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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From Oct. 15, 2002 to Nov. 19, 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
according to OECD and German principles of GLP
Type of assay:
mammalian germ cell cytogenetic assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material: 2-Amino-6-chloro-4-nitrophenol
- TSIN: WR23214
- Substance type: Pure active substance
- Physical state: Brown orange powder
- Storage and stability under test conditions: The substance on storage in dryness and darkness is considered to be stable more than 5 years.
- Solubility: 0.07-0.24% (pH 4.3) in water;8.7 weight% (pH 3.6) in acetone/water 1:1; > 10 weight% in DMSO
- Stability in solution: The stability was tested over a period of seven days.
DMSO solution (approx. 10% w/v): Very good stability (98.5-99.4%)
Acetone/water 1:1 solution (approx. 7% w/v): Very good stability (98.7-100.1%)
Water solution (approx. 0.05% w/v): Very good stability (101.3-104.1%)

Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Cosmital ; n°batch GST009-01/30-07
- Expiration date of the lot/batch:May 08, 2017
- Purity test date: No data
- Purity : 99.9% (HPLC at 254 nm)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature; light protected
- Stability under test conditions: No data
- Solubility and stability of the test substance in the solvent/vehicle: No data
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING No

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd., Biotechnology and Animal Breeding Division; CH-4414 Fullinsdorf
- Age at study initiation: 8-10 weeks (at start of acclimatization)
- Mean body weight at study initiation: 37.9± 3.0 g (males) and 30.6 ± 2.4 g (females)
- Assigned to test groups randomly: Yes, the animals were distributed into the test groups at random and identified by cage number
- Housing: The animals were housed individually in Makrolon Type I cages with wire mesh top. Granulated soft wood bedding was provided.
- Diet: Pelleted standard diet ; ad libitum
- Water: Tap water; ad libitum
- Acclimation period: Minimum 5 days after arrival

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3°C
- Relative humidity: 30-70%
- Photoperiod: Artificial light was provided from 6 am to 6 pm.

EXPERIMENT INITIATION DATE: Oct. 15, 2002
EXPERIMENT COMPLETION DATE: Nov. 19, 2002

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: 1% carboxymethylcellulose (CMC)
- Justification for choice of solvent/vehicle: The vehicle was chosen for its relative non-toxicity for the animals.
- Concentration of test material in vehicle: 1.875, 3.75 and 7.5 mL/kg for 18.75, 37.5 and 75 mg/kg bw dose levels, respectively.
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw

Details on exposure:
PREPARATION OF DOSING SOLUTIONS: On the day of the experiment, the test substance was formulated in the vehicle.
Duration of treatment / exposure:
48 h
Frequency of treatment:
Single administration (intraperitoneal)
Post exposure period:
24 hours (all dose groups) and 48 hours (high-dose group only)
Doses / concentrationsopen allclose all
Dose / conc.:
18.75 mg/kg bw (total dose)
Dose / conc.:
37.5 mg/kg bw (total dose)
Dose / conc.:
75 mg/kg bw (total dose)
No. of animals per sex per dose:
Preliminary study: 2 animals per sex
Main study: 6 animals/sex/dose group/sacrifice time
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
Cyclophosphamide solution was freshly prepared on day of administration by dissolving in deionized water (Supplier: SIGMA-Aldrich Vertriebs-GmbH, D-82041 Deisenhofen)
- Justification for choice of positive control(s): The positive control, Cyclophosphamide, is one of the recommended positive controls by the OECD 474 guideline.
- Route of administration: intraperitoneal (Once)
- Doses / concentrations: 40 mg/kg bw (10 mL/kg bw volume)

Examinations

Tissues and cell types examined:
Tissue: Femoral bone marrow
Cell types: Polychromatic erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The dose levels for the main study were selected on the basis of preliminary toxicity study conducted in a series of 8 experiments (using dose levels of 50, 75, 100, 150, 200 and 500 mg/kg bw). Two animals per sex were treated with the test substance under identical conditions as in the mutagenicity study. The animals were treated intraperitoneally and examined for acute toxic symptoms at intervals of around 1, 2-4, 6, 24, 30 and 48 hours after test substance administration. Based on the results, maximum tolerated dose of 75 mg/kg bw was estimated to be suitable for the main study.

TREATMENT AND SAMPLING TIMES: The animals were treated once (intraperitoneally). The animals of the highest dose group were examined for acute toxic symptoms at intervals of 1, 2-4, 6 and 24 h after administration of the test substance. Bone marrow samples of all dose groups were collected at 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment.

DETAILS OF SLIDE PREPARATION: The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald/Giemsa. Cover slips were mounted with EUKITT (Kindler, D-79110 Freiburg). At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS: Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 2000 polychromatic erythrocytes (PCE) were analyzed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and total erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides. Ten animals (5 males, 5 females) per test group were evaluated.

Evaluation criteria:
The test substance was classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group.
The test substance that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes was considered non-mutagenic in this system.
Statistics:
Statistical significance at the p <0.05 was evaluated by means of the non-parametric Mann-Whitney test. However, the primary point of consideration was the biological relevance of the results.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY On the basis of the pre-experiment toxicity study, 75 mg/kg bw was estimated to be maximum tolerated dose and suitable to be used in main study in terms of toxic effects induced. At higher doses the test substance showed lethal effects. The detailed results are provided in the study report.

RESULTS OF DEFINITIVE STUDY
- Ratio of PCE/NCE: The mean number of PCEs was not affected by the test substance at any test concentration or sampling time as compared to the mean value of PCEs in the vehicle control. Hence, 2-Amino-6-chloro-4-nitrophenol, even tested at systemically toxic doses, showed no clear cytotoxic effect in the bone marrow. However, the occurrence of discolored urine as well as the observed signs of toxicity demonstrates that the substance was systemically distributed and bio-available.
- Clinical signs: The animals treated with 75 mg/kg bw expressed toxic reactions such as reduction of spontaneous activity, abdominal position, eyelid closure and ruffled fur.
- Induction of micronuclei: There was no statistically or biologically significant enhancement in the frequency of the detected micronuclei in comparison to the corresponding vehicle control at any preparation interval after treatment with the test substance at any dose level.

RESULT WITH POSITIVE CONTROL: The positive control Cyclophosphamide (40 mg/kg bw) showed a statistically significant increase of induced micronucleus frequency.

Any other information on results incl. tables

Table 1: Summary of Micronucleus test results of 2-amino-6-chloro-4-nitrophenol (study # 83681)

Test group Dose (mg/kg bw) Sampling time PCEs with micronuclei (%) Range PCE (per 2000 erythrocytes)
MALES
Vehicle (1% CMC) 0 24 0.11 0 – 6 1029
Test substance 18.75 24 0.05 0 – 2 1023
37.5 24 0.05 0 – 3 977
75 24 0.06 0 – 2 1056
48 0.02 948
Positive control (Cyclophosphamide) 40 24 1.44 15 – 56 1123
FEMALES
Vehicle (1% CMC) 0 24 0.05 0 – 3 1063
Test substance 18.75 24 0.14 1 – 6 1046
37.5 24 0.07 0 – 4 1044
75 24 0.06 0 – 5 1076
48 0.04 0 – 3 1106
Positive control (Cyclophosphamide) 40 24 0.76 9 – 20 1011

Table 2: Statistical evaluation of 2-amino-6-chloro-4-nitrophenol at 5% level for induced micronucleus frequency against vehicle control group (study # 83681)

Vehicle control versus test group Significance p
Males Females Males Females
18.75 mg test substance /kg b.w.; 24 h n.t. - n.t. 0.0794
37.50 mg test substance /kg b.w.; 24 h n.t. - n.t. 0.4127
75.00 mg test substance /kg b.w. ; 24 h n.t. - n.t. 0.619
75.00 mg test substance /kg b.w.; 48 h n.t. n.t. n.t. n.t.
40.00 mg Cyclophosphamide /kg b.w.; 24 h + + 0.004 0.04

‘- ‘ denotes not significant

‘+’ denotes significant;

‘n.t.’  denotes not tested, as the mean micronucleus frequency was not above the vehicle control value

Applicant's summary and conclusion

Conclusions:
2-amino-6-chloro-4-nitrophenol was not mutagenic in the in vivo micronucleus test using NMRI mice after a single intraperitoneal administration up to the maximum tolerated dose of 75 mg/kg bw.

Executive summary:

The in-vivo micronucleus test was performed using 2-amino-6-chloro-4-nitrophenol (Chlororange base), following the OECD guideline 474 (Mammalian Erythrocyte Micronucleus Test) and EU Method B.12 (2000).

Adult male and female NMRI mice obtained from RCC Ltd., Biotechnology and Animal Breeding Division; CH-4414 Fullinsdorf were used in this study.

The mean weight of animals at the initiation of the study was 37.9± 3.0 g (males) and 30.6 ± 2.4 g (females).The animals were housed individually on Makrolon Type I with wire mesh top cages and maintained under standard laboratory conditions (temperature: 22 ± 3°C, relative humidity: 30-70%; artificial light from 6 am to 6 pm.). 

The test substance was formulated in 1% CMC (Carboxymethylcellulose) which was used as vehicle control. The volume administered intraperitoneally was 10 mL/kg bw.

Based on the results of a preliminary toxicity study conducted (at dose levels of 50, 75, 100, 150, 200 and 500 mg/kg bw) in a series of 8 experiments using two female and two male mice, 75 mg/kg bw was estimated to be maximum tolerated dose and selected for the main study.

The following dose levels of test substance were investigated with 12 animals (6 male and 6 female)/ dose group:

 24 h preparation interval: 0, 18.75, 37.50 and 75.00 mg/kg bw

 48 h preparation interval: 75 mg/kg bw

Cyclophosphamide (40 mg/kg bw), administered intraperitoneally, was used as a positive control.

24 and 48 h after a single administration of the test substance, the bone marrow cells were collected for micronuclei analysis.

Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei.

The mean number of PCEs was not affected by the test substance at any test concentration or sampling time as compared to the mean value of PCEs in the vehicle control. Hence, 2-Amino-6-chloro-4-nitrophenol, even tested at systemically toxic doses, showed no clear cytotoxic effect in the bone marrow. However, the occurrence of discolored urine as well as the observed signs of toxicity demonstrated that the substance was systemically distributed and bio-available.

The animals treated with 75 mg/kg bw expressed toxic reactions such as reduction of spontaneous activity, abdominal position, eyelid closure and ruffled fur.

There was no statistically or biologically significant enhancement in the frequency of the detected micronuclei in comparison to the corresponding vehicle control at any preparation interval after treatment with the test substance at any dose level. The positive control, Cyclophosphamide, showed a statistically significant increase in induced micronucleus frequency.

Based on the results, 2-amino-6-chloro-4-nitrophenol was not mutagenic in the in vivo micronucleus test using NMRI mice after a single intraperitoneal administration up to the maximum tolerated dose of 75 mg/kg bw.

This mammalian erythrocyte micronucleus test is classified as acceptable, and satisfies the guideline requirements of the OECD 474 method.