Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Diisopropanol-p-toluidin is not mutagenic in vitro in bacteria (two Ames tests) and in mammalian cells (HPRT assay). An in vitro chromosome aberration assay in mammalian V79 cells with diisopropanol-p-toluidin demonstrated that the test sustance has no potential to induce chromosome aberrations.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21.04.1998-26.05.1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
testing lab.
Type of assay:
bacterial reverse mutation assay
Target gene:
S. typhimurium and E. coli
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:
microsomal enzyme system (S-9 mix) from Aroclor 1254 induced Sprague Dawley rat liver
Test concentrations with justification for top dose:
20 µg - 5000 µg/plate (standard plate test and preincubation test)
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-amninoanthracene
Remarks:
with S-9 mix, in TA 1535, TA 100, TA 1537, TA 98, Escherichia coli WP2 uvrA
Positive controls:
yes
Positive control substance:
other: aniline and o-toluidine
Remarks:
with S-9 mix and norharman, in TA 98
Positive controls:
yes
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine
Remarks:
without S-9 mix, in TA 1535, TA 100
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylendiamine
Remarks:
without S-9 mix, TA 98
Positive controls:
yes
Positive control substance:
other: 9-amninoacridine
Remarks:
without S-9 mix, TA 1537
Positive controls:
yes
Positive control substance:
other: 4-Nitroquinoline-N-Oxide
Remarks:
without S-9 mix, E. coli WP uvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION: standard plate and preincubation method

DURATION
- Preincubation period: 20 minutes
- Expression time (cells in growth medium): 48-72 h

NUMBER OF PLATES: 3 per dose or control

DETERMINATION OF CYTOTOXICITY
- Method:
Examination of background lawn and comperison of spontaneous revertants with control
The titer of viable bacteria was >= 10E9/mL


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 nonmutagenic 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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not valid
Positive controls validity:
valid
Additional information on results:
An increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S-9 mix or after the addition of a metabolizing system.
The number of revertant colonies in the negative controls was within the normal range of the historical control data for each tester strain
The sterility controls revealed no indication of bacterial contamination
The positive control articles both with and without S-9 mix induced a significant increase in the number of revertant colonies within the range of the historical control data.
Cytotoxicity:
S.thyphimurium TA 1535, TA 1537, TA 98 and TA 100
standard plate test: slight decrease his+ revertants in the about 5000 µg/plate (TA 1535, TA 100, TA 1537) and 2500 µg/plate onward (TA 98). The preincubation assay: bacteriotoxicity from about 2500 µg/plate (Salmonella strains).
E.coli and WP2 uvr A
Preincubaton assay: bacteriotoxicity at 5000 µg/plate
Precipitation: was not found
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT (hypoxanthine-guanine phosphoribosyl transferase) locus in V79 cells of the Chinese hamster
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (minimal essential medium)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Experiment I (4 hour treatment with and without metabolic activation): 69.7, 139.4, 278.8, 557.5, 1115.0, 2230.0 µg/mL
Experiment II (4 hour treatment with and 24 hour treatment without metabolic acivation): 75.0, 150.0, 300.0, 600.0, 900.0, 1200.0 µg/mL
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Remarks:
ethylmethanesulphonate: without metabolic activation; 7,12-dimethylbenzanthracene: with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: the cells were grown for 24 hours prior to treatment
- Exposure duration: 4 hours with and without S9 mix (first experiment); 24 hours without S9 mix and 4 hours with S9 mix (second experiment)
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days

SELECTION AGENT (mutation assays): 6-thioguanine (6-TG )

NUMBER OF REPLICATIONS: two independent experiments, using two parallel cultures each

NUMBER OF CELLS EVALUATED: the stained colonies with more than 50 cells were counted.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth
A pre-test was performed in order to determine the concentration range for the mutagenicity experiments. The general culture conditions and experimental conditions in this pre-test were the same as described for the mutagenicity experiment below. In this pre-test the colony forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test item was observed and compared to the controls. Toxicity of the test item is indicated by a reduction of the cloning efficiency (CE).

OTHER EXAMINATIONS:
- Other: Each batch was screened for mycoplasm contamination and checked for karyotype stability and spontaneous mutant frequency.
- The osmolarity and pH-value were determined in the solvent control and in the highest concentration of the pre-test without metabolic activation.

Evaluation criteria:
Acceptability of the assay:
The gene mutation assay is considered acceptable if it meets the following criteria:
- The numbers of mutant colonies per 10E6 cells found in the solvent controls falls within the laboratory historical control data.
- The positive control substances should produce a significant increase in mutant colony frequencies.
- The cloning efficiency II (absolute value) of the solvent controls should exceed 50 %. Cloning efficiency II is determined after the expression period to measure viability of the cells without selective ageny.

Evaluation of results:
- 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 were compared to the solvent control groups. A trend was 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:
cytotoxicity
Remarks:
Experiment I: 1115.0 µg/mL and above + S9-mix and at 557.5 µg/mL and above without S 9-mix. Experiment II: 900 µg/mL and above + S 9-mix and at 600 µg/mL and above - S-mix.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Cytotoxic effects:
The recommended cytotoxic range of approximately 10 - 20% relative cloning efficiency (determined immediately after treatment to measure toxicity) or relative cell density was covered with and without metabolic activation.

Mutation frequency:
The threshold of three times the mutation frequency of the corresponding solvent control was exceeded in the second culture of the first experiment with metabolic activation at 557.5 μg/mL. This isolated increase was judged as biologically irrelevant however, as it was not reproduced in the parallel culture under identical experimental conditions and there was no dose dependent increase as indicated by the lacking statistical significance.

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.

Test-species confounding factors:
- Effects of pH: none (pre-test)
- Effects of osmolarity: none (pre-test)
- Precipitation: precipitation occurred at 1115 (without S9-mix) and 2230 µg/mL (with and without S9-mix) following 4 hours treatment in main Experiment I.

Range-finding/screening studies:
In the pre-test, relevant cytotoxic effects were observed at 2230 µg/mL following 4 hours treatment with metabolic activation and at 1115 µg/mL and above following 24 hours treatment without metabolic activation.

Comparison with historical control data:
The numbers of mutant colonies per 10E6 cells for the vehicle and positive control substance compared favourable with the corresponding Laboratory´s historical control data (2009-2010).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (minimal essential medium)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no data
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Without S9-mix:
Experiment I (Preparation interval 18 hrs, Exposure period 4 hrs): 8.7, 17.4, 34.8, 69.7, 139.4, 278.8, 557.5, 1115.0, 2230.0 µg/mL; 139.4, 278.8 and 557.5 µg/mL were evaluated.
Experiment IIA (Preparation interval 18 hrs, Exposure period 18 hrs): 8.7, 17.4, 34.8, 69.7, 139.4, 278.8, 557.5, 1115.0, 2230.0 µg/mL; 139.4, 278.8 and 557.5 µg/mL were evaluated.
Experiment IIA (Preparation interval 28 hrs, Exposure period 28 hrs): 57.17, 34.8, 69.7, 139.7, 278.8, 557.5, 1115.0, 2230 µg/mL; 69.7, 139.4 and 278.8 µg/mL were evaluated.
Experiment IIB (Preparation interval 28 hours, Exposure period 28 hours): 100.0, 200.0, 250.0, 300.0, 325.0, 350.0, 400.0, 600.0 µg/mL; not evaluated, repeated due to the lack of cytotoxicity]
Experiment IIB (Preparation interval 28 hrs, Exposure period 28 hrs): 200.0, 400.0, 500.0, 600.0, 700.0, 750.0, 800.0, 850.0, 1000.0 µg/mL; 400.0, 500.0 and 600.0 µg/mL were evaluated.

With S9-mix:
Experiment I (Preparation interval 18 hrs, Exposure period 4 hrs): 8.7, 17.4, 34.8, 69.7, 139.4, 278.8, 557.5, 1115.0, 2230.0 µg/mL; 278.8, 557.5 and 1115.0 µg/mL were evaluated.
Experiment IIA (Preparation interval 28 hrs, Exposure period 4 hrs): 8.7, 17.4, 34.8, 69.7, 139.4, 278.8, 557.5, 1115.0, 2230.0 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: On the day of the experiment (immediately before treatment), the test item was dissolved in DMSO. The final concentration of DMSO in the culture medium was 0.5 % (v/v).
- Justification for choice of solvent/vehicle:
The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Remarks:
ethylmethanesulphonate (final concentration: 1000 µg/mL Exp. I and 600 µg/mL Exp. IIA & IIB) dissolved in nutrient medium; cyclophoshamide (final concentration 1.4 µg/mL Exp. I and 2.0 µg/mL Exp. IIA & Exp. IIB) dissolved in Saline (0.9% [w/v]).
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: approx. 48 hours for preparation interval 28 hours and approx. 54 hours for preparation interval 18 hours
- Exposure duration: without S9-mix: Exp. I 4 hrs, Exp. IIA 18 hrs and 28 hrs, Exp. IIB 28 hrs; with S9-mix: Exp. I 4 hrs, Exp. IIA 4 hrs.
- Expression time (cells in growth medium): Exp. I 14 hrs (in total 18 hrs), Exp. IIA 0 hrs, the medium was not changed until preparation of the cells (in total 18 hrs and 28 hrs), Exp. IIB 0 hrs, the medium was not changed until preparation of the cells (in total 28 hrs); with S9-mix: Exp. I 14 hrs (in total 18 hrs), Exp. IIA 24 hrs (in total 28 hrs)

SPINDLE INHIBITOR (cytogenetic assays): Colcemid was added to the culture medium (0.2 µg/mL) 2.5 hours prior to cell harvest.
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: In each experimental group two parallel cultures were set up.

NUMBER OF CELLS EVALUATED: The cell numbers were determined by counting 10 defined fields per coded slide. 100 well spread metaphases per culture were evaluated for cytogenetic damage on coded slides, except for the positive controls in Experiment IIA after 18 hours continuous treatment and in Experiment IIB after 28 hours continuous treatment without metabolic activation, where only 50 metaphases were evaluated.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index, reduced cell number
A pre-test on cell growth inhibition was performed in order to determine the toxicity of the test substance, the solubility during exposure and changes in osmolarity and pH value.

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes
Evaluation criteria:
ACCEPTABILITY OF THE TEST
a) The number of structural aberrations found in the solvent controls falls within the range of the laboratory historical control data.
b) The positive control substances produce significant increases in the number of cells with structural chromosome aberrations, which are within the range of the laboratory historical control data.

EVALUATION OF RESULTS
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 historical control data:
- 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 historical control data.
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criterion is valid:
A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of the laboratory historical control data.

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. If the criteria mentioned above for the test item were not clearly met, the classification with regard to the historical data and the biological relevance was discussed and/or a confirmatory experiment was performed.

Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No relevant influence on pH value was observed.
- Effects of osmolarity: No relevant influence on osmolarity was observed.
- Precipitation: In Experiment I, visible precipitation of the test item in the culture medium was observed at 2230.0 µg/mL in the absence of S9 mix and at 1115.0 µg/mL and above in the presence of S9 mix. No relevant influence on osmolarity or pH value was observed.

RANGE FINDING/SCREENING STUDIES
The preliminary test was designated Experiment I.

COMPARISON WITH HISTORICAL CONTROL DATA
The percentage of aberrant and polyploid cells were generally in the range of the Laboratory´s historical control data (2008-2009).

CYTOTOXICITY

- In Experiment I after 4 hours treatment and in Experiment IIA after 18 hours treatment in the absence of S9 mix and after 4 hours treatment in the presence of S9 mix cytotoxicity indicated as reduced cell number or mitotic index was observed at the highest evaluated concentration (42.2 %, 52.7 % (cell number) and 39.6 % (mitotic index), 49.3 % of control (cell number), respectively).

- In Experiment I in the presence of S9 mix no cytotoxicity was observed up to the highest applied concentration. In Experiment IIA after 28 hours treatment in the absence of S9 mix concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage.

- In Experiment IIB after 28 hours treatment in the absence of S9 mix clear cytotoxicity indicated as reduced mitotic index was observed at the highest evaluated concentration (27.5 % of control).

STRUCTURAL CHROMOSOME ABERRATIONS

In the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed.

The aberration rates of the cells after treatment with the test item (0.5 - 3.5 % aberrant cells, excluding gaps) slightly exceeded the range of the solvent control values (0.5 - 2.5 % aberrant cells, excluding gaps) but were within the range of the laboratory historical solvent control data. However, in Experiment I in the absence of S9 mix two statistically significant increases in aberrant cells were observed (3.5% aberrant cells, excluding gaps at 139.4 and 557.5 µg/mL). These values are in the range of the laboratory historical solvent controls and are therefore regarded as being biologically irrelevant.

POLYPLOID METAPHASES

No biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (0.6 - 3.8 %) as compared to the rates of the solvent controls (1.9 - 4.2 %).

ENDOMITOTIC METAPHASES

No biologically relevant increase in the rate of endomitotic metaphases was found after treatment with the test item (0.0 - 0.3 %) as compared to the rates of the solvent controls (0.0 - 0.1 %).

POSITIVE CONTROL

In both experiments, either EMS (600 or 1000.0 µg/mL) or CPA (1.4 or 2.0 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames

In a reverse gene mutation assay the bacteria strains TA 1535, TA 1537, TA 98 and TA 100 of S.TyphimuriumandEscherichia coli WP2 uvrA were exposed to diisopropanol-p-toluidin at concentrations of 20 µg - 5000 µg/plate in the precence and the absence of metabolic activation applying both the standard plate and the pre-incubation method (BASF AG, 1998). A weak bacteriotoxic effect was observed under all test conditions. An increase in the number of his+ or trp+ revertants was not observed in the standard plate test or the preincubation test either without or after the addition of a metabolizing system. According to the results of the present key study, the test substance diisopropanol-p-toluidin is not mutagenic in theS.Typhimurium/Escherichia colireverse mutation assay under the experimental conditions chosen here.

Dipropoxy-p-toluidine was further investigated using the Salmonella/microsome plate incorporation test for point mutagenic effects in doses of up to and including 5000 µg/plate (Bayer AG, 1997). The testedS.Typhimuriumstrains comprised TA 1535, TA 100, TA 1537, TA 98 and TA 102. Doses up to and including 1581 µg/plate did not cause any bacteriotoxic effects. At 5000 µg/plate, the substance had weak, strain-specific bacteriotoxic effect. Due to this effect this dose could only partly be used for assessment purpose. Evidence of mutagenic activity of dipropoxy-p-toluidine was not seen, supporting the results demonstrated in the previous key study.

In vitro mammalian cell gene mutation test (HPRT test)

Diisopropanol-p-toluidin was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster according to OECD guideline 476 (Harlan CCR GmbH, 2012). The assay was performed in two independent experiments with identical experimental procedures, using two parallel cultures each. The first experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment period of 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation. The cell cultures were evaluated at the following concentrations:

 

Experiment I:

without S9 mix: 69.7; 139.4; 278.8; 557.5 µg/mL

with S9 mix: 69.7; 139.4; 278.8; 557.5; 1115.0 µg/mL

Experiment II:

without S9 mix: 75.0; 150.0; 300.0; 600.0; 62.5; 900.0 µg/mL 

with S9 mix: 75.0; 150.0; 300.0; 600.0; 62.5; 900.0 µg/mL 

 

In the presence of metabolic activation test substance precipitation in culture medium at the end of treatment was observed in the first experiment at 11150 µg/mL only. Cytotoxic effects were observed in the first experiment at 1115.0 µg/mL and above with and at 557.5 μg/mL and above without metabolic activation. In the second experiment cytotoxic effects as described above were noted at 900 μg/mL and above with and at 600 μg/mL and above without metabolic activation. The recommended cytotoxic range of approximately 10 - 20% relative cloning efficiency or relative cell density was covered with and without metabolic activation. In this study, 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, 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. In conclusion, under the experimental conditions reported, the test item did not induce gene mutations at the HPRT locus in V79 cells and therefore, diisopropanol-p-toluidin, was considered to be non-mutagenic in this HPRT assay.

In vitro mammalian chromosome aberration test

In a chromosome aberration assay (Harlan CCR GmbH, 2012, OECD 473, GLP compliance) diisopropanol-p-toluidin was tested in Chinese Hamster V79 cells. The highest applied concentration (2230 µg/mL; approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the current guideline. Three independent experiments of the chromosome aberration assay were performed. In Experiment I the exposure period was 4 hours with and without S9 mix. In Experiment IIA the exposure period was 4 hours with S9 mix, 18 and 28 hours without S9 mix. In Experiment IIB the exposure period was 28 hours without S9 mix. The chromosomes were prepared 18 and 28 hours after start of treatment with the test item. In each experimental group two parallel cultures were set up. 100 metaphases per culture were evaluated for structural chromosome aberrations, except for the positive controls in Experiment IIA after 18 hours continuous treatment and in Experiment IIB after 28 hours continuous treatment without metabolic activation, where only 50 metaphases were evaluated. Dose selection for the cytogenetic experiments was performed considering the toxicity data and the occurrence of test item precipitation assessed in a pre-test.

In Experiment I after 4 hours treatment and in Experiment IIA after 18 hours treatment in the absence of S9 mix and 4 hours treatment in the presence of S9 mix cytotoxicity was observed at the highest evaluated concentration. In Experiment I in the presence of S9 mix no cytotoxicity was observed up to the highest applied concentration. In Experiment IIA after 28 hours treatment in the absence of S9 mix concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage. In Experiment IIB after 28 hours treatment in the absence of S9 mix clear cytotoxicity was observed at the highest evaluated concentration. No clastogenicity was observed at the concentrations evaluated either with or without metabolic activation. However, in Experiment I in the absence of S9 mix two statistically significant increases in aberrant cells were observed (3.5% aberrant cells, excluding gaps at 139.4 and 557.5 µg/mL). These values were in the range of the laboratory historical solvent controls and were therefore regarded as being biologically irrelevant. The positive controls induced statistically significant increases in cells with structural chromosome aberrations. In conclusion, under the experimental conditions reported, the test item did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line) in vitro. Diisopropanol-p-toluidin was considered to be non-clastogenic in this chromosome aberration test in the absence and presence of metabolic activation, when tested up to cytotoxic or precipitating or the highest evaluable concentrations.

Justification for classification or non-classification

Based on the available results diisopropanol-p-toluidin was not classified and labelled for genotoxicity according to Regulation (EC) No. 1272/2008 (CLP).