3,3'-[[4-[(2,6-dichloro-4-nitrophenyl)azo]phenyl]imino]bispropiononitrile

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Only bacteria-specific effects were noted in the bacteria reverse mutation assay. The test substance/structural analogue was negative in all other genotoxicity studies.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

From April 03 to May 21, 1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
see justification document in section 13

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source: Disperse Orange 44 / EC 223-765-4 / CAS 4058-30-4
Target: Disperse Yellow 163 / EC 267-758-4 / CAS 67923-43-7

3. ANALOGUE APPROACH JUSTIFICATION
see justification document in section 13

Reason / purpose for cross-reference:

read-across source

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:

other: TA 98, TA100, TA 1535, TA1537 and 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:

Without 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.

Key result

Species / strain:

other: TA 98, TA 100, and TA 1538

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

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:

Mutagenic

Executive summary:

The mutagenic activity of the structural analogue was investigated in a plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98, and TA 100. The test article was tested at the concentrations between 50 and 5000 µg/plate in two independent assays.

No signs of toxicity on bacteria was observed at any concentration tested. Reproducible, dose-dependent increases in revertant colony numbers were obtained with the tester strains TA 98, TA 100 and TA 1538 with and without S9 mix. Strain TA 1537, showed only increased mutation frequencies in the first experiment. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article induced gene mutations by base pair changes and frameshifts in the genome in tested strains.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From April 03 to May 21, 1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

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:

other: TA 98, TA100, TA 1535, TA1537 and 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:

Without 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:

other: TA 98, TA 100, TA 1535, TA 1537, and TA 1538

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

other: TA 98, TA 100, TA 1537, and TA 1538

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

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:

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.

 

Conclusion

Based on the results of these experiments and on standard evaluation criteria, it is concluded that the test substance and its metabolites exerted a strong mutagenic effect in this test system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The test substance/structural analogue did not induce micronuclei in the polychromatic erythrocytes of treated mice nor did it induce DNA repair (as measured by unscheduled DNA synthesis) in rat liver.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

C57BL

Details on species / strain selection:

C57BL/6JfBL10/Alpk male and female mice

Sex:

male/female

Route of administration:

oral: gavage

Duration of treatment / exposure:

Bone marrow samples were taken 24 hours after dosing at 3130 mg/kg and 24, 48 and 72 hours after dosing at 5000 mg/kg.

Frequency of treatment:

Twice at an interval of 24 h

Post exposure period:

Bone marrow samples were taken 24 hours after dosing at 3130 mg/kg and 24, 48 and 72 hours after dosing at 5000 mg/kg.

Dose / conc.:

3 130 mg/kg bw/day

Dose / conc.:

5 000 mg/kg bw/day

No. of animals per sex per dose:

5/sex/dose and killing time

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Route of administration: oral, gavage
- Doses / concentrations: 65 mg/kg bw

Tissues and cell types examined:

Bone marrow erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Based on patterns of lethalities or severe toxicity observed over a 4-d observation period following a single oral dose in a maximum tolerated dose (MTD)-Phase I

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Bone marrow smears were prepared 24 and 48 h after dosing for the vehicle control and treated animals and 24 h after dosing for the cyclophosphamide treated animals.

DETAILS OF SLIDE PREPARATION: The preparations were stained with polychrome methylene blue and eosin to visualise the various cell types.

METHOD OF ANALYSIS: Prior to microscopic assessment, all slides were furnished with code numbers, so that the counting was blind. The following counts were made:
Number of polychromatic erythrocytes (PCE) per slide: 1000 PCE
Percentage of polychromatic erythrocytes in the total erythrocyte population: 1000 Erythrocytes

Evaluation criteria:

A substance is considered positive if there is a significant increase in the number of micronucleated polychromatic erythrocytes compared with the concurrent negative control group

Statistics:

- The incidence of micronucleated PCE and percentage PCE in the erythrocyte sample, were considered by ANOVA.
- All analyses were carried out using the GLM procedure in SAS.
- One-sided Student's t-test:

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

Under the test conditions, test substance is not clastogenic in the mouse micronucleus assay.

Executive summary:

Disperse Orange 44 was tested in C57BL/6JfCD-1/Alpk male and female mice at dose levels of 3130 and 5000 mg/kg body weight, the higher level being the limit dose for this assay. Bone marrow samples were taken 24, 48 and 72 hours after dosing at 5000 mg/kg body weight and 24 hours after dosing at 3130 mg/kg body weight. No statistically or biologically significant increases in the incidence of micronucleated polychromatic erythrocytes, over the vehicle control values, were seen at either dose level at any of the sampling times investigated when the data from both sexes was pooled prior to statistical analysis. Small but statistically significant increases in the incidence of micronucleated polychromatic erythrocytes were noted in male mice 48 hours after being dosed at 5000 mg/kg body weight and in female mice 72 hours after being dosed at 5000 mg/kg body weight. No such increases were observed in any other group of male or female animals. Consideration of the data shows that the increased values are similar to the concurrent vehicle control data and it is considered that the observed increases are not biologically significant. The test system positive control, cyclophosphamide, induced statistically and biologically significant increases in the incidence in micronucleated polychromatic erythrocytes, thus verifying the sensitivity of the test system to a known clastogen.

It is therefore concluded that Disperse Orange 44 under the conditions of test, is not clastogenic in the mouse micronucleus test.

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.

Reference 3

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Species:

rat

Strain:

Fischer 344

Details on species / strain selection:

Fischer 344 (F344) rats were used because of the UDS data available for this strain within CTL and other laboratories.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd, Margate, Kent
- Age at study initiation: 6-8 weeks
- Weight at study initiation: 180-280 g
- Assigned to test groups randomly: the animals were allocated according to the order in which they were removed from the stock cage
- Housing: 5/cage
- Diet (e.g. ad libitum): Porton Combined Diet , ad libitum
- Water (e.g. ad libitum): Filtered tap water, ad libitum
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 40-60
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: Forms good suspension
- Concentration of test material in vehicle: 125 and 200 mg/mL

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Dosing suspensions of the test substance were preparedas a suspension in corn oil by homogenisation. Concentrations prepared for the UDS assay were 125 and 200 mg/mL
All dosing preparations were administered at a volume of 10 mL/kg bw.

Duration of treatment / exposure:

Preparation of hepatocytes took place 2 or 16 hours after dosage. Actual achieved exposure times were in the range of lh 55min to 2h 30min for the 2 hour time point, and 15h 55min to 16h 45min for the 16 hour treatment. In experiment 2, 2-AAF-treated rats were sampled 4h after dosage.

Frequency of treatment:

Single dose

Post exposure period:

2 and 16 h after treatment

Dose / conc.:

1 250 mg/kg bw (total dose)

Dose / conc.:

2 000 mg/kg bw (total dose)

No. of animals per sex per dose:

preliminary study: 5 (treatment group only)
5 males/dose in treatment group
2 males/group in vehicle and positive control group

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive control substances were 2-acetylaminofluorene (2-AAF, CTL reference number Y00892/050) in experiments 1 and 3 and N-nitrosodimethylamine (NDMA, CTL referencenumber Y01468/004) in experiments 2 and 4
- Route of administration: oral, gavage
- Doses / concentrations: AAF: 25 mg/kg bw / 2.5 mg/mL suspension in 0.5% w/v hydroxypropylmethylcellulose in 0.1% w/v PS80 (HPMC).
NDMA: 10 mg/kg bw / 1 mg/mL dissolved in deionised water

Tissues and cell types examined:

Hepatocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Based on a preliminary study, in which the acute MTD for the test substance was >2000 mg/kg

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Single oral dose by oral gavage. Slides from the animals were subsequently analysed. Of the two positive and two vehicle control animals in each experiment, only one of each was scored for induction of UDS

DETAILS OF SLIDE PREPARATION: Preparation of hepatocytes were made 2 or 16 h after dosage. Hepatocytes were prepared from treated animals by a two stage collagenase perfusion technique. Hepatocyte cultures were prepared by allowing cells to attach to plastic cover slips. Medium was removed from the dishes and replaced with fresh medium containing [3H] thymidine. After 4 h incubation at 37 °C within a 5% CO2/95% air (v/v) atmosphere, the medium was removed, the cells washed three times with medium containing unlabelled thymidine and the cultures incubated overnight with the same medium. Cultures were fixed and coverslips mounted onto microscope slides. Slides were coated with photographic emulsion and left for 14 d at 4 °C in the dark. The emulsion was developed, fixed and the cell nuclei and cytoplasm stained with Meyers haemalum and eosin Y phloxine.
Slides were examined microscopically for signs of undue cytotoxicity to enable selection of those to be examined for UDS.


METHOD OF ANALYSIS: Prior to microscopic assessment, all slides were furnished with code numbers, so that the counting was blind. The following counts were made:
- 30 morphologically normal cells/slide and a total of 60 cells/animal.
- The nuclear count (the number of silver grains over the nucleus) and the cytoplasmic count (the number of grains in an adjacent, nuclear sized, most heavily labelled area of cytoplasm) were measured using an automated image analyser (AMS 40-10) and the data captured directly into a computer.
- The mean net grain count (nuclear count - cytoplasmic count), the mean nuclear count and cytoplasmic counts and the percent of cells in repair (net grain count of 5 or greater) to be calculated.

Evaluation criteria:

Criteria for a positive response:
- A mean animal net nuclear grain count [N-C] value of greater than zero represents a biologically significant departure from normal.
- The radiolabelling of the nucleus exceeds that of the cytoplasm, indicating of a real net synthesis of nuclear DNA
Criteria for a negative response:
- The mean net nuclear grain count of all test substance treated animals is less than 0.

Statistics:

No data

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

blue discolouration of the internal organs served as proof for exposure

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: >2000 mg/kg in acute oral study

RESULTS OF DEFINITIVE STUDY
- Animal Toxicity:Over the period of the main UDS experiments (2 or 16 hours post-treatment), no signs of acute toxic effects were observed in treated animals. Although applied at a limit test dose in the main UDS experiments, the observation of blue colouration of the internal organs of treated rats showed that absorbtion and distribution of the test substance or its metabolites had taken place.
- Cytotoxicity: Hepatocytes prepared from animals dosed with the test substance were examined microscopically. No apparent signs of excessive cytotoxicity were observed ie: few cells present, or a high proportion of cells of abnormal morphology or large numbers of pyknotic cells compared with the solvent controls.
- Appropriateness of dose levels and route: the blue colouration of the internal organs of treated rats showed that absorbtion and distribution of the test substance

Conclusions:

Under the test conditions, the test substance did not induce DNA repair in rat liver in vivo up to a limit dose of 2000 mg/kg bw

Executive summary:

Disperse Orange 44 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 of CI Disperse Orange 44 by gavage at 1250 or 2000 mg/kg body weight. The highest test dose, 2000 mg/kg, 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. Hepatocytes from treated rats were exposed to [3H]-thymidine and the amount of radioactivity incorporated into the nucleus and an equal area of cytoplasm determined by autoradiography. The cytoplasmic grain count was subtracted from that of the nucleus. The value obtained, the mean net nuclear grain count [N-C], is an index of UDS activity. In this laboratory no negative control animal has shown a mean net nuclear grain count of greater than zero. An [N-C] value of greater than zero is therefore considered indicative of a UDS response. Each experiment was validated by concurrent control treatments of rats with corn oil, the solvent for Disperse Orange 44 and with the carcinogens 2-acetylaminofluorene [2AAF] at sixteen hours or N-nitrosodimethylamine [NDMA] at two hours. 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. These data showed that background levels of UDS were normal and that the test animals were responsive to known carcinogens requiring metabolic activation for genotoxic activity. Hepatocytes from Disperse Orange 44 treated animals were assessed for UDS at both dose levels. Treatments with Disperse Orange 44 in no case resulted in a mean net nuclear grain count greater than zero, at either time point.

It is concluded, that, when tested up to 2000 mg/kg body weight, the test sample of Disperse Orange 44 did not induce DNA repair (as measured by unscheduled DNA synthesis) in rat liver.

Reference 4

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
see justification document in section 13

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source: Disperse Orange 44 / EC 223-765-4 / CAS 4058-30-4
Target: Disperse Yellow 163 / EC 267-758-4 / CAS 67923-43-7

3. ANALOGUE APPROACH JUSTIFICATION
see justification document in section 13

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

C57BL

Details on species / strain selection:

C57BL/6JfBL10/Alpk male and female mice

Sex:

male/female

Route of administration:

oral: gavage

Duration of treatment / exposure:

Bone marrow samples were taken 24 hours after dosing at 3130 mg/kg and 24, 48 and 72 hours after dosing at 5000 mg/kg.

Frequency of treatment:

Twice at an interval of 24 h

Post exposure period:

Bone marrow samples were taken 24 hours after dosing at 3130 mg/kg and 24, 48 and 72 hours after dosing at 5000 mg/kg.

Dose / conc.:

3 130 mg/kg bw/day

Dose / conc.:

5 000 mg/kg bw/day

No. of animals per sex per dose:

5/sex/dose and killing time

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Route of administration: oral, gavage
- Doses / concentrations: 65 mg/kg bw

Tissues and cell types examined:

Bone marrow erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Based on patterns of lethalities or severe toxicity observed over a 4-d observation period following a single oral dose in a maximum tolerated dose (MTD)-Phase I

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Bone marrow smears were prepared 24 and 48 h after dosing for the vehicle control and treated animals and 24 h after dosing for the cyclophosphamide treated animals.

DETAILS OF SLIDE PREPARATION: The preparations were stained with polychrome methylene blue and eosin to visualise the various cell types.

METHOD OF ANALYSIS: Prior to microscopic assessment, all slides were furnished with code numbers, so that the counting was blind. The following counts were made:
Number of polychromatic erythrocytes (PCE) per slide: 1000 PCE
Percentage of polychromatic erythrocytes in the total erythrocyte population: 1000 Erythrocytes

Evaluation criteria:

A substance is considered positive if there is a significant increase in the number of micronucleated polychromatic erythrocytes compared with the concurrent negative control group

Statistics:

- The incidence of micronucleated PCE and percentage PCE in the erythrocyte sample, were considered by ANOVA.
- All analyses were carried out using the GLM procedure in SAS.
- One-sided Student's t-test:

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

Under the test conditions, test substance is not clastogenic in the mouse micronucleus assay.

Executive summary:

Disperse Orange 44 was tested in C57BL/6JfCD-1/Alpk male and female mice at dose levels of 3130 and 5000 mg/kg body weight, the higher level being the limit dose for this assay. Bone marrow samples were taken 24, 48 and 72 hours after dosing at 5000 mg/kg body weight and 24 hours after dosing at 3130 mg/kg body weight. No statistically or biologically significant increases in the incidence of micronucleated polychromatic erythrocytes, over the vehicle control values, were seen at either dose level at any of the sampling times investigated when the data from both sexes was pooled prior to statistical analysis. Small but statistically significant increases in the incidence of micronucleated polychromatic erythrocytes were noted in male mice 48 hours after being dosed at 5000 mg/kg body weight and in female mice 72 hours after being dosed at 5000 mg/kg body weight. No such increases were observed in any other group of male or female animals. Consideration of the data shows that the increased values are similar to the concurrent vehicle control data and it is considered that the observed increases are not biologically significant. The test system positive control, cyclophosphamide, induced statistically and biologically significant increases in the incidence in micronucleated polychromatic erythrocytes, thus verifying the sensitivity of the test system to a known clastogen.

It is therefore concluded that Disperse Orange 44 under the conditions of test, is not clastogenic in the mouse micronucleus test.

Reference 5

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across from supporting substance (structural analogue or surrogate)

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

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
see justification document in section 13

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source: Disperse Orange 44 / EC 223-765-4 / CAS 4058-30-4
Target: Disperse Yellow 163 / EC 267-758-4 / CAS 67923-43-7

3. ANALOGUE APPROACH JUSTIFICATION
see justification document in section 13

Reason / purpose for cross-reference:

read-across source

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:

The ability of the structural analogue, to induce cytogenetic damage and/or disruption of the mitotic apparatus in mouse bone marrow was investigated measuring the induction of micronuclei in polychromatic erythrocytes. Three groups of mice (5 males and 5 females each) were treated orally once with the maximum tolerated dose (MTD) of 5000 mg/kg (as determined in the tolerability test). In the three dose-groups, 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. 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. 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.

Reference 6

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
see justification document in section 13

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source: Disperse Orange 44 / EC 223-765-4 / CAS 4058-30-4
Target: Disperse Yellow 163 / EC 267-758-4 / CAS 67923-43-7

3. ANALOGUE APPROACH JUSTIFICATION
see justification document in section 13

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Species:

rat

Strain:

Fischer 344

Details on species / strain selection:

Fischer 344 (F344) rats were used because of the UDS data available for this strain within CTL and other laboratories.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd, Margate, Kent
- Age at study initiation: 6-8 weeks
- Weight at study initiation: 180-280 g
- Assigned to test groups randomly: the animals were allocated according to the order in which they were removed from the stock cage
- Housing: 5/cage
- Diet (e.g. ad libitum): Porton Combined Diet , ad libitum
- Water (e.g. ad libitum): Filtered tap water, ad libitum
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 40-60
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: Forms good suspension
- Concentration of test material in vehicle: 125 and 200 mg/mL

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Dosing suspensions of the test substance were preparedas a suspension in corn oil by homogenisation. Concentrations prepared for the UDS assay were 125 and 200 mg/mL
All dosing preparations were administered at a volume of 10 mL/kg bw.

Duration of treatment / exposure:

Preparation of hepatocytes took place 2 or 16 hours after dosage. Actual achieved exposure times were in the range of lh 55min to 2h 30min for the 2 hour time point, and 15h 55min to 16h 45min for the 16 hour treatment. In experiment 2, 2-AAF-treated rats were sampled 4h after dosage.

Frequency of treatment:

Single dose

Post exposure period:

2 and 16 h after treatment

Dose / conc.:

1 250 mg/kg bw (total dose)

Dose / conc.:

2 000 mg/kg bw (total dose)

No. of animals per sex per dose:

preliminary study: 5 (treatment group only)
5 males/dose in treatment group
2 males/group in vehicle and positive control group

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive control substances were 2-acetylaminofluorene (2-AAF, CTL reference number Y00892/050) in experiments 1 and 3 and N-nitrosodimethylamine (NDMA, CTL referencenumber Y01468/004) in experiments 2 and 4
- Route of administration: oral, gavage
- Doses / concentrations: AAF: 25 mg/kg bw / 2.5 mg/mL suspension in 0.5% w/v hydroxypropylmethylcellulose in 0.1% w/v PS80 (HPMC).
NDMA: 10 mg/kg bw / 1 mg/mL dissolved in deionised water

Tissues and cell types examined:

Hepatocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Based on a preliminary study, in which the acute MTD for the test substance was >2000 mg/kg

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Single oral dose by oral gavage. Slides from the animals were subsequently analysed. Of the two positive and two vehicle control animals in each experiment, only one of each was scored for induction of UDS

DETAILS OF SLIDE PREPARATION: Preparation of hepatocytes were made 2 or 16 h after dosage. Hepatocytes were prepared from treated animals by a two stage collagenase perfusion technique. Hepatocyte cultures were prepared by allowing cells to attach to plastic cover slips. Medium was removed from the dishes and replaced with fresh medium containing [3H] thymidine. After 4 h incubation at 37 °C within a 5% CO2/95% air (v/v) atmosphere, the medium was removed, the cells washed three times with medium containing unlabelled thymidine and the cultures incubated overnight with the same medium. Cultures were fixed and coverslips mounted onto microscope slides. Slides were coated with photographic emulsion and left for 14 d at 4 °C in the dark. The emulsion was developed, fixed and the cell nuclei and cytoplasm stained with Meyers haemalum and eosin Y phloxine.
Slides were examined microscopically for signs of undue cytotoxicity to enable selection of those to be examined for UDS.


METHOD OF ANALYSIS: Prior to microscopic assessment, all slides were furnished with code numbers, so that the counting was blind. The following counts were made:
- 30 morphologically normal cells/slide and a total of 60 cells/animal.
- The nuclear count (the number of silver grains over the nucleus) and the cytoplasmic count (the number of grains in an adjacent, nuclear sized, most heavily labelled area of cytoplasm) were measured using an automated image analyser (AMS 40-10) and the data captured directly into a computer.
- The mean net grain count (nuclear count - cytoplasmic count), the mean nuclear count and cytoplasmic counts and the percent of cells in repair (net grain count of 5 or greater) to be calculated.

Evaluation criteria:

Criteria for a positive response:
- A mean animal net nuclear grain count [N-C] value of greater than zero represents a biologically significant departure from normal.
- The radiolabelling of the nucleus exceeds that of the cytoplasm, indicating of a real net synthesis of nuclear DNA
Criteria for a negative response:
- The mean net nuclear grain count of all test substance treated animals is less than 0.

Statistics:

No data

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

blue discolouration of the internal organs served as proof for exposure

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: >2000 mg/kg in acute oral study

RESULTS OF DEFINITIVE STUDY
- Animal Toxicity:Over the period of the main UDS experiments (2 or 16 hours post-treatment), no signs of acute toxic effects were observed in treated animals. Although applied at a limit test dose in the main UDS experiments, the observation of blue colouration of the internal organs of treated rats showed that absorbtion and distribution of the test substance or its metabolites had taken place.
- Cytotoxicity: Hepatocytes prepared from animals dosed with the test substance were examined microscopically. No apparent signs of excessive cytotoxicity were observed ie: few cells present, or a high proportion of cells of abnormal morphology or large numbers of pyknotic cells compared with the solvent controls.
- Appropriateness of dose levels and route: the blue colouration of the internal organs of treated rats showed that absorbtion and distribution of the test substance

Conclusions:

Under the test conditions, the test substance did not induce DNA repair in rat liver in vivo up to a limit dose of 2000 mg/kg bw

Executive summary:

Disperse Orange 44 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 of CI Disperse Orange 44 by gavage at 1250 or 2000 mg/kg body weight. The highest test dose, 2000 mg/kg, 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. Hepatocytes from treated rats were exposed to [3H]-thymidine and the amount of radioactivity incorporated into the nucleus and an equal area of cytoplasm determined by autoradiography. The cytoplasmic grain count was subtracted from that of the nucleus. The value obtained, the mean net nuclear grain count [N-C], is an index of UDS activity. In this laboratory no negative control animal has shown a mean net nuclear grain count of greater than zero. An [N-C] value of greater than zero is therefore considered indicative of a UDS response. Each experiment was validated by concurrent control treatments of rats with corn oil, the solvent for Disperse Orange 44 and with the carcinogens 2-acetylaminofluorene [2AAF] at sixteen hours or N-nitrosodimethylamine [NDMA] at two hours. 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. These data showed that background levels of UDS were normal and that the test animals were responsive to known carcinogens requiring metabolic activation for genotoxic activity. Hepatocytes from Disperse Orange 44 treated animals were assessed for UDS at both dose levels. Treatments with Disperse Orange 44 in no case resulted in a mean net nuclear grain count greater than zero, at either time point.

It is concluded, that, when tested up to 2000 mg/kg body weight, the test sample of Disperse Orange 44 did not induce DNA repair (as measured by unscheduled DNA synthesis) in rat liver.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

There are no data regarding genotoxicity for the test item Disperse Yellow 163. However, a close structural analogue, Disperse Orange 44, was tested positive in the Bacteria Reverse Mutation Assay (Ames test) in Salmonella strains. Due to the nitro-group in para-position to the azo-bond in both compounds, it is most likely that also Disperse Yellow 163 would be tested positive in the Ames test.This positive effect in the bacterial mutation assay is a bacteria-specific effect due to bacterial nitro-reductases, which are highly effective in these bacterial strains, but not in mammalian cells. This is shown to be true by the negative test results with Disperse Orange 44 in one in-vivounscheduled DNA synthesis (UDS) test in rats and two in-vivo micronucleus test (MNT) in mice.

This effect was also in-depth investigated in the close structural analogueDisperse Blue 291and another dye with a similar properties, Disperse Blue 367.

Disperse Blue 291 was tested positive in an Ames test with nitroreductase andO‑acetyltransferase positive Salmonella typhimurium strains, but negative with nitroreductase and O-acetyltransferase negative strains andin a test for unscheduled DNA synthesis.

Disperse Blue 367was tested positive in an Ames test, but negative in the following tests:

Study Type	Metabolic activation	Result
Mitotic recombination assay withSaccharomyces cerevisae	Rat liver S9-mix	Negative
Point mutation assay with Saccharomyces cerevisae	Rat liver S9-mix	Negative
HPRT assay with V79 hamster cells	Rat liver S9-mix	Negative
UDS assay with rat hepatocytes	Metabolic competent hepatocytes were used	Negative
Micronucleus assay in vivo (mouse)	In vivo assay	Negative

The nitroreductase family comprises a group of flavin mononucleotide (FMN)- or flavin adenine dinucleotide (FAD) -dependent enzymes that are able to metabolize nitroaromatic and nitroheterocyclic derivatives (nitrosubstituted compounds) using the reducing power of nicotinamide adenine dinucleotide (NAD(P)H). These enzymes can be found in bacterial species and, to a lesser extent, in eukaryotes. The nitroreductase proteins play a central role in the activation of nitro-compounds. Type I nitroreductases can transfer two electrons from NAD(P)H to form the nitroso and hydroxylamino intermediates and finally the amino group. Type II nitroreductases transfer a single electron to the nitro group, forming a nitro anion radical, which in the presence of oxygen generates the superoxide anion in a futile redox cycle, regenerating the nitro group [de Oliveira et al. 2010].

The positive effect in the bacterial reverse mutation test (Ames) was clearly related to a bacteria-specific metabolism of the test substance, as it is well-known for aromatic nitro compounds to be positive in the Ames assay resulting from metabolism by the bacteria-specific enzyme nitro-reductase [Tweats et al. 2012]. This could be also be proved to be true in studies with Disperse Blue 291, which was tested for mutagenic activity in the Salmonella assay with strains with different levels of nitroreductase and O-acetyltransferase[Umbuzeiro et al. 2005]. In this study,Disperse Blue 291 showed mutagenic activity with all standard strains of Salmonella typhimurium tested (TA1537, TA1538, TA98 and TA100), except for TA1535.In nitroreductase and O-acetyltransferasenegative strains (TA98NR, TA98DNP6) not mutagenic activity was observed in the absence of S9, whereas themutagenic activity was increased with the nitroreductaseand/or O‑acetyltransferaseoverproducing strains, (YG1021, YG1024 and YG1041) This shows the importance of the bacterial acetyltransferase enzyme in the activation of Disperse Blue 291. Because of the remarkable increase in the response with the nitroreductase and O‑acetyltransferase overproducing strain (YG1041), it is assumed that the product of the nitroreductaseis a substrate for the O-acetyltransferase. As there was a very slight increase in mutagenicity with TA98NR, TA98, YG1021, TA98DNP6, and YG1024 in the presence of S9, it was assumed that P450 enzymes have also a role in the activation of Disperse Blue 291, besides the bacterial enzymes. This could however not proven true in studies withDisperse Blue 367 in mammalian cells or in in-vivo studies with Disperse Blue 291, Disperse Orange 44 or Disperse Blue 367.

It has also been demonstrated in various other publications that this mutagenic activity is a bacteria-specific effect and that these Ames positive nitro-substances are not mutagenic in mammalian assays.

That the reduction of these nitro-compounds to mutagenic metabolites is a bacteria-specific effect is demonstrated in the following by means of the two compounds AMP397 and fexinidazole.

• AMP397 is a drug candidate developed for the oral treatment of epilepsy. The molecule contains an aromatic nitro group, which obviously is a structural alert for mutagenicity. The chemical was mutagenic in Salmonellastrains TA97a, TA98 and TA100, all without S9, but negative in the nitroreductase-deficient strains TA98NR and TA100NR. Accordingly, the ICH standard battery mouse lymphomatkand mouse bone marrow micronucleus tests were negative, although a weak high toxicity-associated genotoxic activity was seen in a micronucleus test inV79 cells [Suter et al. 2002].The amino derivative of AMP397 was not mutagenic in wild type TA98 and TA100. To exclude that a potentially mutagenic metaboliteis released by intestinal bacteria, a Muta^TMMouse study was done in colon and liver with five daily treatments at the MTD, and sampling of 3, 7 and 21 days post-treatment. No evidence of a mutagenic potential was found in colon and liver. Likewise, a comet assay did not detect any genotoxic activity in jejunum and liver of rats, after single treatment with a roughly six times higher dose than the transgenic study, which reflects the higher exposure observed in mice. In addition, a radioactive DNA binding assay in the liver of mice and rats did not find any evidence for DNA binding. Based on these results, it was concluded that AMP397 has no genotoxic potential in vivo. It was hypothesized that the positive Ames test was due to activation by bacterial nitro-reductase, as practically all mammalian assays including fourin vivoassays were negative, and no evidence for activation by mammalian nitro-reductase or other enzymes were seen. Furthermore, no evidence for excretion of metabolites mutagenic for intestinal cells by intestinal bacteria was found.
• Fexinidazolewas in pre-clinical development as a broad-spectrum antiprotozoal drug by the Hoechst AG in the 1970s-1980s, but its clinical development was not pursued. Fexinidazole was rediscovered by the Drugs for Neglected Diseases initiative (DNDi) as drug candidate to cure the parasitic disease human African trypanomiasis (HAT), also known as sleeping sickness. The genotoxicity profile of fexinidazole, a 2-substituted 5-nitroimidazole, and its two active metabolites, the sulfoxide and sulfone derivatives were investigated [Tweats et al. 2012]. All the three compounds are mutagenic in the Salmonella/Ames test; however, mutagenicity is either attenuated or lost in Ames Salmonella strains that lack one or more nitroreductase(s). It is known that these enzymes can nitroreduce compounds with low redox potentials, whereas their mammalian cell counterparts cannot, under normal conditions. Fexinidazole and its metabolites have low redox potentials and all mammalian cell assays to detect genetic toxicity, conducted for this study either in vitro (micronucleus test in human lymphocytes) or in vivo (ex vivo unscheduled DNA synthesis in rats; bone marrow micronucleus test in mice), were negative.

Based on these data and the common mechanism between the reduction of these nitro-compounds, which is widely explored in literature [de Oliveira et al. 2010], it is concluded, that the mutagenic effects observed in the Ames test with Disperse Orange 44 is a bacteria specific effect and not relevant to mammalians. Due to the close structural similarity, this is considered to be also true for Disperse Yellow 163. The test substance was not genotoxic in the in-vivo UDS and MNT test. Therefore, a direct genotoxic effect as well as a metabolisation towards genotoxic structures by mammalian species can be excluded.

References

De Oliveira IM, Bonatto D, Pega Henriques JA. Nitroreductases: Enzymes with Environmental Biotechnological and Clinical Importance. InCurrent Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology; Mendez-Vilas, A., Ed.; Formatex: Badajoz, Spain, 2010:1008–1019.

Suter W, Hartmann A, Poetter F, Sagelsdorff P, Hoffmann P, Martus HJ. Genotoxicity assessment of the antiepileptic drug AMP397, an Ames-positive aromatic nitro compound. Mutat Res. 2002 Jul 25;518(2):181-94.

Umbuzeiro GA, Freeman H, Warren SH, Kummrow F, Claxton LD. Mutagenicity evaluation of the commercial product CI Disperse Blue 291 using different protocols of the Salmonella assay. Food and Chemical Toxicology 2005;43:49–56.

Tweats D, Bourdin Trunz B, Torreele E. Genotoxicity profile of fexinidazole--a drug candidate in clinical development for human African trypanomiasis (sleeping sickness). Mutagenesis. 2012 Sep;27(5):523-32.

Additional information

Bacteria reverse mutation

The mutagenic activity of Disperse Orange 44 (100% purity),was investigated in a plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98, and TA 100. The test article was tested at the concentrations between 50 and 5000 µg/plate in two independent assays.

No signs of toxicity on bacteria was observed at any concentration tested. Reproducible, dose-dependent increases in revertant colony numbers were obtained with the tester strains TA 98, TA 100 and TA 1538 with and without S9 mix. Strain TA 1537, showed only increased mutation frequencies in the first experiment. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article induced gene mutations by base pair changes and frameshifts in the genome in tested strains.

UDS in-vivo

Disperse Orange 44 (98% purity) 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 of CI Disperse Orange 44 by gavage at 1250 or 2000 mg/kg body weight. The highest test dose, 2000 mg/kg, 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. Hepatocytes from treated rats were exposed to [3H]-thymidine and the amount of radioactivity incorporated into the nucleus and an equal area of cytoplasm determined by autoradiography. The cytoplasmic grain count was subtracted from that of the nucleus. The value obtained, the mean net nuclear grain count [N-C], is an index of UDS activity. In this laboratory no negative control animal has shown a mean net nuclear grain count of greater than zero. An [N-C] value of greater than zero is therefore considered indicative of a UDS response. Each experiment was validated by concurrent control treatments of rats with corn oil, the solvent for Disperse Orange 44 and with the carcinogens 2-acetylaminofluorene [2AAF] at sixteen hours or N-nitrosodimethylamine [NDMA] at two hours. 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. These data showed that background levels of UDS were normal and that the test animals were responsive to known carcinogens requiring metabolic activation for genotoxic activity. Hepatocytes from Disperse Orange 44 treated animals were assessed for UDS at both dose levels. Treatments with Disperse Orange 44 in no case resulted in a mean net nuclear grain count greater than zero, at either time point.

It is concluded, that, when tested up to 2000 mg/kg body weight, the test sample of Disperse Orange 44 did not induce DNA repair (as measured by unscheduled DNA synthesis) in rat liver.

MNT in-vivo

The ability of Disperse Orange 44 (100% purity), to induce cytogenetic damage and/or disruption of the mitotic apparatus in mouse bone marrow was investigated measuring the induction of micronuclei in polychromatic erythrocytes. Three groups of mice (5 males and 5 females each) were treated orally once with the maximum tolerated dose (MTD) of Disperse Orange 44, 5000 mg/kg (as determined in the tolerability test). In the three dose-groups, 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 Disperse Orange 44 is resorbed and that the test material and/or its metabolites reach the blood circulation and thereby also the bone marrow. 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. It is concluded that under the given experimental conditions no evidence for clastogenic or aneugenic effects was obtained in mice treated with Disperse Orange 44.

In a second test, Disperse Orange 44 was tested in C57BL/6JfCD-1/Alpk male and female mice at dose levels of 3130 and 5000 mg/kg body weight, the higher level being the limit dose for this assay. Bone marrow samples were taken 24, 48 and 72 hours after dosing at 5000 mg/kg body weight and 24 hours after dosing at 3130 mg/kg body weight. No statistically or biologically significant increases in the incidence of micronucleated polychromatic erythrocytes, over the vehicle control values, were seen at either dose level at any of the sampling times investigated when the data from both sexes was pooled prior to statistical analysis. Small but statistically significant increases in the incidence of micronucleated polychromatic erythrocytes were noted in male mice 48 hours after being dosed at 5000 mg/kg body weight and in female mice 72 hours after being dosed at 5000 mg/kg body weight. No such increases were observed in any other group of male or female animals. Consideration of the data shows that the increased values are similar to the concurrent vehicle control data and it is considered that the observed increases are not biologically significant. The test system positive control, cyclophosphamide, induced statistically and biologically significant increases in the incidence in micronucleated polychromatic erythrocytes, thus verifying the sensitivity of the test system to a known clastogen.

It is therefore concluded that Disperse Orange 44 under the conditions of test, is not clastogenic in the mouse micronucleus test.

Justification for classification or non-classification

Based on the results of in vivo testing, no classification for genotoxicity is required for the test substance according to CLP (EC 1272/2008) criteria.