4-[[2-methoxy-4-[(4-nitrophenyl)azo]phenyl]azo]phenol

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:

From September 09, 2015 to December 02, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

but considered to have not affected the integrity of the study

Principles of method if other than guideline:

This study is included in a combined repeated dose toxicity study with the reproduction/developmental toxicity screning test in rats. Here, only the assessment of genotoxicity will be adressed. The ability of the test substance to induce cytogenetic damage and/or disruption of the mitotic apparatus in rat bone marrow was investigated measuring the induction of micronuclei in polychromatic erythrocytes.

GLP compliance:

yes

Type of assay:

other: Mammalian Erythrocyte Micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

4-[[2-methoxy-4-[(4-nitrophenyl)azo]phenyl]azo]phenol

EC Number:

243-325-5

EC Name:

4-[[2-methoxy-4-[(4-nitrophenyl)azo]phenyl]azo]phenol

Cas Number:

19800-42-1

Molecular formula:

C19H15N5O4

IUPAC Name:

4-[[2-methoxy-4-[(4-nitrophenyl)azo]phenyl]azo]phenol

Test material form:

solid: particulate/powder

Remarks:

Dark brown

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

- A total of 130 Hsd: Sprague Dawley SD rats (65 males and 65 virgin females), 6 to 7 weeks old and weighing 176 to 200 g for males and 151 to 175 g for females, were ordered from Harlan Italy s.r.l., San Pietro al Natisone (UD), Italy. 5 animals per cage.
- Acclimatisation period of approximately 3 to 9 weeks, depending on the type of treatment.
- Temperature and relative humidity: 22+/- 2°C and 55 +/- 15%, respectively.
- Artificial light for 12 hours.
- Feed (commercially available laboratory rodent diet (4 RF 21) and water: ad libitum.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

The test substance was administered orally by gavage to animals at 5 mL/kg bw. The oral route was selected as it is a possible route of exposure of the test substance in man.

Duration of treatment / exposure:

Main groups:
- Males animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing, through the mating period and thereafter at least until the minimum total dosing period of 28 days has been completed including the day before necropsy. Dose volumes will be adjusted once per week for each animal according to the last recorded body weight.
- Females animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until at least up to, and including, Day 3 post partum or the day before sacrifice. Dose volumes will be adjusted once per week for each animal according to the last recorded body weight. During the gestation period, dose volumes will be calculated according to individual body weight on Days 0, 7, 14 and 20 post coitum and on Day 1 post partum. Thereafter individual dose volumes will remain constant.
Positive control group:
- Animals will receive a single dose approximately 24 hours before sacrifice.

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Mitomycin-C (2.00 mg/kg)

Examinations

Tissues and cell types examined:

- Bone marrow
- Erythrocytes

Details of tissue and slide preparation:

Samples of bone marrow were collected approximately 24 hours following the final treatment and approximately 48 hours following the second last treatment from 5 males and 5 females of the main groups randomly selected and from all animals of the positive control group. One femur of each animal was removed and bone marrow cells obtained by flushing with foetal calf serum. The cells were centrifuged and a concentrated suspension prepared to make smears on slides. These slides were air-dried, fixed with methanol and then stained with haematoxylin and eosin solutions and mounted with Eukitt. At least three slides were made from each animal.
At first, only slides from male animals were examined. Subsequently, in order to obtain bone marrow toxicity information, slides from two female animals treated at the high dose level and two females from the vehicle control group, were scored.

Evaluation criteria:

- The slides were randomly coded by a person not involved in the subsequent microscope scoring and examined under low power to select one or more slides from each animal according to staining and quality of smears. Four thousand polichromatic erythrocytes (PCEs) per animal were examined for the presence of micronuclei at high power (x 100 objective, oil immersion). At the same time, the numbers of normal and micronucleated normochromatic erythrocytes (NCEs) were also recorded.
- The test substance was considered to induce micronuclei if a statistically significant increase in the micronucleus incidence of polychromatic erythrocytes (at P<0.05) was observed in any treatment group and a dose-effect relationship was demonstrated. Where statistically significant increases in the incidence of micronucleated PCEs were observed, but all results were inside the distribution of negative control values within this laboratory, then historical control data were used to demonstrate that these increases did not have any biological significance.

Statistics:

Only counts obtained from polychromatic cells were subjected to statistical analysis and the original observations (and not micronucleus frequencies per 1000 cells) were used. The variation between individual animals within each treatment group was assessed by χ2 calculation. In case of no significant heterogeneity within either group, the χ2 test was employed to compare treated groups with the vehicle control. If at least one of the groups was not homogeneous, the variance ratio (F) value was calculated from the betweengroup and within-group χ2 values to show the significance of any difference between treated and vehicle control groups. In addition, a test for a linear trend (Snedecor and Cochran) was performed in order to evaluate dose-effect relationship.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- Following treatment with the test substance, no relevant increase in the number of micronucleated PCEs over the concurrent negative control (where values were within the historical control range) was observed at any dose level. A marked increase in the frequency of micronucleated PCEs was noticed in the positive control group.
- Bone marrow cell toxicity. The ratio of mature to immature erythrocytes and the proportion of immature erythrocytes among total erythrocytes were analysed to evaluate the bone marrow cell toxicity. Based on these results, no relevant inhibitory effect on erythropoietic cell division was observed at any dose level.

Any other information on results incl. tables

No relevant differences in clinical signs were observed between male and female animals from the main and recovery groups. The only difference was seen in the post-partum phase, probably due to the stress of delivery and weaning. Also the preliminary scoring of slides from female animals did not show substantial differences between sexes, in terms of bone marrow toxicity and incidence of micronucleated cells. Based on these results, the genotoxicity assessment was performed including male animals only.

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the test substance did not induce micronuclei in the polychromatic erythrocytes of treated rats.

Executive summary:

A combined study was conducted to determine the repeated dose toxicity, the reproductive and the in vivo genetic toxicity of the test substance, according to OECD Guidelines 422 and 474. The ability of the test substance to induce cytogenetic damage and/or disruption of the mitotic apparatus in rat bone marrow was investigated measuring the induction of micronuclei in polychromatic erythrocytes. Male rats (5 in each groups) were exposed to the test substance at concentrations of 0, 100, 250 and 600 mg/kg bw/day for periods ranging from 29 to 42 days (depending on the dosing, the symptoms, and the timing of sacrifice). A positive control group (mitomycin-C, 2.0 mg/kg) was also tested. Following treatment with the test substance, no relevant increase in the number of micronucleated PCEs over the concurrent negative control (where values were within the historical control range) was observed at any dose level. A marked increase in the frequency of micronucleated PCEs was noticed in the positive control group. The ratio of mature to immature erythrocytes and the proportion of immature erythrocytes among total erythrocytes were analysed to evaluate the bone marrow cell toxicity. No relevant inhibitory effect (no toxicity) on erythropoietic cell division was observed at any dose level. The values for the positive and negative controls were within the expectation ranges. The experiment was therefore considered valid. Under the study conditions, the test substance did not induce micronuclei in the polychromatic erythrocytes of treated rats (Rossiello, 2016).