2-(4-amino-2-nitroanilino)ethanol

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 3 June 2002 to 30 September 2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

HC Red n° 3 did not have cytotoxic properties in the bone marrow and that systemic distribution and thus bioavailability of HC Red n° 3 in bone marrow is not confirmed. Four consecutive administration were performed and sampling was made 4 hours after the last exposure and 24 hours after the third administration. According to OECD method, sampling time should be done between 24 and 48 hours.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

Four consecutive administration were performed and sampling was made 4 hours after the last exposure and 24 hours after the third administration. According to OECD method, sampling time should be done between 24 and 48 hours.

GLP compliance:

yes

Type of assay:

other: In Vivo Micronucleus Assay

Test material

Test material information

Constituent 1

Reference substance name:

2-(4-amino-2-nitroanilino)ethanol

EC Number:

220-701-7

EC Name:

2-(4-amino-2-nitroanilino)ethanol

Cas Number:

2871-01-4

Molecular formula:

C8H11N3O3

IUPAC Name:

2-(4-amino-2-nitroanilino)ethanol

Test material form:

solid: particulate/powder

Remarks:

dark green powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by Wella AG , Batch No. 71 barrel 922169
- Expiration date of the lot/batch: August 2005
- Purity test date: 7 November 2002

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in dryness and darkness
- Stability under test conditions: stable more than 8 years in dryness and darkness conditions
- Solubility and stability of the test substance in the solvent/vehicle: The substance showed over a period of 7 days in solution (ca 5 weight% in 1% CMC in 0.9% brine) in the dark at room temperature a good stability. Solubility : 0.2-0.4 weight% in water (pH 6.4)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Doses were prepared fresh on each day of dosing as a suspension in 1% carboxymethylcellulose (CMC) dissolved in n-saline each day prior to dosing. After mixing with an homogenizer, the dose formulations were sonicated to ensure a homogeneous suspension. During dosing, formulations were maintened at room temperature while slowly being stirred on a stir plate to ensure homogeneity

Test animals

Species:

mouse

Strain:

B6C3F1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Age at study initiation: 7 weeks
- Weight at study initiation: 22.9 to 26 g
- Assigned to test groups randomly: yes
- Fasting period before study: not specified
- Housing:housed in polycarbonate shoebox cages measuring 17cm wide, 28 cm long, 13 cm high
- Diet (e.g. ad libitum): Purina Certified Rodent Chow 5002 ad libitum
- Water (e.g. ad libitum): water ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 26 °C
- Humidity (%): 30 to 70% relative humidity
- Air changes (per hr): rate of 10-12 exchanges per hour
- Photoperiod (hrs dark / hrs light): 12-hours light/12-hours dark

IN-LIFE DATES: From: 10 June 2002 To: 29 August 2002

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used CMC (carboxymethyl cellulose)
- Justification for choice of solvent/vehicle: not specified
- Concentration of test material in vehicle: 2.5, 5.0, 10.0%
- Amount of vehicle (if gavage or dermal): 10mL/kg
- Type and concentration of dispersant aid (if powder): magnetic stirrer and sonication

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Doses were prepared fresh on each day of dosing as a suspension in 1% carboxymethylcellulose (CMC) dissolved in n-saline each day prior to dosing. After mixing with an homogenizer, the dose formulations were sonicated to ensure a homogeneous suspension. During dosing, formulations were maintened at room temperature while slowly being stirred on a stir plate to ensure homogeneity.

Duration of treatment / exposure:

4 Days

Frequency of treatment:

Once Daily

Post exposure period:

24 hours for the first, second and 20 hours after the third administration and 4 hours for the last administration

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

6 animals per condition were used

Control animals:

yes

yes, concurrent vehicle

other: positive control

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): no justification
- Route of administration: oral gavage
- Doses / concentrations: 50mg/kg

Examinations

Tissues and cell types examined:

Bone Marrow Erythrocytes and Blood Erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Based on historical data, previous toxicology and carcinogenesis studies in the litterature provided by the sponsor

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The test article was administered by oral gavage one daily (24 hours apart) on 3 consecutive days. The first day of dosing was designated as Day 1. On Day
4, all the animals were dosed 20 hours after the dose received on Day 3, four hour prior to necropsy. Animals were killed by exsanguination. Following sacrifice, the bone marrow were sampled. Blood samples were taken by the vena cava.

DETAILS OF SLIDE PREPARATION: Bone marrow slides intended for micronucleus analysis were fixed with methanol and stained with acridine orange.

METHOD OF ANALYSIS:
For each dose, 2000 polychromatic erythrocytes (PCEs) per animal were scored for the presence of micronuclei. To assess if the test article inhibits erythropoesis (signified by a reduction in the percentage of PCEs among erythrocytes), the number of PCEs among a total of 200 erythrocytes per animal were determined at 1000x magnification for the presence of micronuclei. Except for the 1000 mg/kg bw dose group in which slides from 5 animals were analyzes, each dose group analyzed consisted of 6 animals.




OTHER:

Evaluation criteria:

Where data were obtained at multiple dose levels, an experiment was considered positive if both a statistically significant dose dependent increase and a statistically significant increase for at least one treatment dose were detected. An experiment was considered equivocal if either but not both of these responses were detected. An experiment was considered negative if neither of these responses was detected. An equivocal test might require a replicate test using the same or adjusted doses to resolve the equivocal finding.

Statistics:

The statistical analysis was based on the number of micronucleated PCEs. Based on a inspection of normality, analysis involved a one tailed parwise comparison of each treatmen group against the concurrent control group and a one-tailed trend test using the appropriate parametric or non parametric statistics.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Based on both a one tailed linear trend test and a one tailed pairwise comparison of each dose group against the concurrent control, the number of micronucleated PCEs in the bone marrow of animals dosed with the test article was not significantly increased nor was the percentage of PCEs present in the bone marrow significantly decreased. The positive control chemical, cyclophosphamide , induced a significant increase in the number of micronucleated PCEs in the bone marrow while also decreasing the percentafe PCEs.

Any other information on results incl. tables

Table 1 :Summary of results

	Dose (mg/kg)	% PCE	Micronucleated PCEs per 1000 PCEs	P-value	Numbersof animal
CP	50	28.8	33.92	<0.001*	6
Vehicle	0	63.3	3.33		6
Test Item	250	60.0	2.33	0.923	6
Test Item	500	58.3	1.92	0.986	6
Test Item	1000	60.0	2.60	0.781	5

 *p<0.05

%PCE :the number of PCEs per 100 erythrocytes

P-value :Pairwise comparison using student t-test

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions used HC Red n° 3 did not induce an increase in the number of bone marrow cells with micronuclei in treated mice and, consequently, HC Red n° 3 was not genotoxic (clastogenic and/or aneugenic) in these micronucleus test with mice. However, The OECD 474 guideline indicates that cells should be sampled 18-24 hours after the last dose in a multiple dosing study. In this study, the cells were collected 4 hours ± 19 minutes after the last dose and 28 hours after the third dose. This variation in harvest time from the 18-24 hours recommended time point is considered too small to affect the validity of this multi-dose study.

Executive summary:

The purpose of this GLP compliant study was to evaluate the potential for the test article HC Red No. 3 to induce DNA damage as well as bone marrow, after oral administration of the test article to male mice.

HC Red n° 3 was evaluated for its potential to induce micronuclei in bone marrow after oral administration of the test article to male mice. Groups of 6 mice were treated with test material dissolved in 1% CMC in normal saline, orally by gavage once daily (24 hr apart) for three consecutive days, and then were treated with a fourth dose 20 hr after the previous dose. The test article was administered by oral gavage at 250, 500, and 1000 mg/kg bw/ day. During the acclimatization period each animal was observed at least once daily for mortality and moribundity. Prior to dosing, within 1 h of dosing and at the end of each workday each animal was observed for health concerns. Tissue sampling occurred 4 hours ± 19 minutes after the final treatment on Day 4. At the time of necropsy, blood samples were collected when the vena cava was cut to exsanguinate the animals. Following exsanguinations duplicate bone marrow slides were prepared for micronucleus analysis. Bone marrow slides intended for micronucleus analysis were fixed with methanol and stained with acridine orange. For each animal, the number of PCE among a total of 200 erythrocytes was determined to assess if HC Red no 3 inhibits erythropoesis. For micronuclei evaluation, 2000 PCE per animal were evaluated. The statistical analysis was based on the number of micronucleated PCEs.

Following oral administration of dose levels up to 1000 mg/kg bw/day administered on four consecutive days, bone marrow was collected. Analysis of micronuclei did not sustain an increase in the number of micronucleated PCEs. The % PCE present in bone marrow was not substantially changed in the treated animals indicating that HC Red n° 3 did not have cytotoxic properties in the bone marrow and that systemic distribution and thus bioavailability of HC Red n° 3 in bone marrow is not confirmed.

Under the experimental conditions used HC Red n° 3 did not induce an increase in the number of bone marrow cells with micronuclei in treated mice and, consequently, HC Red n° 3 was not genotoxic (clastogenic and/or aneugenic) in these micronucleus test with mice. However, The OECD 474 guideline indicates that cells should be sampled 18-24 hours after the last dose in a multiple dosing study. In this study, the cells were collected 4 hours ± 19 minutes after the last dose and 28 hours after the third dose. This variation in harvest time from the 18-24 hours recommended time point is considered too small to affect the validity of this multi-dose study.