4-amino-3-nitrophenol

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 2004-12-20 to 2005-05-05

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted 21 July 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

other: In vivo rats micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

4-amino-3-nitrophenol

EC Number:

210-236-8

EC Name:

4-amino-3-nitrophenol

Cas Number:

610-81-1

Molecular formula:

C6H6N2O3

IUPAC Name:

4-amino-3-nitrophenol

Test material form:

solid: particulate/powder

Remarks:

Dark red powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: No. 0508916
- Expiration date of the lot/batch: 09/2005
- Purity test date: 31 August 2004

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Approximately +4°C, protected from light and under nitrogen gas. Deviation: Storage of neat test article under a nitrogen atmosphere was not recorded in MTTS upon receipt of the material . Therefore, it could not be verified that this was donc . The test article, however, was maintained under a nitrogen atmosphere during preparation and dosing.
- Stability under test conditions: Stability and homogeneity of the formulated test article were determined in CIT study No. 26965 AHS
- Solubility and stability of the test substance in the solvent/vehicle: according te stability results achieved in CIT study No. 26965 A13S. Solutions of B051 in the concentration range 1-100 mg/mL in 0.5% aqueous carboxymethylcellulose were stable for 6 hours in the aforementioned storage conditions.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The top stock of the test article was ground to a fine powder using a mortar and pestle, mixed with the required quantity of vehicle, and homogenized thereafter using a magnetic stirrer.
- Final dilution of a dissolved solid, stock liquid or gel: 50, 100 and 200 mg/ml
- Final preparation of a solid: test item was ground with mortar and pestle

Test animals

Species:

rat

Strain:

other: Crl:CD (SD)BR

Details on species / strain selection:

This is an outbred strain that maximizes genetic heterogeneity and therefore tends to eliminate strain-specific response to test articles. The rat has been routinely utilized as an animal model of choice for the mammalian bone marrow erythrocyte micronucleus assay .

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories,North Carolina
- Age at study initiation: Dose Rangefinding Assay : 9 weeks old; Micronucleus assay : 8 weeks old
- Weight at study initiation: Dose Rangefinding assay : male : 275 to 309 g / female 202 to 225g; Micronucleus assay : male : 257 to 313g / female : 182 to 221 g
- Assigned to test groups randomly: Cyscore version 2 was used to randomize animals
- Fasting period before study: not specified
- Housing: The animais were housed in sanitary, stainless-steel, hanging, wire cages . The animais were housed, separated by gender, up to two animais per cage during acclimation and individually a lter randomization .
- Diet (e.g. ad libitum): PMI Certified Rodent Dieto #5002 was available ad libitum, unless otherwise noted. The manufacturer analyzed the diet for nutritional components and environmental contaminants.
- Water (e.g. ad libitum): Tap water, by an automatic watering system, was available ad libitum unless otherwise noted. Water samples are routinely analyzed for specified microorganisms and environmental contaminants. No contaminants were known to be present in the diet or water at levels which might interfere with this study .
- Acclimation period: For at least 5 days before initiation of study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.8°C to 26.1°C
- Humidity (%): relative humidity 30 to 70%
- Air changes (per hr): 10 or greater air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hours light/12-hours dark

IN-LIFE DATE: Dose rangefinding assay : From 10 or 11 December 2004 to 17 or 18 December 2004
Micronucleus assay : From 31 December 2004 to 6 January 2005

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Vehicle(s)/solvent(s) used: CMC (carboxymethyl cellulose)
- Justification for choice of solvent/vehicle: Stability and homogeneity of the formulated test article were determined in CIT study No. 26977 AHS. In that study, solutions of B051 in the concentration range 1-100 mg/mL in 0.5% aqueous carboxymethylcellulose were stable for 6 hours (room temperature) protected from light and under nitrogen atmosphere.
- Concentration of test material in vehicle: 50,100 and 200 mg/ml.
- Amount of vehicle (if gavage or dermal): 0.5% of carboxymethyl cellulose
- Type and concentration of dispersant aid (if powder): magnetic stirrer
- Lot/batch no. (if required): 034KO120
- Purity: not specified

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: All mixing containers were purged with nitrogen gas prior to use . Before preparation, the vehicle was degassed by sonication for at least 15 minutes, then saturated with nitrogen gas. The vehicle was kept under nitrogen atmosphere for at least 15 minutes prior to dosing and during the dosing procedure. The test article was kept under a nitrogen atmosphere during preparation .The top stock of the test article was ground to a fine powder using a mortar and pestle, mixed with the appropriate volume of the vehicle, and homogenized thereafter using a magnetic stirrer. The test article was administered as a suspension in the vehicle. Lower concentrations were obtained by dilution with the vehicle. The formulations were held at room temperature, protected from light in a nitrogen atmosphere prior to dosing. Dosing was performed within 6 hours of test article preparation. The treatment regimen was single oral gavage
dose administration. The oral route of administration was selected because this is a relevant route of administration for this test article to utilize in this assay .
Dosing volume : 10 ml/kg bodyweight

Duration of treatment / exposure:

24 and 48 hours

Frequency of treatment:

single oral gavage dose administration

Post exposure period:

24 and 48 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

limit test dose

No. of animals per sex per dose:

In dose rangefinding assay, 3 males and 3 females were used in each dose group. 5 males and 5 females per treatments groups for 24 hours and for 48 hours for micronucleus assay.

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control : cyclophosphamide
- Justification for choice of positive control(s): not specified
- Route of administration: oral gavage
- Doses / concentrations: 60 mg/kg

Examinations

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Based on the results of the dose rangefinding assay, the top dose selected for the micronucleus assay was 2000 mg/kg, the limit dose for this assay based on OECD guidelines.
Extraction of Bone Marrow:
The hind limb bones (tibias) were removed for marrow extraction from five surviving animais in each treatment and control group. For each animal, the marrow from the bone was flushed into an individual centrifuge tube containing 3 to 5 mL fetal bovine serum (one tube per animal).
Preparation of Slides:
Following centrifugation to pellet the marrow, the supernatant was removed by aspiration and portions of the pellet were spread on slides and air-dried. The slides were fixed in methanol, stained in acridine orange, and analyzed under fluorescent microscopy. For control of bias, ail slides were coded prior to analysis.
Slide Analysis:
Slides prepared from the bone marrow collected from five animals per group at the designated harvest timepoints were scored for micronuclei and the PCE to NCE cell ratio. The micronucleus frequency (expressed as percent micronucleated cells) was determined by analyzing the number of micronucleated PCEs from at least 2000 PCEs per animal. The PCE:NCE ratio was determined by scoring the number of PCEs and NCEs observed while scoring at least 500 erythrocytes per animal.

Evaluation criteria:

The criteria for the identification of micronuclei are those of Schmid (1976). Micronuclei were darkly stained and generally round, although almond and ring-shaped micronuclei occasionally occurred. Micronuclei were sharp bordered and were generally between 1/20 and 1/5 the size of the PCE. The unit of scoring is the micronucleated cell, not the micronucleus ; thus, the occasional cell with more than one micronucleus was counted as one micronucleated PCE, not two (or more) micronuclei.
The staining procedure permits the differentiation by color of polychromatic and normochromatic erythrocytes (bright orange and ghost-like, dark green, respeclively).
The criteria for a positive response was the detection of a statistically significant increase in micronucleated PCEs for at least one dose level, and a statistically significant dose related response. A test article that did not induce both of these responses was considered negative. Statistical significance was not the only determinant of a positive response; the Study Director also considered the biological relevance of the results in the final evaluation.

Statistics:

The following statistical methods were used to analyze the micronucleus data:
• Assay data analysis was performed using an analysis of variance (Winer, 1971) on untransformed proportions of cells with micronuclei per animal and on untransformed PCE:NCE ratios when the variances were homogeneous. Ranked proportions were used for heterogeneous variances.
• If the analysis of variance was statistically signif cant (p < 0 .05), Dunnett's t-test (Dunnett, 1955 ; 1964) was used to determine which dose groups, if any, were statistically significantly different from the vehicle control. Analyses were performed separately for cach sampling time.
For each sex, the 500, 1000, and 2000 mg/kg dose groups, as well' as the positive control group, were compared with the vehicle control group at the 5%, one-tailed probability level.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

cytotoxic to the bone marrow (i.e., a statistically significant decrease in the PCE :NCE ratio) in males at 2000 mg/kg at the 48- hour harvest timepoint,

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Results of the range finding test:
The test article, 4-amino-3-nitrophenol (B051), induced mortality in one out of three females treated at 2000 mg/kg. Clinical observations in animals treated at 1000 or 2000 mg/kg included slight hypoactivity, hunched posture, ataxia, irregular respiration, piloerection, squinted eyes, orange discoloration of urine, and orange stains on various parts of the body. Based on these results, the top dose selected for the micronucleus assay was the dose limit for this assay, 2000 mg/kg.
Results of the Micronucleus Assay:
The test article, 4-amino-3-nitrophenol (B051), induced mortality in one male treated at 1000 mg/kg and one female at 2000 mg/kg. Clinical observations included squinted eyes, slight hypoactivity/hypoactivity, hunched posture, tremors, orange genital discharge, red oral discharge, yellow ocular discharge, orange oral discharge, and/or red to orange discoloration of various parts of the body in animals treated at 1000 or 2000 mg/kg. Orange genital discharge observed in animals treated at 1000 or 2000 mg/kg is considered to be evidence of systemic exposure following administration of the test article.
4-amino-3 -nitrophenol (B051) did not induce statistically significant increases in micronucleated PCEs at any test article dose examined (500, 1000 and 2000 mg/kg). However, 4-amino-3-nitrophenol (B051) was cytotoxic to the bone marrow (i.e., a statistically significant decrease in the PCE:NCE ratio) in males at 2000 mg/kg at the 48 hour harvest timepoint, indicating that the bone marrow was exposed to the test compound.
The vehicle control group mean was less than the maximum published value of approximately 0 .4% micronucleated PCEs. The group mean was consistent with the historical control range of 0.085 ± 0 .009 % (males) and 0.068 ± 0 .014 % (females) for the 24-hour timepoint and 0.073 ± 0 .008 % (males) and 0.040 % (females) for the 48-hour timepoint. The actual values for the vehicle control group mean were 0 .07 ± 0 .03 % (males) and 0.04 ± 0.02 % (females) for the 24-hour timepoint and 0.05 ± 0.03 % (males) and 0.04 ± 0.01 % (females) for the 48-hour timepoint.
The positive control, cyclophosphamide, induced a statistically significant increase in micronucleated PCEs as compared to that of the vehicle control, with a mean and standard error of 1.74 ± 0.12% for the males and 1.36 ± 0.17% for the females.

Any other information on results incl. tables

Clinical Observations - Dose Range finding Assay

Target Dose level (mg/kg)	Sex	Animal ID	Time after dosing
			IPD	Approx. 1 hour	1 day	2 days
500	M	7928	0	0	0	0
		7931	0	0	0	0
		7933	0	0	0	0
	F	7937	0	0	0	0
		7939	0	0	0	0
		7942	0	0	0	0
1000	M	7923	0	0	0	0
		7926	0	0	0	0
		7934	0	0	0	0
	F	7935	0	1, 2, 3	0	0
		7944	0	0	0	0
		7945	0	1	0	0
2000	M	7925	0	1, 3	3	9, 10, 11, 12
		7929	0	1, 3	3	9, 10, 12
		7932	0	0	3	9, 10, 11, 12
	F	7938	0	1, 2, 4, 5, 6, 7	3	9, 11, 12
		7940	0	2, 3	3	0
		7946	0	1, 3	8	-

 

0 = Normal, 1 = slightly hypoactive, 2 = hunched posture, 3 = orange urine, 4 =ataxie, 5 = squinted eyes, 6 = irregular respiration, 7 = piloerection, 8 = found dead, 9 = orange stain on feet, 10 = orange stain on scrotum, 11 = orange fur on lower abdomen, 12 = orange stain on tail.

IPD _ Immediately post dose

 

 

Clinical Observations - Micronucleus Assay - Males

Target Dose level (mg/kg)	Harvest Timepoint	Animal ID	Time after dosing
			IPD	Approx. 1 hour	Approx. 6-6.5 hours	1 day	2 days
Vehicle Control 0	24	All	0	0	NP	0	NA
	48	All	0	0	NP	0	0
Positive control 60	24	All	0	0	NP	0	NA
500	24	8386	0	0	NP	0	NA
		8405	0	0	NP	0	NA
		8411	0	0	NP	0	NA
		8416	0	0	NP	0	NA
		8419	0	0	NP	0	NA
1000	24	8381	0	1	1,11	0	NA
		8402	0	1	1,11	0	NA
		8404	0	1	1,11	0	NA
		8408	0	1,5,8,9,10	12	-	NA
		8412	0	2,5	1,11	11	NA
2000	24	8388	0	1	1,11	11,13	NA
		8389	0	0	1,11	11,13	NA
		8401	0	0	1,11	11,13	NA
		8409	0	0	1,11	2,3,11,13	NA
		8414	0	2, 3	1,11	11,13	NA
	48	8390	0	1,3	1,11	13	13
		8391	0	1,3	1,11	13	13
		8403	0	1	1,11	13	13
		8417	0	2,5,8	1,11	13	13
		8421	0	1	1,11	13	13
	Replacement animals	8393	0	1	1,11	13	13
		8394	0	1,2	1,11	13	13
		8395	0	1	1,11	13	13
		8399	0	1,2	1,11	13	13
		8406	0	1,2	1,11	13	13
		8420	0	1,2	1,11	13	13

 

0 = Normal, 1 = orange genital discharge, 2 = squinted eyes, 3 = slightly hypoactive, 4 = hunched posture, 5 = hypoactive, 6 = red oral discharge, 7 = red stained paws, 8 = yellow ocular discharge, 9 = orange oral discharge, 10 = tremors, 11 = orange stained paws, 12 = found dead, 13 = orange stained tail

IPD - Immediately post dose.

NA = not applicable, animal harvested at the 24-hour harvest timepoint.

NP = observation not performed.

Applicant's summary and conclusion

Conclusions:

The test article, 4-amino-3-nitrophenol (B051), was evaluated as negative in the rat bone marrow micronucleus assay under the conditions of this assay, when tested up to the testing limit of 2000 mg/kg, which gave evidence of bone marrow toxicity .

Executive summary:

This GLP-compliant study was assessed to determine damage induced by the test item to the chromosomes or the mitotic apparatus of erythocytes sampled from bone marrow of rats. The study method was perform according to OECD 474 method for Mammalian Erythrocyte Micronucleus Test.

In the micronucleus assay, the test article was formulated in 0.5% aqueous carboxymethylcellulose and administered once by oral gavage at 500, 1000, or 2000 mg/kg (at 10 ml/kg volume) to male and female rats. Cyclophosphamide (60 mg/kg) served as a positive control treatment. Bone marrow was extracted and at least 2000 PCEs per animal were analyzed for the frequency of micronuclei. Cytotoxicity was assessed by scoring the number of PCEs and normochromatic erythrocytes (NCEs) in at least 500 total erythrocytes for each animal.

Under the experimental conditions of this study, the test item did not induce damage to chromosomes or mitotic apparatus leading to micronucleus formations on erythrocytes from treated rats bone marrow at the limited dose 2000 mg/kg body weight. Some clinical signs were observed, as systemic exposure.