1,1'-(methylenedi-4,1-phenylene)bis(3-butylurea)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

HAT ISO was tested for genetic toxicity in three in vitro tests. None of these tests showed evidence for mutagenic activity. In summary, it can be concluded that HAT ISO has no mutagenic properties.

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:

1993-07-12 to 1994-01-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Analytical purity: >= 99.65 %
Lot/batch No.: 93.166

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- => his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA 1535, TA 100) and frameshift (TA 1537, TA 98, TA 1538) mutations.

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain / cell type:

S. typhimurium TA 1537

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 8 ... 5000 µg/plate
Concentration range in the main test (without metabolic activation): 8 ... 5000 µg/plate

Vehicle / solvent:

Solvent: Dimethylsulphoxyde (DMSO)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA98, TA1537 without S9-mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2-A)

Remarks:

TA98, TA100, TA1535, TA1537, TA1538 with S9-mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA100 without S9-mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

TA1535 without S9-mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537 without S9-mix

Details on test system and experimental conditions:

Preliminary study:
A preliminary study is performed on one bacterial strain TA100 (or TA98), without metabolic activation, in order to evaluate the toxicity of the test article to bacteria.
Increasing concentrations of the test article are used; examples for which are as follows: 8 - 40 - 200 - 1000 - 5000 mg/plate. The highest dose is 5000 µg/plate if the formulation type allows this dose level to be reached. A minimum of 5 dose levels are used.
Each concentration is tested in duplicate. At the end of the incubation period, the plates are observed for signs of toxicity. The colonies which appears in the presence of the test article are counted and the intensity of the bacterial lawn examined. These observations are compared with those performed in the presence of the vehicle. The signs of toxicity (reduction in bacterial lawn or reduction of the number of colonies) are noted.
Main study:
Two studies are conducted, the second study being performed to confirm or to complement the results of the first one for example by using a closer range near to the top limit dose.
For each study, each concentration of the test article is tested in triplicate with and without metabolic activation on each bacterial strain.
Concentration of the test substance resulting in precipitation: 1000 µg/plate

Evaluation criteria:

The test article will be considered to be clearly mutagenic if:
- The assay is valid
- The number of revertants in presence of the test article is greater than or equal to twice the number of revertants in the negative control at one dose level (the upper dose before toxicity) or more with a dose correlation
- The positive trends/effects are reproducible.

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

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

Positive controls validity:

valid

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

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

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

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:

NA

The test article PATE HAT was tested on 5 strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537, TA1538) with and without metabolic activation. A range of sub-toxic concentrations was determined in a preliminary study on the strain TA100 without metabolic activation.

Five concentrations (5000, 1000, 200, 40 and 8 µg/plate) were tested in triplicate on the strains mentioned above with and without metabolic activation. The results were confirmed in a second independent study, using a closer range near the top of limit dose (5000, 2500, 1250, 625 and 313 µg/plate). A negative control (vehicle) and a positive control (specific standard mutagen) were included in each study.

Conclusion: Under the experimental conditions employed, it can be concluded that the test article PATE HAT did not cause an increase in the number of revertants per plate of any of the tester strains used, either in presence or absence of a metabolic activation system in both of the two independent studies performed

Conclusions:

HAT ISO was tested for mutagenic activity in the Ames-test with the bacterial strains S. typhimurium TA98, TA100,TA1535, TA1537, TA1538 with and without metabolic activation in two independent studies. No increase of the number of revertants per plate was observed. No classification and labelling for genetic toxicity is required according to Regulation 1272/2008/EC (CLP).

Executive summary:

PATE HAT was tested on 5 strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537, TA1538) with and without metabolic activation.

Five concentrations (5000, 1000, 200, 40 and 8 µg/plate) were tested in triplicate on the strains mentioned above with and without metabolic activation. The results were confirmed in a second independent study, using a closer range near the top of limit dose (5000, 2500, 1250, 625 and 313 µg/plate). A negative control (vehicle) and a positive control (specific standard mutagen) were included in each study.

PATE HAT did not cause an increase in the number of revertants per plate of any of the tester strains used, either in presence or absence of a metabolic activation system in both of the two independent studies performed

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1993-07-12 to 1994-04-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1983

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Analytical purity: >= 99.65 %
Lot/batch No.: 93.166

Target gene:

To evaluate the clastogenic potential of a test article by its effects on the chromosomes of cultured peripheral blood lymphocytes treated in the absence and in the presence of a metabolising system.

Species / strain / cell type:

mammalian cell line, other: human lymphocytes

Details on mammalian cell type (if applicable):

The lymphocytes are obtained from a human donor not suspected of any virus infection nor exposed to high levels of radiation or hazardous chemicals.
One donor will be used in each experiment.
A appropriate volume of whole blood will be drawn from the peripheral circulation into heparinised tubes, and if not used within the day, may be stored (refrigerated), for up to 2 days prior to establishing cultures

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 27 ... 1600 µg/mlL
Concentration range in the main test (without metabolic activation): 27 ... 1600 µg/mL

Vehicle / solvent:

Dimethylsulphoxyde (DMSO)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

with and without S9-mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with and without S9-mix

Details on test system and experimental conditions:

Exposure period (with metabolic activation): 3 hours
Exposure period (without metabolic activation): 48 hours

Selection time:
24 and 48 hours

Fixation time:
2 hours with colcemid.

Evaluation criteria:

A test substance will be considered clearly positive in this assay if:
1) statistically significant increases in the proportion of cells with structural aberrations (excluding gaps) occur at one or more concentrations
2) the incidence of aberrations at such data points exceeds the normal range.

Species / strain:

mammalian cell line, other: human lymphocytes

Metabolic activation:

with

Genotoxicity:

not specified

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 µg/mlL

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

mammalian cell line, other: human lymphocytes

Metabolic activation:

without

Genotoxicity:

not specified

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 µg/mL

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

mammalian cell line, other: human lymphocytes

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1600 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

mammalian cell line, other: human lymphocytes

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1600 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: other: preliminary test

The test article PATE HAT was tested in an in vitro-cytogenetic assay using duplicate human lymphocytes cultures from two donors in 2 independent experiments.

 

The test article was dissolved in dimethylsulfoxide (DMSO). Concentrations of 50 to 5000 μg/ml were tested in range-finding assay with and without metabolic activation. Under nonactivation conditions ( - S₉), severe toxicity (mitotic inhibition of 74 %) was observed in the culture

treated with 5000 μg/ml and a reduction of 30 % as compared with the solvent control culture was observed in the culture treated with 1600 μg/ml. Under activation conditions (+S₉), severe toxicity (mitotic inhibition of 76 %) was noted in the culture treated at 5000 μg/ml and a reduction of 57 % as compared with the solvent control culture was observed in the culture treated at 1600 μg/ml.

 After incorporation of dosing solutions, a precipitate was observed in the culture medium at the dose levels of 500, 1600 and 5000 μg/ml.

 

Experiment 1:

Based on these data, replicate cultures were incubated with different concentrations of the test article for about 24 or 48 hours in the assay without and with S₉. Cultures were harvested about 24 or 48 hours after the initiation of dosing. In the first instance, chromosome aberrations were analysed in the 24 hour assays for the cultures treated at the three highest dose levels that could be analysed according to the mitotic index reduction (M.I. red). In the absence of significant effects (NS) in the 24 hour assay, chromosome aberrations were analysed in the 48 hour assays for the highest dose level.

 

The dose level of 1600 μg/ml was not selected in the first instance in experiment 1 because of the presence of precipitate on the slides observed in the preliminary study.

 

24 hour sampling time

 

- S9	Dose level (μg/ml M.I. red (%) Significance (p <=)^(1)		27	49	88	157	280	500
			23	42	19	16	44	38
						NS	NS	NS

 

+ S9	Dose level (μg/ml M.I. red (%) Significance (p <=)^(1)		27	49	88	157	280	500
			7	21	13	9	2	10
						NS	NS	NS

 

48 hour sampling time

 

- S9	Dose level (μg/mL M.I. red (%) Significance (p <=)^(1)		88	157	280	500
			36	18	30	42
						NS

 

- S9	Dose level (μg/mL M.I. red (%) Significance (p <=)^(1)		88	157	280	500
			0	0	0	18
						NS

 

⁽¹⁾_ statistical significance of the structural chromosome aberrations increase, NS : not statistically significant (p > 0.05).

 

After incorporation of the test article, a precipitate was noted in the culture medium at 157, 281 and 500 μg/ml. This precipitate was also noted on the slides at 500 μg/ml in the absence of S₉.

 

Experiment 2: independent study performed with a closer and higher range of dose levels including 1600 μg/ml.

 

24 hour sampling time

 

- S9	Dose level (μg/mL M.I. red (%) Significance (p <=)^(1)		157	280	500	896	1600
			10	0	17	24	38
				NS	NS	NS

 

+ S9	Dose level (μg/mL M.I. red (%) Significance (p <=)^(1)		157	280	500	896	1600
			18	15	30	20	51
				NS	NS	NS

 

48 hour sampling time

 

- S9	Dose level (μg/mL M.I. red (%) Significance (p <=)^(1)		500	896	1600
			36	16	48
				NS

 

+ S9	Dose level (μg/mL M.I. red (%) Significance (p <=)^(1)		500	896	1600
			0	0	15
				NS

 

After the incorporation of the test article, a precipitate was noted in the culture medium with all tested dose levels.

This precipitate was also observed on the slides at 500, 896 and 1600 μg/ml in the absence of S₉ and at 896 and 1600 μg/mL in the presence of S₉.

 

Appropriate negative (solvent) control cultures were included in the test system in both experiments at both sampling times. The proportions of cells with structural aberrations in these cultures were within usual solvent control ranges.

 

4-Nitroquinoline 1-oxide (NQO) and cyclophosphamide (CP) were employed as positive control chemicals in the absence and presence of S₉ respectively. Cells receiving the positive controls were sampled in each experiment 24 hours after the start of treatment ; both compounds induced statistically significant increases in the proportion of cells with structural aberrations.

 

All criteria for a valid study were met in both experiments.

 

No significant increase in the number of cells with structural chromosome aberrations was noted in the absence or presence of S₉in both experiments at both sampling times (24 and 48 hours).

 

No abnormal values in the number of numerical aberrations were observed in all cultures in the presence of the test article.

Conclusions:

PATE HAT was tested for genetic toxicity in a chromosome aberration test in culture human peripheral blood lymphocytes. The test article did not induce structural chromosome aberrations when tested to the highest practicable concentrations with and without metabolic activation. No classification and labelling for genetic toxicity is required according to Regulation 1272/2008/EEC (CLP).

Executive summary:

PATE HAT was tested in an in vitro cytogenetic assay using duplicate human lymphocytes cultures from two donors in 2 independent experiments. The test article was dissolved in dimethylsulfoxide (DMSO). Concentrations of 50 to 5000 mg/mL were tested with and without metabolic activation.

All criteria for a valid study were met in both experiments.

No significant increase in the number of cells wit structural chromosome aberrations was noted in the absence or presence of S9 -mix in both experiments and both sampling times (24 and 48 hours). No abnormal values in the number of numerical aberrations were observed in all cultures in the presence of the test article.

PATE HAT did not induce structural chromosome aberrations in cultured human peripheral blood lymphocytes when tested to its highest practicable concentrations in both the absence or presence of a metabolic activation system.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

199-11-08 to 2000-02-07

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

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

Lot/batch No.: 99-3117

Target gene:

TK Locus in L5178Y Mouse Lymphoma Cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 15.6; 31.3, 62.5; 125 µg/mlL
Concentration range in the main test (without metabolic activation): 15.6; 31.3; 62.5; 125 µg/mL

Vehicle / solvent:

Dimethylsulfoxyde (DMSO)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9-mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9-mix

Details on test system and experimental conditions:

Exposure period (with metabolic activation): 3 hours
Exposure period (without metabolic activation): 24 hours

Expression time:
2 days after treatment

Selection time:
24 and 48 hours

Fixation time:
---

Evaluation criteria:

A compound is considered as mutagenic in this test system if the following conditions are fulfilled:
1) A multiplication by two of the number of spontaneous mutants has to be provoked by at least one dose.
2) A dose/effect relationship that is an increase in the number of mutants with the dose, must be observed, and does not necessarily imply a proportional increase (in the case of positive responses, a decrease in the number of mutants at the highest dose, is frequently observed linked with cytotoxicity).
3) The results have to be reproducible in an independent study, at least from a qualitative point of view.
None of the three criteria mentioned above must be fulfilled for a compound to be considered as non-mutagenic in this system of study.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

not specified

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

125 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

not specified

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

125 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

125 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

125 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Both in two independent assays using 3-hour and 24-hour treatments without metabolic activation and in two separate or combined assays with metabolic activation, no significant increase in the frequency of total induced mutants (small and large colonies ) was noted at any dose tested in the presence of HAT Paste.

Remarks on result:

other: other: preliminary test

Cytotoxic activity by determining % RTG (Relative total growth)

Doses μg/mL		0	7.8	15.6	31.3	62.5	125
Without S9 mix	3 h treatment	100.0	71.2	78.1	100.0	122.4	89.7
	24 h treatment	100.0	131.6	78.4	113.9	119.0	123.8
With S9 mix	3 h treatment	100.0	99.2	105.6	90.2	119.0	105.8

The investigation of a mutagenic activity of the component HAT Paste dried was tested in a gene mutation test at the TK locus in L5178 mouse lymphoma cell culture. The test was performed at the maximum concentration compatible with its solubility, i.e. 125 µg/mL, with and without metabolic activation, as well as three doses below this concentration. In parallel, tests were carried out with reference mutagenic compounds. These assays demonstrated the sensitivity of the studied cell strain to this type of mutation and the efficiency of the metabolic activation system.

Both in two independent assays using 3-hour and 24-hour treatments without metabolic activation and in two separate or combined assays with metabolic activation, no significant increase in the frequency of total induced mutants (small and large colonies ) was noted at any dose tested in the presence of HAT Paste dried (see tables).

Mutagenic activity:

		Mutation frequency x 10^-6cells
Doses μg/ml		0	15.6	31.3	62.5	125	MMS *
Without S9	ASSAY 1 3 h	149.0	117.9	146.4	148.0	119.6	586.0
	ASSAY 2 24 h	84.9	110.3	90.3	97.5	77.4	378.7

* 10 μg/ml (3 h treatment), 2 μg/ml (24 h treatment)

 

		Mutation frequency x 10^-6cells
Doses μg/ml		0	15.6	31.3	62.5	125	CPA 2 μg/ml
With S9	ASSAY 1	127.4	100.5	94.9	108.2	99.5	957.1
	ASSAY 2	122.5	157.2	125.6	128.9	113.5	584.6

 

Conclusions:

HAT Paste dried was tested for genetic toxicity in the mouse lymphoma cell culture assay. The test article showed no mutagenic activity with and without metabolic activation. Hat Paste dried needs no classification and labelling for genetic toxicity according to Regulation 1272/2008/EC (CLP).

Executive summary:

The investigation of mutagenic activity of the component HAT Paste dried was tested in a gene mutation test at the TK locus in L5178 mouse lymphoma cell culture.

Both in two independent assays using 3-hour and 24-hour treatments without metabolic activation and in two separate or combined assays with metabolic activation, no significant increase in the frequency of total induced mutants (small and large colonies ) was noted at any dose tested in the presence of HAT Paste dried.

HAT Paste dried did not induce any mutagenic activity both with and without metabolic activation in this test. No classification for genetic toxicity is required.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

HAT ISO was tested for genetic toxicity in one in vivo test. Results of this test showed no evidence for mutagenic activity. In summary, it can be concluded that HAT ISO has no mutagenic properties.

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

Study period:

2008-09-02 to 2009-05-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

Analytical purity: >= 99.4 %
Lot/batch No.: 05-12-06

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River; Saint-Germain-sur-l'Arbresle, France;
- Age at study initiation: 5 to 6 weeks;
- Weight at study initiation: males: 150 to 180 g; females: 150 to 187 g;
- Assigned to test groups randomly: yes;
- Fasting period before study: animals were not fasted before study begin;
- Housing: in polypropylene cages, in groups of 2 or 3; by random distribution;
- Diet: No. A04C10 irradiated rat/mouse feed; Supplier: SAFE;
- Water: drinking water softened, treated by osmosis and filtered over 0.2 µm membrane filter; ad libitum;
- Acclimation period: at least 5 days;


ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 3 °C;
- Humidity: 55 +/- 15 % R.H.;
- Air changes: 20 air changes per hour;
- Photoperiod: 12 hours light/12 hours dark from 8:00 a.m. to 8:00 p.m.;

Route of administration:

subcutaneous

Vehicle:

corn oil

Details on exposure:

Limit test: N

Duration of treatment / exposure:

2 successive administrations at 24-hour intervals;

Frequency of treatment:

2 times;

Post exposure period:

24 hours

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

2000 mg/kg bw: 5 males, 5 females;
1000 mg/kg bw: 5 males, 5 females;
500 mg/kg bw: 5 males, 5 females;

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide;
25 mg/kg bw/day;

Tissues and cell types examined:

Bone marrow;
immature (polychromated) erythrocytes;
micronucleated immature erythrocytes;
immature among total erythrocytes;

Details of tissue and slide preparation:

Bone marrow was extracted with foetal calf serum. Cell supensions were centrifugated for 5 minutes at 1000 rpm. Centrifugate was spread on slides. Smears were stained to make possible to distinguish between polychromatic (PCE) and normochromatic (NCE) erythrocytes. PCE are purple whereas NCE are red.

Evaluation criteria:

The mean number of micronuclei observed in the negative control animals must be within the range of the historical values for control animals. The mean number of micronuclei observed in the positive control animals treated with cyclophosphamide must show a statistically significant increase from the negative control animals and higher than the minimal historical value for positive animals. If deaths are observed at the tested dose, the mortality rate must be less than 20 % per group. The dead animals are replaced by those in parallel treatment.

Statistics:

The statistical comparison for the proportion of polychromatic among total erythrocytes and for the weight homogeneity within the sex of each group was performed using the Student's test. Statistical analysis was performed for micronucleus number using a non-parametric test, the Mann Whitney U rank test. An analysis of a large number of control results has shown that the distribution of the numbers of micronuclei does not correspond to a Gaussian distribution, but to a Poisson-type distribution.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

GENOTOXICITY ASSAY
Number of sampling times :
one: for negative control and treated groups: 24 hours after the second treatment
one: for positive control group: 24 hours after single treatment

Reference substance :
cyclophosphamide, 25 mg/kg bw, intraperitoneal administration;

Number of polychromatic erythrocytes observed for each animal :
2000 for micronuclei ;
1000 for fraction of PCE among total erythrocytes (PCE+NCE).

No statistically significant decrease in the fraction of PCE among total erythrocytes (PCE+NCE) was observed (this finding provides no evidence that bone marrow cell exposure has occurred). No statistically significant increase in the number of micronuclei was noted at the doses of 2000, 1000 or 500 mg/kg bw/day (x2) in male or in female rats.

SAMPLING TIME (24 H after last treatment)	TEST ITEM   DOSES in mg/kg bw/day (x2)	SEX	Fraction of PCE* (%)		MICRONUCLEI FOR 1000 PCE
			Mean	Student’s t Test (p)	Mean	Mann Whitney (p)
Negative control Group	VEHICLE 10 mL/kg bw	M F M + F	62.9 54.2 58.5		0.70 0.60 0.65
Positive control Group	Cyclosphosphamide 25 mg/kg bw/day (x1)	M F M + F	44.4 37.8 41.1	<0.01 <0.001 <0.001	9.60 8.90 9.25	P<0.01 P<0.01 P<0.001
HAT ISO     TREATED     GROUPS	2000	M F M + F	63.3 48.0 55.7	N.S. N.S. N.S.	0.40 0.80 0.60	N.S. N.S. N.S.
	1000	M F M + F	58.7 53.8 56.3	N.S. N.S. N.S.	0.70 0.60 0.65	N.S. N.S. N.S.
	500	M F M + F	55.1 53.7 54.4	N.S. N.S. N.S.	0.60 0.50 0.55	N.S. N.S. N.S.

N.S.: Non significant at the threshold of p=0.05

PCE: Polychromatic erythocytes

NCE: Normochromatic erythocytes

*Fraction of PCE among total erythrocytes = 100xPCE /(PCE+NCE)

Conclusions:

HAT ISO was tested for mutagenic activity in the in vivo Micronucleus test in rats. Under the experimental conditions of this test, HAT ISO induced no genotoxic activity. No classification and labelling for mutagenic activity is required according to Regulation 1272/2008/EC (CLP).

Executive summary:

HAT-ISO was investigated by means of the in vivo micronucleus test, in male and female OFA Sprague Dawley rats. Animals were treated by subcutaneous route twice with 2000, 1000 or 500 mg/kg bw/day (x2). Treatment was followed by one sampling time 24 hours after the last treatment. In conclusion, HAT-ISO induced no genotoxic activity under these experimental conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The genetic toxicity of HAT ISO was tested in three in vitro tests and one in vivo test. The in vitro tests were a bacterial reverse mutation assay (Ames-test) according to EU Method B.13/14, an in vitro mammalian chromosome aberration test according to EU Method B.10, and an in vitro mouse lymphoma test according to OECD Guideline 476. HAT ISO showed no signs of mutagenic activity in these three tests. An in vivo mutagenicity assay was performed with HAT ISO in the rat micronucleus test according to EU Method B.12. HAT ISO showed no signs of mutagenic activity in this test. Based on the results obtained in four tests on mutagenicity and genotoxicity, genotoxic activity of HAT ISO was excluded.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genotoxicity the test item does not require classification according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.