3,3'-[[4-[(2-chloro-4-nitrophenyl)azo]phenyl]imino]bis[propiononitrile]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Non mutagenic 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

April 24, 1987

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Qualifier:

no guideline available

Principles of method if other than guideline:

No information about guideline followed

GLP compliance:

not specified

Type of assay:

bacterial forward mutation assay

Target gene:

histidine operon

Species / strain / cell type:

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

Remarks:

TA 1538

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction

Test concentrations with justification for top dose:

No data

Vehicle / solvent:

No data

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Details on test system and experimental conditions:

No data

Evaluation criteria:

No data

Statistics:

No data

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

other: see results in additional information

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

other: see results in additional information

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

other: see results in additional information

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

other: see results in additional information

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

other: see results in additional information

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

Strains TA 98, TA 100, TA 1535, TA 1537, TA 1538 result positives with and without metabolic activation in the present test. The positivity to the test is due to the presence of nitro groups. An expert assessment based on a collection of reliable data on substances containing these functional groups describes in an exhaustive way the mechanism of action that underlies the positive results. Therefore, based on the available information the substance is not classified as mutagenic

Conclusions:

Non mutagenic

Executive summary:

Method:

The substance was tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimunum, TA 98, TA 100, TA 1535, TA 1537 and TA 1538.No information about guideline followed.

 

Conclusion:

Non mutagenic.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

From April 03 to May 21, 1992

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine operon

Species / strain / cell type:

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

Remarks:

TA 1538

Details on mammalian cell type (if applicable):

Source of strains:
The histidine-auxotrophic strains of Salmonella typhimurium (TA 98, TA 100, TA 1535, TA 1537 and TA 1538) were obtained from Prof. B. Ames, Berkeley, CA., U.S.A.
Preparation of the bacterial cultures:
Inoculates from frozen master copies were set up monthly. They were grown in liquid NB-medium overnight and then plated on NBagar. After incubation, single colonies were taken from the plates, grown overnight in liquid NB-medium and then used for the experiment.
Control of the genotype of the strains:
The characteristics of the strains were checked monthly. Histidine-auxotrophy of the strains was demonstrated by the requirement for L-histidine. The presence of the rfa character was assayed by the sensitivity for crystal-violet. The deletion of the uvrB gene was demonstrated by the sensitivity for UV-light. The salmonella strains containing the R-factor (TA 98 and TA 100) were additionally checked for ampicillin resistance. Furthermore, all strains were checked for their characteristic reversion properties with known mutagens (positive controls).

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat-liver microsomal fraction S9

Test concentrations with justification for top dose:

With and Without microsomal activation: 50, 158, 500, 1581, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Remarks:

Vehicle

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

Remarks:

whitout metabolic activation

Untreated negative controls:

yes

Remarks:

Vehicle

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: 2-aminoanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Preliminary Toxicity/Ranqe-Finding test
A toxicity test (check for reduction in the number of revertant colonies) was carried out with strain TA 100 without and with microsomal activation at six concentrations of the test substance and one negative control according to Standard Operating Procedures of Genetic Toxicology. The highest concentration applied was 5000 µg/plate. The five lower concentrations decreased by a factor of 3. The plates were inverted and incubated for about 48 hours at 37 ± 1.5 °C in darkness. Thereafter, they were evaluated by counting the colonies and determining the background lawn. One plate per test substance concentration, as well as each negative control was used.

Mutagenicity test
The mutagenicity test was performed with strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 without and with microsomal activation according to Standard Operating Procedures of Genetic Toxicology. Each of the five concentrations of the test substance, a negative and a positive control were tested, using three plates per test substance concentration as well as each positive and negative control with each tester strain. The highest concentration applied was 5000 µg/plate (because of weak toxicity in the range finding test) and the four lower concentrations were each decreased by a factor of V10 (3.1623). The plates were inverted and incubated for about 48 hours at 37 ± 1.5 °C in darkness. Thereafter, they were evaluated by counting the number of colonies and determining the background lawn.

Colony counting and scoring of the plates
Colonies were counted electronically with an Artek counter. The results were sent online to a computer. They were checked on a random basis by the operator.Observations indicating precipitates of the test substance in the top agar or a reduced or absent bacterial background lawn were registered additionally. Means and standard deviations for all mutagenicity assays were calculated by a previously validated computer program.

Evaluation criteria:

Assay acceptance criteria
A test is considered acceptable if the mean colony counts of the control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response. In either case the final decision is based on the scientific judgement of the Study Director.
Criteria for a positive response
The test substance is considered to be mutagenic in this test system if the following conditions are met:
At least a reproducible meaningful increase of the mean number of revertants per plate above that of the negative control at any concentration for one or more of the following strains:
S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538.
Generally a concentration-related effect should be demonstrable.

Statistics:

In deviation to the OECD guideline a statistical analysis was not performed. At present the use of statistical methods concerning this particular test system is not generally recommended.
No appropriate statistical method is available.

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

other: see results in additional information

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

other: see results in additional information

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

other: see results in additional information

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

other: see results in additional information

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

other: see results in additional information

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

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:

Strains TA 98, TA 100, TA 1537, TA 1538 gave positive results with and without metabolic activation. While strain TA 1535 gave a positive result only in the presence of metabolic activation. The positivity to the test is due to the presence of nitro groups. An expert assessment based on a collection of reliable data on substances containing these functional groups describes in an exhaustive way the mechanism of action that underlies the positive results. Therefore, based on the available information the substance is not classified as mutagenic.

Toxicity test/Range finding test:

Six concentrations of the test substance ranging from 20.6 - 5000 µg/plate were tested with strain S. typhimurium TA 100 to determine the highest concentration to be used in the mutagenicity assay. The experiments were performed with and without microsomal activation. Normal background growth was observed. The numbers of revertant colonies were not reduced. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000 µg/plate without and with activation.

 

Mutagenicity test, original experiment:

In the experiment performed without microsomal activation, treatment with the test substance lead to a concentration dependend increase in the number of revertant colonies with strains TA 98, TA 100, TA 1537, and TA 1538. A slight increase was registered with strain TA 1537 at the concentrations of 1581.1 to 5000 µg/plate, a distinct increase with strain TA 100 at the concentrations of 500 to 5000 µg/plate and a strong increase with strains TA 98 and TA 1538 at the concentrations of 158.1 and 5000 µg/plate. In the experiments with activation a similar effect occurred on the same strains. A slight increase was seen with strain TA 1537 at the concentration of 5000 µg/plate, a distinct increase with strain TA 1538 at the concentration of 5000 µg/plate, a strong increase with strain TA 98 at the concentrations of 500 to 5000 µg/plate and with strain TA 100 at the concentrations of 1581.1 to 5000 µg/plate.

 

Mutagenicity test, confirmatory experiment:

In the experiments performed without microsomal activation, treatment with the test substance lead to increased back-mutant counts with strains TA 98, TA 100 and TA 1538. A distinct increase was observed with strain TA 100 at the concentrations of 500 to 5000 µg/plate, and a strong increase with strain TA 98 at the concentrations of 500 to 5000 µg/plate and with strain TA 1538 at the concentrations of 158.1 and 5000 µg/plate. In the experiments with activation increased numbers of revertants occurred on strains TA 98, TA 100 TA 1535 and TA 1538. A slight increase was seen with strain TA 1535 at the concentration of 5000 µg/plate, a distinct increase with strain TA 100 at the concentrations of 1581.1 to 5000 µg/plate, with strain TA 1538 at the concentration of 5000 µg/plate and strong increase with strain TA 98 at the concentrations of 50 to 5000 µg/plate. In the mutagenicity tests normal background growth was observed with all strains at all concentrations. The numbers of revertant colonies were not reduced. The test substance exerted no toxic effect on the growth of the bacteria. The various mutagens, promutagens, sterility checks, sensitivity and resistance tests, etc., employed to ensure the test system was acceptable, all produced results within our established limits. There were no known circumstances or occurrences in this study that were considered to have affected the quality or integrity of the data.

Conclusions:

Non mutagenic

Executive summary:

Method:

The substance was tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimunum, according to the OECD Guideline 471. The following strains of Salmonella typhimunum were used: TA 98, TA 100, TA 1535, TA 1537 and TA 1538.

The concentration range of the test substance to be tested in the mutagenicity test was determined in a preliminary toxicity test . Thus, the test substance was tested for mutagenic effects without and with metabolic activation at five concentrations in the range of 50 to 5000 µg/plate. In order to confirm the results, the experiments were repeated in an independent experiment with the same concentrations.

 

Results:

Toxicity test/Range finding test

In this test no signs of toxicity of the test substance on the bacteria were observed up to the concentration of 5000 µg/plate.

Mutagenicity test, original experiment

In the original experiment performed without and with metabolic activation, treatment with the test substance led to an increase of revertant growth with strains TA 98, TA 100, TA 1537 and TA 1538 at the upper concentrations.

Mutagenicity test, confirmatory experiment

In the confirmatory experiment performed without metabolic activation, an increase of revertant growth was observed with strains TA 98, TA 100, and TA 1538 at the upper concentrations. In the experiment carried out with activation this effect was seen with strains TA 98, TA 100, TA 1535, and TA 1538.

 

The positivity to the test is due to the presence of nitro groups. An expert assessment based on a collection of reliable data on substances containing these functional groups describes in an exhaustive way the mechanism of action that underlies the positive results. Therefore, based on the available information the substance is not classified as mutagenic.

 

Conclusion:

Based on the results of these experiments and on standard evaluation criteria, it is concluded that the test substance is not mutagenic.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

other: expert assessment

Adequacy of study:

key study

Study period:

July 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline required

Principles of method if other than guideline:

Expert statement

GLP compliance:

no

Type of assay:

other: expert statement

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: TA 98NR, TA 100NR

Metabolic activation:

with and without

Metabolic activation system:

Prival - Hamster liver S9 Mix

Test concentrations with justification for top dose:

Refer to the attached expert statement

Vehicle / solvent:

Refer to the attached expert statement

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Species / strain:

other: refer to the attached expert statement

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Based on the expert statement, the test substance is not considered as genotoxic in gene mutation studies on bacteria.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Micronucleus test

No clastogeinc in mice.

 

DNA damage and/or repair test

No DNA repair in rat liver.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

October 21, 1991.

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

abstract

Qualifier:

no guideline available

Principles of method if other than guideline:

In Vivo Rat liver Unscheduled DNA Synthesis Assay

GLP compliance:

not specified

Type of assay:

unscheduled DNA synthesis

Species:

rat

Strain:

Fischer 344

Sex:

male

Route of administration:

oral: gavage

Dose / conc.:

1 250 mg/kg bw/day

Dose / conc.:

2 000 mg/kg bw/day

Control animals:

yes, concurrent no treatment

Positive control(s):

2-acetylaminofluorene and N-dimethylnitrosamine

Tissues and cell types examined:

Hepatocytes

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Solvent treated rats gave rise to mean net nuclear grain counts of less than zero, whilst hepatocytes from 2AAF or NDMA treated animals had mean net nuclear grain counts of greater than +5.

Conclusions:

The test substance did not induce DNA repair in rat liver.

Executive summary:

Method:

The test substance was tested for the ability to induce unscheduled DNA synthesis (UDS) in an in vivo rat hepatocyte assay. Male Fischer 344 (F344) rats were treated with a single oral dose by gavage at 1250 or 2000 mg/kg bw. The highest test dose, 2000mg/kg bw, was the limit test dose for a non-toxic test agent in this assay. Animals were killed and hepatocytes prepared two hours and sixteen hours following administration of the chemical. Two independent experiments were carried out for each time point.

 

Results:

Treatments with the test substance in no case resulted in a mean net nuclear grain count greater than zero, at either time point. Solvent treated rats gave rise to mean net nuclear grain counts of less than zero, whilst hepatocytes from 2AAF or NDMA treated animals had mean net nuclear grain counts of greater than +5.

 

Conclusion:

It is concluded that, when tested up to 2000 mg/kg bw, the test sample did not induce DNA repair (as measured by unscheduled DNA synthesis) in rat liver.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From January 11 to February 16, 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)

Deviations:

not specified

Principles of method if other than guideline:

SOP N°300111

GLP compliance:

yes

Type of assay:

mammalian germ cell cytogenetic assay

Species:

mouse

Strain:

Tif:MAGf

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Animal species: MOUSE (Tif: MAGf (SPF) ) , males and females, reared at the Animal Farmof CIBA-GEIGY, Sisseln, Switzerland
- Number of animals per group: 5 females + 5 males
- Number of polychromatic erythrocytes scored per animal: 1000
- Randomization : The animals were random-selected.
- Identification : The animals were housed in groups of two. They were marked individually using colour pens.
- Period of acclimatization: The animals were kept on location for about one week prior to being used in the study. Shortly before use the health status of the animals was checked by the laboratory personnel according to veterinary/scientific standards.
- Diet: Pelleted, certified standard diet (Nafag No 890) was administered ad libitum up to 12 hours before dosing. All batches were assayed for nutritive ingredients and contaminant level by the manufacturer.
- Water: Tap water ad libidum, drinking water quality according to the specifications of the "Schweizerisches Lebensmittelbuch, Edition 1972". Results of the routine chemical examination of water at source (Grundwasserfassung Basel) conducted periodically by the water authority (Baudepartement des Kanton Basel, Abteilung Gewässerschutz).
- Range of the weights: - Tolerability test: Females: 23-24 g Males: 30-32 g; - Micronucleus test: Females: 20-25 g Males: 23-30 g
- Age range: 4 to 5 weeks
- Housing: The animals were housed 2/cage, individually marked with colour pens

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 - 22.5 °C
- Humidity (%): 38 - 62 %
- Photoperiod (hrs dark / hrs light): 12 hours per day

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Carboxymethylcellulose, 0.5 % (Suspension)

Duration of treatment / exposure:

16, 24 and 48 hours

Dose / conc.:

2 500 mg/kg bw/day (nominal)

Remarks:

Dosed at a volume of 10 ml/kg bw by gavage

Dose / conc.:

5 000 mg/kg bw/day (nominal)

Remarks:

Dosed at a volume of 10 ml/kg bw by gavage

No. of animals per sex per dose:

5 females + 5 males

Control animals:

not specified

Positive control(s):

cyclophosphamide (CPA, 64 mg/kg)

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

The animals were sacrificed by dislocation of the cervical vertebrae. Bone marrow was harvested from the shafts of both femurs with fetal calf serum. Bone marrow was washed by centrifugation and the cells were resuspended in fetal calf serum. Thereof smears were made. They were air-dried and then stained with May-Grünwald/Giemsa solution. After rinsing with distilled water and airdrying, the slides were cleared in Xylene and mounted.

Evaluation criteria:

Criteria for scoring micronuclei
Micronuclei are uniform, darkly stained, more or less round bodies in the cytoplasm of erythrocytes. Inclusions which are reflective, improperly shaped or stained, or which are not in the focal plain of the cell are judged to be artifacts and are not scored as micronuclei. Cells containing more than one micronucleus are only counted once.
Prior to analysis the slides were coded. The slides of five animals/sex/dose, showing good differentiation between mature and polychromatic erythrocytes, were scored by a laboratory technician. From each animal the ratio of polychromatic to normochromatic erythrocytes was determined and 1000 polychromatic erythrocytes were scored for micronuclei.

Statistics:

The significance of differences was assessed by the Chi-Squaretest (p<0.05).

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Tolerability test:
In the tolerability test the doses of 5000 and 2500 mg/kg bw the test substance were administered to each one male and one female animal. All animals survived the treatment and showed no signs of toxicity. The urine of the animals treated with the test material was yellow to orange in colour. Based on these results the dose of 5000 mg/kg bw was chosen as the highest dose to be administered in the micronucleus test.

Micronucleus test:
The test was performed with the dose of 5000 mg/kg bw at the three sampling times of 16, 24 and 48 hours. In the three dosage group all animals survived the treatment and showed no signs of toxicity. The urine of the animals treated with the test material was yellow to orange in colour. This finding implicates that the test substance is resorbed and that the test material and/or its metabolites reach the blood circulation and thereby also the bone marrow. At all sampling times (16, 24 and 48 hours) there was no statistically significant increase in the number of micronucleated polychromatic erythrocytes in the animals (females and males pooled) treated with the test substance as compared with the negative control animals. The mean percentage values of micronucleated PCEs of the animals treated with the test substance were 0.06 at 16 hours, 0.09 at 24 hours and 0.05 at 48 hours after treatment. The negative control value (24 hours) was 0.08. In the positive control (24 hours) the percentage of micronucleated cells within polychromatic erythrocytes was clearly increased. The mean percentage of micronucleated PCEs was 0.82. In comparison with the negative control (0.08) this value is highly significant (p<0.05).

Conclusions:

Not clastogenic

Executive summary:

Method:

The substance was tested for its genetic toxicity effects according to OECD Guideline 474, in male and female Tif:MAGf mice.

 

Observations:

Three groups of mice (5 males and 5 females each) were treated orally once with the maximum tolerated dose (MTD) of the test substance, 5000 mg/kg (as determined in the tolerability test). The animals were sacrificed 16, 24 and 48 hours thereafter. Subsequently femoral bone marrow cells were prepared and polychromatic erythrocytes were scored for micronuclei. In all groups assessed after the different treatment periods, no significant increase in the number of micronucleated polychromatic erythrocytes was observed when compared with the negative control group.

 

Conclusion:

It is concluded that under the given experimental conditions no evidence for clastogenic or aneugenic effects was obtained in mice treated with the test substance.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

GERM CELL MUTAGENICITY:

This hazard class is primarily concerned with substances that may cause mutations in the germ cells of humans that can be transmitted to the progeny. However, the results from mutagenicity or genotoxicity tests in vitro and in mammalian somatic and germ cells in vivo are also considered in classifying substances and mixtures within this hazard class.

Category 1: Substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans. Substances known to induce heritable mutations in the germ cells of humans.

Categoty 2: Substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

The classification in Category 2 is based on:

— Positive evidence obtained from experiments in mammals and/or in some cases from in vitro experiments, obtained from:

— Somatic cell mutagenicity tests in vivo, in mammals; or

— Other in vivo somatic cell genotoxicity tests which are supported by positive results from in vitro mutagenicity assays.

 

Classification for heritable effects in human germ cells is made on the basis of well conducted, sufficiently validated tests as:

- In vivo somatic cell mutagenicicty tests such as these indicated in paragraph 3.5.2.3.5:

- Mammalian bone marrow chromosome aberration test;

- Mouse spot test;

- Mammalian erythrocyte micronucleus test.

- In vitro mutagenicity tests such as these indicated in 3.5.2.3.8:

- In vitro mammalian chromosome aberration test;

- In vitro mammalian cell gene mutation test;

- Bacterial reverse mutation tests.

 

Different studies are available that give information about the mutagenic potential of the test substance:

- Different strains gave positive results in the Ames test. The positivity to the test is due to the presence of nitro groups. An expert assessment based on a collection of reliable data on substances containing these functional groups describes in an exhaustive way the mechanism of action that underlies the positive results. Therefore, based on the available information the substance is not classified as mutagenic.

- An in vivo Micronucleus Assay in rats, as required per REACH Regulation (EC 1907/2006), is available. The test substance in this test doesn't show clastogenic effects. This result is confirmed by a second study where similar results has been obtained.

- Moreover a further in vivo study, carried out in order to evaluate the ability of the test substance to induct DNA Damage and/or Repair, is available. Even in this study the test sample did not induce DNA repair (as measured by unscheduled DNA synthesis) in rat liver.

Therefore, it is possible to conclude that the test substance is Not Classified for Mutagenic Toxicity.