N,N''-(4-methyl-m-phenylene)bis[N',N'-dimethylurea]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The mutagenic potential of TDI-Urone was assessed in a standard battery of genotoxicity tests according to REACH Annex VII/VIII. All required guideline studies (OECD 471 (AMES), OECD 473 (CA), OECD 476 (HPRT) were performed.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001-04-04 to 2001-05-07

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 (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): N,N"(4-Methyl-m-phenylen)bis(N',N'­dimethylharnstoff)
- Substance type: organic
- Physical state: colourless solid
- Analytical purity: 100 %
- Purity test date: 01 March 2000
- Lot/batch No.: 0232 07
- Expiration date of the lot/batch: March 2003
- Storage condition of test material: store dry in closed containers
- Other: stable at dry storage conditions for at least 2 years

Target gene:

Gene involved in histidine synthesis.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

n.a.

Additional strain / cell type characteristics:

other: deep rough (rfa) mutation; uvrB gene deletion (BER)

Metabolic activation:

with and without

Metabolic activation system:

Mammalian Microsomal Fraction S9 Mix

Test concentrations with justification for top dose:

Experiments I and II: 31.6; 100.0; 316.2; 1000.0; 2500.0 and 5000.0 µg per plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:The test item was dissolved in DMSO and diluted prior to treatment. The solvent was compatible with the survival of the bacteria and the S9 activity.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-NOPD

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-AA

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation;

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency;

Evaluation criteria:

The mutation factor is calculated by dividing the mean value of the revertant count through the mean values of the solvent control.

A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to crystal violet and ampicillin
- the control plates without S9 mix are within the following ranges (range of spontaneaus reversion frequencies-historical control- data range):
TA 1535: 5-30
TA 1537: 4-32
TA 98: 18-63
TA 100: 79-197
TA 102: 173-396
-corresponding background growth on both negative control and test plates is observed.
- the positive controls show a distinct enhancement over the control plate.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

Interpretation of results:
negative

TDI-Urone has no mutagenic potential under the conditions of this study.

Executive summary:

The test item TDI-Urone was investigated 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 100, TA 98 and TA 102.

In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation (S9 mix). The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:

Experiment I and II: 31.6; 100.0; 316.2; 1000.0; 2500.0 and 5000.0 µg//plate

No toxic effects of the test item (indicated by a reduction of the background lawn or by a reduction of the spontaneaus rate) were observed in both experiments with or without metabolic activation.

No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with TDI-Urone at any concentration level, either in the presence or absence of metabolic activation (S9-mix) in experiment I and II. There was also no tendency of higher mutation rates with increasing concentrations in the range beyond the generally acknowledged border of biological relevance.

Therefore, TDI-Urone is not considered to be mutagenic in bacterial cells.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-03-22 until 2012-06-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Principles of method if other than guideline:

first experiment 4 hours treatment with and without metabolic activation
second experiment 24 hours treatment without metabolic activation, 4 hours treatment with metabolic activation

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

Identity: TDI-Urone
CAS Number: 17526-94-2
Batch No.: 126815
Molecular Weight: 264.33 g/mol
Purity: >/= 95 %, dose calculation adjusted to purity
Physical State: White, solid
Storage: At room temperature
Stability in Solvent: Stable
Solubility in water (20°C): 20 g/L
Expiration Date: June 01, 2013
On the day of the experiment (immediately before treatment), the test item was dissolved in deionised water. The solvent was chosen according to its solubility properties and its non-toxicity to the cells. The final concentration of deionised water in the culture medium was 10% v/v.

Target gene:

HPRT (hypoxanthine-guanine phosphoribosyl transferase)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-Naphtoflavone induced Rat liver S9

Test concentrations with justification for top dose:

Experiment I (4 hours treatment):
without metabolic activation: 87.5; 175; 350; 700; 1400; 2800 µg/mL
with metabolic activation: 87.5; 175; 350; 700; 1400; 2800 µg/mL
Experiment II (24 hours treatment):
without metabolic activation: 87.5; 175; 350; 700; 1400; 2800 µg/mL
Experiment II (4 hours treatment)
with metabolic activation. 87.5; 175; 350; 700; 1400; 2800 µg/mL
The cultures at the lowest concentration of 87.5 µg/mL were not analysed as a minimum of only four concentrations are required.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation, Experiment II: 24 hours without metabolic activation, 4 hours with metabolic activation
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): 6-Thioguanine

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: >1,5x10exp. 6

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces reproducibly with one of the concentrations a mutation frequency that is three times higher than the spontaneous mutation frequency 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.
In a case by case evaluation this decision depends on the level of the corresponding solvent control data.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT (R) 11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for the groups treated with the test item 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 relevance and statistical significance were considered together.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not effected (pH 7.37 measured in the solvent control and at 2800 µg/mL)
- Effects of osmolality: No relevant increase (282 measured in the solvent control versus 293 measured at 2800 µg/mL)
- Evaporation from medium: Not examined
- Water solubility: Soluble
- Precipitation: No
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES:
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). The pre-experiment was prematurely terminated due to microbial contamination. The experiment was repeated as pre-experiment 2. The data of pre-experiment 2 are reported as pre-experiment.

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.

In the range finding pre-experiment test item concentrations between 21.9 and 2800 µg/mL (≈10 mM) were used to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation.No relevant toxic effects were observed up to the maximum concentration with and without metabolic activation. The test medium was checked for precipitation or phase separation at the end of each treat-ment period (4 or 24 hours) prior to removal to the test item. Neither precipitation nor phase separation occurred up to the maximum concentration with and without metabolic activation.
There was no relevant shift of pH and osmolarity of the medium even at the maximum con-centration of the test item.
Based on the results of the pre-experiment, the individual concentrations of the main experi-ments were selected. The maximum concentration was again, 2800 µg/mL approximately equal to 10 mM. The individual concentrations were spaced by a factor of 2.


COMPARISON WITH HISTORICAL CONTROL DATA: Complies

ADDITIONAL INFORMATION ON CYTOTOXICITY: None

Remarks on result:

other:

Summary Table

				relative	relative	relative	mutant		relative	relative	relative	mutant
	conc.	P/	S9	cloning	cell	cloning	colonies/	induction	cloning	cell	cloning	colonies/	induction
	µg/mL	PS	mix	efficiency I	density	efficiency II	10^6cells	factor	efficiency I	density	efficiency II	10^6cells	factor
				%	%	%			%	%	%
Column	1	2	3	4	5	6	7	8	9	10	11	12	13
Experiment I / 4 h treatment				culture I					culture II
Solvent control			-	100.0	100.0	100.0	7.1	1.0	100.0	100.0	100.0	3.9	1.0
Positive control (EMS)	150.0		-	98.5	100.2	87.6	118.9	16.7	98.6	97.6	94.6	106.5	27.4
Test item	87.5		-	98.2	culture was not continued^#				97.4	culture was not continued^#
Test item	175.0		-	98.2	82.8	96.2	12.9	1.8	100.3	97.7	98.4	5.2	1.3
Test item	350.0		-	95.7	99.0	87.6	6.5	0.9	95.0	100.6	93.1	5.5	1.4
Test item	700.0		-	99.9	95.2	100.2	17.9	2.5	100.4	102.7	108.7	7.4	1.9
Test item	1400.0		-	99.5	102.0	98.2	10.4	1.5	97.7	100.4	112.3	2.9	0.7
Test item	2800.0		-	100.9	84.1	104.5	15.7	2.2	100.8	76.9	99.5	7.5	1.9
Solvent control			+	100.0	100.0	100.0	9.3	1.0	100.0	100.0	100.0	6.2	1.0
Positive control (DMBA)	1.1		+	31.7	47.4	91.8	649.9	69.6	27.1	68.8	64.3	489.9	78.5
Test item	87.5		+	95.5	culture was not continued^#				96.1	culture was not continued^#
Test item	175.0		+	96.0	88.0	99.9	13.3	1.4	95.1	93.1	75.6	3.8	0.6
Test item	350.0		+	96.2	89.9	98.4	6.6	0.7	92.8	113.2	81.8	3.4	0.5
Test item	700.0		+	92.5	73.6	112.8	7.5	0.8	95.7	89.3	82.5	5.4	0.9
Test item	1400.0		+	95.8	92.4	110.7	9.9	1.1	94.7	93.1	84.2	5.7	0.9
Test item	2800.0		+	92.8	82.0	100.5	19.5	2.1	94.2	83.2	80.6	1.5	0.2
Experiment II / 24 h treatment				culture I					culture II
Solvent control			-	100.0	100.0	100.0	11.8	1.0	100.0	100.0	100.0	19.5	1.0
Positive control (EMS)	150.0		-	101.5	84.3	81.0	342.3	29.0	91.1	87.1	119.2	448.4	23.0
Test item	87.5		-	100.1	culture was not continued^#				91.4	culture was not continued^#
Test item	175.0		-	99.5	76.8	73.7	14.2	1.2	97.6	105.3	86.2	23.9	1.2
Test item	350.0		-	102.5	77.6	93.2	26.4	2.2	92.5	97.6	110.7	10.4	0.5
Test item	700.0		-	98.6	77.5	90.6	14.0	1.2	86.8	91.9	116.5	10.3	0.5
Test item	1400.0		-	92.6	80.1	88.4	6.1	0.5	90.4	91.6	99.2	27.2	1.4
Test item	2800.0		-	95.4	65.3	87.8	15.1	1.3	102.9	83.0	113.1	15.1	0.8
Experiment II / 4 h treatment
Solvent control			+	100.0	100.0	100.0	11.5	1.0	100.0	100.0	100.0	16.7	1.0
Positive control (DMBA)	1.1		+	29.4	66.7	85.8	899.2	78.2	27.5	51.4	89.0	991.1	59.5
Test item	87.5		+	100.4	culture was not continued^#				106.0	culture was not continued^#
Test item	175.0		+	99.7	121.4	122.0	11.9	1.0	105.4	65.6	114.7	16.4	1.0
Test item	350.0		+	104.1	145.4	112.4	16.1	1.4	102.3	85.0	116.4	23.7	1.4
Test item	700.0		+	101.0	98.5	116.8	11.1	1.0	98.9	106.9	116.2	15.6	0.9
Test item	1400.0		+	94.5	98.2	124.2	26.3	2.3	101.5	106.9	106.8	14.6	0.9
Test item	2800.0		+	95.7	83.2	104.3	21.4	1.9	92.8	111.2	112.8	16.2	1.0

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

P/PS = precipitation/phase separation

Conclusions:

The test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, TDI-Urone is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The test item TDI-Urone 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 second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The maximum concentration of the test item in the main experiments equals a molar concentration of approximately 10 mM, with respect to the purity of the test item (95 %).

No precipitation of the test item was observed up to the maximum concentration with and without metabolic activation.

No relevant cytotoxic effect occurred up to the maximum concentration of 2800 µg/mL with and without metabolic activation.

No relevant and reproducible increase in mutant colony numbers/10⁶cells 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 did not reach or exceed the threshold of three times the mutation frequency of the corresponding solvent control.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT^â11 statistics software. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 3.8 up to 23.6 mutant colonies per 10⁶cells; the range of the groups treated with the test item was from 1.5 up to 27.2 mutant colonies per 10⁶cells.

EMS (150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-12-01 to 2012-01-18

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Name of test material (as cited in study report): TDI urone
- Substance type: organic
- Physical state: solid
- Analytical purity: >= 95%;
- Purity test date:
- Lot/batch No.: 126815
- Expiration date of the lot/batch: June 01, 2012
- Stability under test conditions: stable in water
- Storage condition of test material: at room temperature

Target gene:

n.a.

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) containing Hank´s salts, glutamine and Hepes (25 mM)
- 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 specified

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

With respect to the molecular weight and the purity (95 %) of the test item, 2782.0 µg/mL of TDI-Urone was applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations between 10.9 and 2782.0 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity.
Test concentrations: 10.9, 21.7, 43.5, 86.9, 173.9, 347.8, 695.5, 1391.0, 2782.0 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionised water
- 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:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;

DURATION
- Preincubation period: no preincubation
- Exposure duration: Experiment I 4 hours; experiment II: with S9: 4 hours; without S9: 18 hours
- Expression time (cells in growth medium): 18 hours
- Selection time (if incubation with a selection agent): n.a.
- Fixation time (start of exposure up to fixation or harvest of cells): 15.5 hours

SELECTION AGENT (mutation assays): n.a.
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): no stain

NUMBER OF REPLICATIONS: 1.5 replications

NUMBER OF CELLS EVALUATED: 100 metaphases per culture are eveluated for structural chromosomal aberrations

DETERMINATION OF CYTOTOXICITY: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: the number of polyploid cells in 500 metaphases per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype)
- Determination of endoreplication: the number of endomitotic cells evaluated at the evaluation of polyploid cells was noticed and reported (% endomitotic metaphases)

Evaluation criteria:

A test item is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of the laboratory historical control data and
- 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 and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

Statistics:

Statistical significance was confirmed by means of the Fisher’s exact test (p < 0.05).

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: At the selected doses (maximum 2782.0 µg/ml) no influence on solubility, pH value, or osmolarity was detected.
- Evaporation from medium: no
- Water solubility: the test item is watewr soluble
- Precipitation: No precipitation of the test item was observed
- Other confounding effects: not reported

RANGE-FINDING/SCREENING STUDIES: A pre-test on cell growth inhibition was performed in order to determine the toxicity of the test item,the solubility during exposure and changes in osmolarity and pH value at experimental conditions. At the selected dose no influence on solubility, pH value, or osmolarity was detected.The experimental conditions in this pre-experiment were identical to those required and described below for the mutagenicity assay.

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: the test item is not cytotoxic

Conclusions:

TDI-Urone did not induce structural chromosomal aberrations in V79 cells of the Chinese hamster in vitro, when tested up to the highest required concentration under conditions of this study.

Executive summary:

In a chromosome aberration assay V79 Chinese hamster cells cultured in vitro were exposed to TDI-Urone, which was dissolved in deionized water, at concentrations of 10.9, 21.7, 43.5, 86.9, 173.9, 347.8, 695.5, 1391.0, 2782.0 µg/ml in the presence and absence of mammalian metabolic activation (S9).

The highest treatment concentration in this study, 2782.0 µg/mL (approx. 10 mM) was chosen with regard to the molecular weight and the purity (95 %) of the test item and with respect to the OECD Guideline for in vitro mammalian cytogenetic tests. The positive controls did induce the appropriate response. There was no evidence of induced structural chromosomal aberrations in comparison to the controls. In both experiments, 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 - 3.0 % aberrant cells, excluding gaps) were slightly above the range of the solvent control values (1.0 - 2.5 % aberrant cells, excluding gaps) and within the range of the laboratory historical solvent control data.

This study is classified as acceptable and satisfies the requirement for Test Guideline OECD 473 for in vitro cytogenicity study in mammalian cells. Therefore, TDI-Urone is not considered to be clastogenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

TDI-Urone was tested negative for genotoxic effects in GLP guideline studies according to OECD 471 (AMES), OECD 473 (CA) and OECD 476 (HPRT). Concentrations up to the limit dose were used. No cytotoxic effects were observed.

Justification for selection of genetic toxicity endpoint
Last assay in a series of gentox studies. All result negative.

Justification for classification or non-classification

TDI-Urone was tested negative for genotoxic effects in GLP guideline studies according to OECD 471 (AMES), OECD 473 (CA) and OECD 476 (HPRT).