Reaction mass of hydrogen [1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1) and hydrogen bis[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1) and hydrogen bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1)

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 January 2005 to 21 March 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of hydrogen [1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1) and hydrogen bis[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1) and hydrogen bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1)

IUPAC Name:

Reaction mass of hydrogen [1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1) and hydrogen bis[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1) and hydrogen bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1)

Test material form:

solid: particulate/powder

Details on test material:

- Description: Black powder
- Storage Conditions: Room temperature

Test animals

Species:

mouse

Strain:

NMRI

Remarks:

NMRI BR

Details on species / strain selection:

The NMRI BR mouse is used as test system because it is a readily available rodent species, which is commonly used for this purpose, with documented susceptibility to a wide range of toxic substances. These mice are recommended by international guidelines (e.g. OECD, EEC).

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: Young adult animals were selected (6-8 weeks old).
- Weight at study initiation: Mean body weight immediately prior to dosing with test material ranges from 33.2 ± 1.6 to 35.2 ± 3.6. The body weights of the mice at the start of the treatment were within 20 % of the sex mean.
- Assigned to test groups randomly: Yes, as they came to hand from the delivery boxes.
- Fasting period before study: No
- Housing: Group housing of 5 animals per sex per cage in labelled polycarbonate cages containing Woody Clean bedding. Paper bedding was provided as nest material.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: At least 5 days before start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): Approximately 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours dark per day.

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: Corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was suspended in corn oil. Test material concentrations were blended and treated with ultra-sonic waves to obtain a homogeneous suspension. Test material concentrations were dosed within 2.5 h after preparation.

TREATMENT
The mice received an intraperitoneal injection of a maximum tolerated (high), an intermediate and a low dose of test material. The route of administration was chosen to maximize the chance of the test material reaching the target tissue.
The dosing volume was 10 mL/kg body weight. The route and frequency of administration and the volume administered of the negative and the positive control was the same as those of the test material.

Duration of treatment / exposure:

The test material was administered as a single intraperitoneal injection.

Frequency of treatment:

All test groups received a single intraperitoneal injection.

Post exposure period:

Bone marrow of the groups treated with the test material was sampled 24 or 48 hours after dosing.
Bone marrow of the negative control group was isolated 24 hours after dosing and bone marrow of the positive control group was isolated 48 hours after dosing.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

62.5 mg/kg bw (total dose)

Doses / concentrations 2

Dose / conc.:

125 mg/kg bw (total dose)

Doses / concentrations 3

Dose / conc.:

250 mg/kg bw (total dose)

No. of animals per sex per dose:

5 animals per sex per dose group. There were two test groups for the animals dosed at 250 mg/kg body weight.

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control: Cyclophosphamide dissolved in physiological saline was used to treat the positive control group.
- Route of administration: A single intraperitoneal injection.
- Doses / concentrations: 50 mg/kg body weight.

Examinations

Tissues and cell types examined:

Bone marrow erythrocytes.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Selection of an adequate dose range for the micronucleus main test was based on a dose range finding study. Five dose groups, three comprising 1 male and 1 female, and two groups comprising 3 males and 3 females received a single dose of the test material. The study duration per dosing was two to three days. During this period mortality and physical condition were recorded at least once daily.

DETAILS OF SLIDE PREPARATION:
The animals were sacrificed by cervical dislocation. Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with approximately 2 mL of foetal calf serum. The cell suspension was collected and centrifuged at 1 000 rpm (approximately 100 g) for 5 min.
The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the serum by aspiration with the remaining serum. A drop of the cell suspension was placed on the end of a slide, which was previously cleaned (24 h immersed in a 1:1 mixture of 96 % (v/v) ethanol/ether and cleaned with a tissue) and marked (with the study identification number and the animal number). The drop was spread by moving a clean slide with round-whetted sides at an angle of approximately 45 ° over the slide with the drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100 % methanol and air-dried overnight. Two slides were prepared per animal.
The slides were automatically stained using the "Wright-stain-procedure" in an "Ames" HEMAtek slide stainer. The dry slides were dipped in xylene before they were embedded in MicroMount and mounted with a coverslip.

METHOD OF ANALYSIS:
All slides were randomly coded before examination. An adhesive label with study identification number and code was stuck over the marked slide. At first the slides were screened at a magnification of 100x for regions of suitable technical quality, i.e. where the cells were well spread, undamaged and well stained. Slides were scored at a magnification of 1 000 x. The number of micronucleated polychromatic erythrocytes was counted in 2 000 polychromatic erythrocytes. The ratio polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1 000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated.

Evaluation criteria:

A micronucleus test is considered acceptable if it meets the following criteria:
a) The positive control substance induced a statistically significant (Wilcoxon Rank Sum Test, two-sided test at P < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes.
b) The incidence of micronucleated polychromatic erythrocytes in the control animals should reasonably be within the laboratory historical control data range (mean ± three times the standard deviation):
Males: 1 .4 ‰ ± 4.1 ‰ indicated are means for n = 231.
Females: 1.5 ‰ ± 3.7 ‰ indicated are means for n = 160.
Equivocal results should be clarified by further testing using modification of experimental conditions.

Statistics:

A test material is considered positive in the micronucleus test if:
- It induces a statistically significant (Wilcoxon Rank Sum Test; two sided test at P < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) in the combined data for both sexes or in the data for male or female groups separately.

A test material is considered negative in the micronucleus test if:
- None of the tested concentrations or sampling times showed a statistically significant (P < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes neither in the combined data for both sexes nor in the data for male or female groups separately.

The preceding criteria are not absolute and other modifying factors may enter into the final evaluation decision.

Results and discussion

Test results

Key result

Sex:

female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 250, 500, 750, 1 000 and 2 000 mg/kg.
- Solubility: The test material was suspended in corn oil and treated with ultrasonic waves to obtain a homogenous suspension.
- Clinical signs of toxicity in test animals: There were deaths and clinical signs including lethargy, hunched posture, rough coat and hairless body parts dark coloured.


RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity in test animals: The animals of the negative and positive control groups showed no abnormalities. During the first two hours after dosing two male animals and one female animal of the groups treated with 250 mg/kg body weight had a hunched posture. One of these male animals also had a rough coat. In the groups treated with 125 mg/kg body weight two female animals had a hunched posture and a rough coat. In the groups treated with 62.5 mg/kg body weight the animals showed no reaction to treatment.
Within 20 hours after dosing all animals treated with the test material had dark coloured hairless body parts. Two male animals treated with 250 mg/kg body weight also had a hunched posture and one of these animals also had a rough coat.
Within 43 hours after dosing all animals of the groups treated with 250 mg/kg body weight had a hunched posture, a rough coat and their hairless body parts were dark coloured. Two male animals were also lethargic.

- Induction of micronuclei: The mean number of micronucleated polychromatic erythrocytes scored in test material treated groups were compared with the corresponding solvent control group.
No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of test material treated animals compared to the vehicle treated animals.
The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical solvent control data range.
Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes. Hence, the acceptability criteria of the test were met.

- Ratio of PCE/NCE: The animals of the groups which were treated with the test material showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which reflects a lack of toxic effects of this compound on the erythropoiesis. The animals of the groups treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes, demonstrating toxic effects on erythropoiesis.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the test, the test material is not mutagenic.

Executive summary:

The test material was assessed in the Micronucleus Test in mice, to evaluate its genotoxic effect on erythrocytes in bone marrow according to OECD Test Guideline 474 and EU Method B.12.

The test material was a black powder with a purity of >85.5 %, and was suspended in corn oil.

Five male and five female animals were used in each of the six treatment groups, including negative and positive controls. All groups received a single intraperitoneal injection. The negative and positive control groups were treated with vehicle and 50 mg/kg body weight of cyclophosphamide (CP), respectively. Animals were dosed with the test material at 250 (two groups), 125 (one group), and 62.5 (one group) mg/kg body weight. After dosing the animals of the dose level of 250 mg/kg body weight showed the following toxic signs: Lethargy (2 male animals), hunched posture (7 male and 6 female animals), rough coat (5 male and 5 female animals) and dark coloured hairless body parts (all animals). All animals of the dose level of 125 and 62.5 mg/kg body weight had dark coloured hairless body parts after dosing.

Two female animals dosed with 125 mg/kg body weight had a rough coat and a hunched posture.

Bone marrow of the groups treated with the test material was sampled 24 or 48 (highest dose only) hours after dosing. Bone marrow of the negative and positive control groups was harvested 24 and 48 hours after dosing, respectively.

No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with the test material.

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical solvent control data range. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes. Hence, both criteria for an acceptable assay were met.

The groups that were treated with the test material showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, which reflects a lack of toxic effects of this compound on the erythropoiesis. The groups that were treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, demonstrating toxic effects on erythropoiesis.

It is concluded that the test material is not mutagenic in the micronucleus test under the experimental conditions described.