was considered questionable (?) if the dose-response was judged insufficiently high to support a call of “ +W,” if only a single dose was elevated over the control, or if the increase seen was not dose related. The distinctions between a questionable mutagenic response and a nonmutagenic or weak mutagenic response, and between a weak mutagenic response and mutagenic response are highly subjective. It was not necessary for a response to reach
twofold over background for a chemical to be judged mutagenic. A chemical was judged mutagenic (+) or weakly mutagenic (+ W) if it produced a reproducible dose-related reponse over the solvent control in replicate trials.
A chemical was judged questionable (?) if the results of individual trials were not reproducible, if increases in his+ revertants did not meet the criteria for a “+W” response, or if only single doses produced increases in his+ revertants in repeat trials. Chemicals were judged nonmutagenic (-) if they did not meet the criteria for a mutagenic or questionable response.
Statistics:
no data
Key result
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Triisopropanolame was negative (non-mutagenic) in all assays with and without metabolic activation.

Strain TA1535

Dose

No Activation
(Negative)

10% HLI
(Negative)

10% RLI
(Negative)

Dose units

Mean

± SEM

Mean

± SEM

Mean

± SEM

0         

42

3.8

21

2.7

11

1.3

100         

33

1.2

13

1.9

13

0.3

333         

34

1.9

15

1.2

15

2.3

1000         

32

6.4

13

0.3

12

4.6

3333         

24

3.5

16

1.8

11

1

10000         

28

5.5

15

0.3

10

0.9

Positive Control

1162

4.5

115

9.1

99

0.9

 

Strain TA100 

Dose

No Activation
(Negative)

10% HLI
(Negative)

10% RLI
(Negative)

Dose units

Mean

± SEM

Mean

± SEM

Mean

± SEM

0         

151

9

141

0.7

151

10.2

100         

141

9

147

4.8

139

0.6

333         

131

0.6

141

5.6

146

2.4

1000         

144

8.6

146

3.2

149

4.3

3333         

152

5

151

10.3

143

7.9

10000         

148

2.5

146

4.7

139

5.8

Positive Control

1406

62.8

1040

19.8

1025

37.8

 

Strain TA98 

Dose

No Activation
(Negative)

10% HLI
(Negative)

10% RLI
(Negative)

Dose units

Mean

± SEM

Mean

± SEM

Mean

± SEM

0         

20

1

26

4.5

30

1.9

100         

19

1.8

28

0.9

33

4.9

333         

19

2.9

31

1.5

29

2

1000         

21

0

31

4.1

30

0.9

3333         

15

0.9

31

4.3

30

0.6

10000         

16

3.1

28

1.2

25

2.3

Positive Control

1320

21.1

864

24.3

697

11.5

 

Strain TA1537 

Dose

No Activation
(Negative)

10% HLI
(Negative)

10% RLI
(Negative)

Dose units

Mean

± SEM

Mean

± SEM

Mean

± SEM

0         

8

0.3

8

0.7

6

2.3

100         

4

0.6

7

1

10

2.9

333         

6

0.6

11

3.2

9

0.6

1000         

8

0.9

11

1.8

9

2

3333         

6

1.3

10

1.5

8

1.7

10000         

7

0.3

11

0.6

10

0.9

Positive Control

282

11.2

88

7

82

6

 

Abbreviations:
RLI = induced male Sprague Dawley rat liver S9
HLI = induced male Syrian hamster liver S9

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EPA OTS 798.5300 (Detection of Gene Mutations in Somatic Cells in Culture)
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
other: EEC In vitro mammalian cell gene mutation test Official Journal of the European Communities, L133, 31, 61-63
Deviations:
not specified
GLP compliance:
yes
Type of assay:
other: in vitro mammalian cell gene mutation tests using the hprt and/or xprt genes
Specific details on test material used for the study:
Purity was 99.21%
Analysis of Dose Solutions by HPLC with Refractive Index Detection
Target gene:
HGPRT
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
In cultured chinese hamster ovary (CHO) cells
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S9-mix
Test concentrations with justification for top dose:
50, 167, 500, 1667 and 5000 µg/mL
Vehicle / solvent:
water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
CHO/HPGRT Mutation Assays

In this study, Triisopropanolamine 99 was tested in two independent assays. Duplicate cultures seeded with 5x10E5 cells/flask were used at each dose level.

For parallel toxicity determination, three plates with 200 cells/plate were used at each dose level. The cells were treated for 5 hours with and without metabolic activation.

In the mutation assay, the cells were treated at test article concentrations of 50, 167, 500, 1667, and 5000 µg/mL both with and without metabolic activation. Appropriate positive, solvent, and untreated controls were also included in the assays. After the exposure time, the cells were washed with HBSS, refed with complete Hx-free medium, and allowed to grow for 18-24 hours. The cells were then subcultured to initiate cultures for the expression of mutant phenotype. The cytotoxicity determination was performed as described in the rage finding test. The cytotoxicity was expressed as RCS.

For the expression of 6-thioguanine-resistant mutants (HGPRT locus mutants), the cells from each of the duplicate culture flasks were subcultured with 5% dialyzed HIFBS, 2mM L-glutamine, 50 Units/mL of penicillin and 50 µg/ml streptomycin (cloning medium) at a density of 1x10E6 cells/75 cm2 flask. The cells were subcultured as described above at 2- to 3-day intervals for a period of 9 days in the first assay and 8 days in the confirmatory assay prior to selecting the mutant phenotypes. The cells were grown as attached cultures in T-75 cm2 tissue culture flasks.

After the expression period, the cells from each of the treatment replicates were harvested and seeded in five 100 mm2 tissue culture plates at a density of 2x10E5 cells/plate in 10 mL of cloning medium containing 10 µM of 6-tioguanine (TG). To determine the cloning efficiency of the cells at the time of selection, 200 cells/60 mm2 dish were plated in triplicate in the cloning medium. All clonable test doses and appropriate positive and solvent controls were cloned for mutant selection. The cultures were then incubated for 7 days without disturbing the plates to minimize the formation of satellite colonies. The colonies were then washed with PBS, fixed, stained and counted for cloning efficiency and mutant selection. The average number of clones from the triplicate plates was calculated and the percent clonable cells (cloning efficiency) for each treatment condition was determined. The number of TG-resistant mutants for 1x10E6 cells seeded was calculated by totaling the number of mutants from the five replicate plates. Based on the cloning efficiency, the number of TG-resistant mutants per 1x10E6 surviving cells was calculated for each dose.
Evaluation criteria:
The test article is considered positive in the assay if it induces a statistically significant and reproducible increase in mutation frequency at more than one of the dose levels tested. The final interpretation of the data also takes into consideration such factors as the mutation frequency and cloning efficiencies in the negative controls and dose-response relationships.
Statistics:
The following method of statistical analysis was performed: The frequencies of mutants per 1x10E6 clonable cells were analyzed by the Cochran-Armitage test for trend (≤ 0.025, one-sided, increasing) and the Fisher-Irwin exact test for group comparisons for proportions (≤ 0.01, one sided). The within-group replicates were pooled for the final analysis.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Range Finding Test: In the range finding test, the test article was tested at dose levels of 0.1, 0.5, 1.0, 5.0, 10, 50, 100, 500, 1000 and 5000 µg/mL.

The test cultures seeded approximately 18-24 hours earlier. Triplicate culture plates were used at each test article and solvent control dose level.

In the activated system, the medium was removed and 5 ml of serum-free culture medium containing appropriate concentrations of S-9 mixture and test article was added to each of the culture plates. The cells were exposed to the test article for 5 hours. After the exposure period, the cells were washed with HBSS and refed with complete Hx-free medium.

In the non-activated system, the medium was removed, and 5 ml of test article containing serum-free culture medium was added to each of the culture plates. The cells were then exposed to the test article for 5 hours, washed with HBSS, and refed with complete Hx-free medium.

The cells were allowed to grow for a period of 7 days without any disturbance to minimize the formation of satellite colonies. The colonies were then washed with phosphate buffered saline (PBS), fixed with methanol, stained with Giemsa and counted. A cluster of more than 50 cells growing within a confined area was considered a colony. The average number of colonies per plate was calculated, and the relative cell survival (RCS) was determined by the following formula:
RCS = Average No. of Colonies in Test Plates/Average No. of Colonies in Solvent Plates x 100


Analysis of Dose Solutions by HPLC with Refractive Index Detection
Expt. B1 Expt. B2
Target Observed Target Observed
mg/mL mg/mL mg/mL mg/mL
500 551 500 542
167 189 167 184
50 60.5 50 57.8
17 20.5 16.7 20.0
5 7.22 5 6.97

CHO/HGPRT Mutation Assay - Exp. 1

                Without Activation     With S-9 Activation
Conc. (ug/ml)
   Mutants/10-6 Cells     Mutants/10-6 Cells
 
Sol. A
                4                       3
Sol. B
               14                      25

50 A
                 21*                      5
50 B
                 27*                      8

167 A
                12                      28*
167 B
                13                      19*

500 A
                20*                      6
500 B
                51*                      4

1667 A
               14                      10
1667 B
                6                      17

5000 A
               13                       3
5000 B
                0                        2
 
* Statistically Significant Dose (p<0.01)

CHO/HGPRT Mutation Assay - Exp. 2

                Without Activation     With S-9 Activation
Conc. (ug/ml)
   Mutants/10-6 Cells     Mutants/10-6 Cells
 
Sol. A
                2                       4
Sol. B
                2                       1

50 A
                  9                       3
50 B
                  3                       1

167 A
                 6                       4
167 B
                 1                       7

500 A
                 4                       0
500 B
                 5                       0

1667 A
                8                       8
1667 B
                3                       2

5000 A
                3                       3
5000 B
                0                       1



Based upon the frequency of the TG-resistant mutants recovered in cultures treated with the test material, it was concluded that TIPA 99 did not induce a mutagenic response in the assay system employed.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993
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)
GLP compliance:
yes
Type of assay:
other: in vitro mammalian chromosomal aberration test
Specific details on test material used for the study:
Purity 99.21%
Target gene:
chromosomal aberrations
Species / strain / cell type:
lymphocytes: rat
Details on mammalian cell type (if applicable):
Sprague Dawley rat
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9-mix
Test concentrations with justification for top dose:
0, 50, 167, 500, 1670 and 5000 µg/mL
Vehicle / solvent:
water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
Toxicity and Chromosomal Aberration Assay:
Two independent assays were conducted under each activation condition. In Assay 1 without activation, cultures were treated with various concentrations of the test chemical (167 to 5000 µg/mL), the selected concentrations of EMS, and the solvent (negative control). In Assay 1 with activation, cultures were treated with 50-500 µg/mL of TIPA 99, the selected concentration of CP, and the solvent (negative control). Cultures were harvested approximately 24 h after termination of the treatment. In Assay 2, cultures were treated with three concentrations of the test chemical (500, 1670, and 5000 µg/mL), selected concentrations of the positive control chemicals, and the solvent (negative control) and harvested approximately 24 and 48 hr after termination of the treatment. The positive control treated cultures were harvested at 24 h after termination of treatment. Colcemid was added approximately 3 h prior to harvest at a final concentration of 0.2 µg/mL. The cells were swollen by hypotonic treatment (0.075 M KCl), fixed with methanol:acetic acid (3:1), dropped on microscope slides, and stained in Giemsa. Mitotic indices were determined as the number of cells in metaphase among 1000 cells/replicate and expressed as percentages. One hundred metaphases/replicate were examined from coded slides at each selected concentration of the test chemical (a total of 200 cells/treatment) for structural abnormalities. In the positive control cultures, 50 metaphases/replicate (a total of 100 metaphases/treatment) were examined. The microscopic coordinates of each metaphase were recorded. Only those metaphases that contained 42 centromeres were scored with the exception of cells with multiple aberrations, in which case accurate counts of the chromosomes were not possible. Those cells having 5 or more aberrations/cell were classified as cells with multiple aberrations. Gaps were not included in calculations of total cytogenetic aberrations.
Evaluation criteria:
For a test to be acceptable, the chromosomal aberration frequency in the positive control cultures should be significantly higher than the negative controls. The aberration frequency in the negative control should be within reasonable limits of the laboratory historical values. A test chemical is considered positive in this assay if it induces a significant dose-related, and reproducible increase in the frequency of cells with aberrations.
Statistics:
The frequencies of cells with aberrations (excluding gaps) were compared by the following statistical methods. At each dose level, data from the replicates were pooled. A two way contingency table was constructed to analyze the frequencies of cytogenetic abnormalities. An overall Chi-square statistic, based on the table, was partitioned into components of interest. Specifically, statistics were generated to test the two global hypotheses of (1) no differences in average number of cells with aberrations among the dose groups, and (2) no linear trend of increasing number of cells with aberrations with increasing dose. An ordinal metric (0, 1,2, etc.) was used for the doses in the statistical evaluation. If either statistic was found to be significant at alpha=0.01 versus a one-sided increasing alternative, pairwise tests (i.e., control vs treatment) were performed at each dose level and evaluated at alpha=0.01 again versus a one-sided alternative.
Key result
Species / strain:
mammalian cell line, other: rat lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Assay 1
The average mitotic index (M.I.) of cultures treated with the highest dose level (5000 µg/mL) in the absence of S-9 was 6.6% as compared to the negative control value of 8.1%. In the presence of S-9, the average M.I. for cultures treated with 5000 µg/ml was 15.2% compared to the negative control value of 17.4% . Hence cultures treated with the three top concentrations (i.e., 500, 1670, and 5000 µg/mL) were selected for determining the chromosomal aberration frequencies. In the non-activation assay, the frequencies of cells with aberrations in the test chemical treated cultures ranged from 1.0 to 2.0% . In the activation assay, the frequencies of cells with aberrations in the test chemical treated cultures ranged from 0 to 2.0%. These values were not significantly different from the concurrent negative controls and were within the background aberration rate for rat lymphocytes.

Assay 2
In a confirmatory assay, rat lymphocyte cultures were treated with 500, 1667, and 5000 µg/ml of the test compound in the absence and presence of S-9 activation and were harvested at 24 and 48 h following treatment. The additional 48 h harvest time was included in this assay in order to accommodate such factors as treatment induced cell cycle delays. All cultures were found to be suitable for the scoring of aberrations at the 24 and 48 h harvest times. Slides from only the highest concentration of the test material assayed (i.e., 5000 µg/mL) were selected for determining the chromosomal aberration frequencies at the 48 h harvest in the absence and presence of S-9. There were no significant increases in the incidence of cells with chromosomal abnormalities in cultures treated with the test material either in the absence or presence of S-9 and harvested at 24 h or 48 h.

In both Assays 1 and Assay 2, significant increases in aberration rates were observed in cultures treated with the positive control chemicals.


Concentration Check
Assay 1 with S9 Assay 1 without S9 Assay 2
Target Observed Target Observed Target Observed
mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL
500 507 500 503 500 533
167 156 167 154 167 167
50 50.9 50 48.1 50 50.5
16.7 15.8 16.7 15.5 --- ---
--- --- 5 4.85 --- ---
0 ND 0 ND 0 ND

No significant increase in the incidence of abnormal cells was noticed at any of the treatment levels with or without S-9 activation when compared to the negative controls. The positive controls had significantly higher incidences of abnormal cells. Hence, TIPA 99 was considered to be negative in the in vitro chromosomal aberration assay utilizing rat lymphocytes.

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

Genetic toxicity in vivo

Description of key information

In an in vivo micronucleus test in mice (OECD TG 474) no clastogenic effects were observed afer single oral administration of TIPA at doses up to 2000 mg/kg bw.

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
Type of assay:
other: mammalian erythrocyte micronucleus test
Specific details on test material used for the study:
- Analytical purity: 92.0 %
- Lot/batch No.: 10-4852
- Storage condition of test material: room temperature (N2 conditions)
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River GmbH, WIGA, Sulzfeld,
- Weight at study initiation: 27.5 g (mean)
- Housing: individually
- Diet: Standardized pelleted feed (Kliba Haltungsdiaet, Klingentalmühle AG, Kaiseraugst, Switzerland) ad libitum
- Water: botteled tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: water
Duration of treatment / exposure:
24 h or 48 h
Frequency of treatment:
single application
Post exposure period:
24 h or 48 h
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide (CCP); vincristine (VCR)
Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: pretests

DETAILS OF SLIDE PREPARATION: The bone marrow was prepared according to the method described by Schmid, W. One drop of cells was dropped onto clean microscopic slide. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained. The slides were stained in eosin and methylene blue solution for 5 minutes, rinsed in purified water and then placed in fresh purified water for 2 or 3 minutes. They were finally stained in Giemsa solution for 12 minutes. After being rinsed twice in purified water and clarified in xylene, the preparations were embedded in Corbit-Balsam.




Evaluation criteria:
In general, 1000 polychromatic erythrocytes (PCE) from each of the male and female animals of every test group are evaluated and investigated for micronuclei (MN). The normochromatic erythrocytes (NCE), which occur, are also scored. The following parameters are recorded:
- Number of polychromatic erythrocytes
- Number of polychromatic erythrocytes containing micronuclei
The increase in the number of micronuclei in polychromatic erythrocytes of treated animals as compared with the solvent control group provides an index of a chromosome-breaking (clastogenic) effect or of a spindle activity of the substance tested.
- Number of normochromatic erythrocytes
- Number of normochromatic erythrocytes containing: micronuclei
The number of micronuclei in normochromatic erythrocytes at the early sacrifice intervals represents the situation before test substance administration and may serve as a control value. A substance-induced increase in the number of micronuclei in normocytes may be found with an increase in the duration of the sacrifice intervals.
- Ratio of polychromatic to normochromatic erythrocytes
This ratio indicates an influence of the test substance specifically on the bone marrow.
- Number of small micronuclei (d < D/4) and of large micronuclei (d > D/4) (d = diameter of micronucleus, D = cell diameter)
The size of micronuclei may give an indication on the possible mode of action of the test substance, i.e. a clastogenic or a spindle poison effect.
Statistics:
The statistical evaluation of the data was carried out using the program system MUKERN (BASF AG ). A comparison of the dose group with the vehicle control was carried out using the Wilcoxon test for the hypothesis of equal medians. Here, the relative frequencies of cells with micronuclei of each animal were used. If the results of this test were significant, labels (+ for p < 0.05, ++ for p < 0.01) were printed with the group means in the tables. This test was performed one-sided. Analysis was done separately for each sex and combined for both sexes.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Oral administration of the test substance led to irregular respiration and piloerection at all three dose levels after about 30 minutes.
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid

Polychromatic and normochromatic erythrocytes:

    24 h           48 h                                                                
Dose (mg/kg) Total No. of PCE´s NCE´s/PCE´s  MN in PCE´s (‰) MN in NCE´s (‰)  Total No. of PCE´s NCE´s/PCE´s  MN in PCE´s (‰) MN in PCE´s (‰)                                                        
control  10000  4928  2.4  4.1  10000  4419  2.7  2.9                                                        
500  10000  4641  3.2  3.2  -  -                                                        
1000  10000  5404  2.0  2.4  -  -  -  -                                                        
2000  10000  5708  3.4  5.1  10000  6389  2.3  3.8                                                        
CCP 20  5000  2875  21.4*  5.9  -  -  -                                                        
VCR 0.15  5000  5054  112.6* 14.2  -  -  -  -                                                        
 

*: p<0.01

CCP: cyclophosphamide

VCR: vincristine

The oral administration of the test substance led to irregular respiration and piloerection at all three dose levels after about 30 minutes. In the dose groups of 1,000 mg/kg and 2,000 mg/kg squatting posture was additionally observed. After 2-4 hours these signs were not observed any longer.

The single administration of the vehicle in a volume of 10 mL/kg body weight led to 2.4‰ polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval or to 2.7‰ after the 48-hour sacrifice interval.

After the single administration of the highest dose of 2,000 mg/kg body weight, 3.4‰ polychromatic erythrocytes containing micronuclei were found after 24 hours and 2.3‰ after 48 hours. In the two lower dose groups rates of micronuclei of about 2 ‰ (1,000 mg/kg group) and 3.2‰ (500 mg/kg group) were detected after a sacrifice interval of 24 hours in each case.

With 21.4‰ the positive control substance cyclophosphamide for clastogenicity, as expected, led to a clear increase in the number of polychromatic erythrocytes containing mainly small  micronuclei at a dose level of 20 mg/kg body weight. With 112.6‰ the positive control vincristine for spindle poison effects also led to a clearly enhanced number of micronuclei cntaining polychromatic erythrocytes with the expected amount of large micronuclei, i.e. 15.6‰. The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the negative control or in the various dose groups at any of the sacrifice intervals.  

Thus, the test substance Triisopropanolamin did not lead to any increase in the rate of micronuclei. The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) or large micronuclei (d ≥ D/4) did not deviate from the solvent control value at any of the sacrifice intervals. No inhibition of erythropoiesis induced by the treatment of mice with Triisopropanolamin was detected. The ratio of polychromatic to normochromatic erythrocytes was always in the same range as that of the control values in all dose groups .

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

Mode of Action Analysis / Human Relevance Framework

No further details.

Additional information

TIPA was tested in the Ames reverse mutation assay using S. typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 at 100 to 10,0000 µg/plate with and without metabolic activation. Treatment with TIPA was not associated with reverse mutations in any of the strains tested (Zeiger et al., 1987). TIPA was also evaluated at concentrations of 50 to 5000 µg/mL in a HGPRT assay according to OECD TG 476 using Chinese hamster ovary cells with and without metabolic activation. No cytotoxicity was observed and negative, vehicle and positive control responses were valid. TIPA was not associated with increased gene mutations (Sitek, 1993). Induction of chromosomal aberrations was tested in an OECD TG 473 study in which rat lymphocytes were exposed to 50 to 5000 µg/mL with and without metabolic activation. No cytotoxicity was observed and negative, vehicle and positive controls exhibited appropriate responses. No significant increases in chromosomal aberrations upon treatment with TIPA were observed (Dow, 1993).

In an in vivo micronucleus test according to OECD TG 474, NMRI mice (5/sex/dose) received a single oral dose of 500, 1000 or 2000 mg/kg bw TIPA (BASF, 1995). 24 and 48 hours after treatment, polychromatic erythrocytes were prepared from bone barrow and investigated for micronuclei. 30 minutes after treatment, irregular respiration and piloerection was observed at all dose levels, and squatting posture was observed in the 1000 and 2000 mg/kg bw dose groups. After 2-4 hours these signs were no longer observed. Moreover, treatment with TIPA did not induce any increase in the rate of micronuclei and no inhibition of erythropoiesis was observed. Vehicle and positive controls were valid.

In summary, these data indicate that TIPA is not genotoxic.

Justification for classification or non-classification

Based on the results of the in vitro and in vivo genetic toxicity studies, TIPA does not need to be classified according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.