4-amino-3-nitrophenol

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 28 Match 1991 to 23 June 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

GLP study, method comparable to OECD 407 guideline method.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

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: flakes

Remarks:

bown red flakes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:Batch Op 238
- Expiration date of the lot/batch: not specified
- Purity test date:not specified

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in brown glass jar, stored at room temperature in the dark
- Stability under test conditions: Confirmation that the test article is both stable and mixes homogenously in the vehicle was obtained before the start of the study at the test facility.
- Solubility and stability of the test substance in the solvent/vehicle: Dose solutions prepared were found to contain within 9% of nominal concentration.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:The test article was formulated freshly each day in the vehicle.

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Remarks:

CR1:CD(SD)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation:28 Days old
- Weight at study initiation:167-20g for male and 128-154g for female
- Fasting period before study: not specified
- Housing:animals were housed by group of 5 in grid stainless steel cagemeasuring 56-35-20 cm.
- Diet (e.g. ad libitum): pelleted diet (SQC rat and Mouse Maintenance Diet No. 1 Expanded) ad libitum
- Water (e.g. ad libitum): water ad libitum
- Acclimation period: seven days before the start of treatment.

DETAILS OF FOOD AND WATER QUALITY: Supplier's certificates of analysis for the batch of diet was provided in the report.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):19-23°C
- Humidity (%): 37-61%
- Air changes (per hr): air conditioned room
- Photoperiod (hrs dark / hrs light): 12h/12h

IN-LIFE DATES: From: 28 March 1991 To: 7 May 1991

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
A weighed amount of test article was made up to a known volume and mixed.

VEHICLE
- Justification for use and choice of vehicle (if other than water): No justification provided
- Concentration in vehicle: 10 and 20 mg/mL
- Amount of vehicle (if gavage): 20 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Additional stability analysis were performed with sampled formulations. Stability was quantified by HPLC approach. The dose solution prepareds during week 1 and 4 were found to contain within 9% of nominal concentrations.

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

250 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

600 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 animals per dose per sex per group were used.

Control animals:

yes

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked : mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of dosing, weekly thereafter and at necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
The food consumed by each cage was recorded weekly

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: The water consumed by each cage of animals was recorded daily over a period of 5 working days before treatment started and again during week 3 of treatment.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before the treatment and at the week 3.
- Dose groups that were examined: all animals before the treatment, In week 3 the eyes of all control and high dose group were examined

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the week 4
- Anaesthetic used for blood collection: Yes (halothane)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked : Haemoglobin concentration, Red blood cell count, packed cell volume, mean cell volume, mean cell haemoglobin volume, total leucocyte count, platelet count, leucocyte differential count, reticulocyte count, fibrinogen, prothrombin time, partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the week 4
- Animals fasted: Yes
- How many animals: all the animals
- Parameters checked : blood urea nitrogen, glucose, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, total protein, protein differential, albumin/globulin ratio (A/G ratio), Sodium, potassium, calcium, chloride, inorganic phophorus, total bilirubin, creatinine, cholesterol, triglyceride, gamma glutamyl transferase

URINALYSIS: Yes
- Time schedule for collection of urine: at the week 4
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes
- Parameters checked : volume, specific gravity, pH, protein, glucose, ketones, bilirubin, blood pigments, urobilinogen, sodium, potassium, deposit, micrscopic examination of erythrocytes, leucocytes, crystals, debris, casts.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All animals were weighed and were then examined externally. A macroscopic examination was then performed by opening the cranial, thoracic and visceral cavities and by observing the appearance of the tissues in situ. The location, colour, shape and size of any abnormalities were recorded.

HISTOPATHOLOGY: Yes
Whole organs or samples were preserved in buffered formol saline : adrenals, aorta, brain, caecum , colon, duodenum, epididymes, eyes, exoorbital lacrimal gland, femur, harderian gland, heart, ileum, jejunum, kidneys, mesenteric lymph node, oesophagus, ovaries, pancreas, pituitary, prostate, rectum, salivary glands, seminal vesicles, sciatic nerve, skeletal muscle, skin, spinal cord, spleen, sternum, stomach, submandibular lymph node, testes, thymus, thyroids, trachea, tongue, urinary bladder, uterus, vagina.

Statistics:

Bodyweight, haematological and organ weight data were evaluated by analysis of variance and, if a between groups difference significant at the 5% level occured, by pairwise t-tests betweenthe control and treamtent groups. Blood chemistry were analysed by kruskal wallis testing and if a between groups difference significant at the 5% level occured, by pairwise Multiple Comparison test.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs noted included, laboured, noisy respiration observed in one male animal and one female animal dosed at 600 mg/kg/day on day 27 and 19 respectively. The female animal showed mild convulsion in the day 23 too. Post dose salivation was observed in one male and several females (3) dosed at 600 mg/kg/day.

Mortality:

mortality observed, treatment-related

Description (incidence):

There were two deaths during the treatment period. A group 4 female died during blood sampling which was not considered as related to treatment. A female of the high dose group was killed in extremis on day 23. Clinical signs included noisy breathing, post dose salivation and mild convulsion. Macroscopic findings for bopth animals revealed orange furstaining of the skin and all tissues of the died female of the high dose group showed yellow discolouration.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Bodyweight gain was reduced in male animals dosed at 600 mg/kg/day (16%).

Food consumption and compound intake (if feeding study):

no effects observed

Water consumption and compound intake (if drinking water study):

effects observed, non-treatment-related

Description (incidence and severity):

Water consumption was reduced by 18% in female animals dosed at 600 mg/kg/day and by 12% in female dosed at 250 mg/kg/day. These decreases were considered to be within normal variations and to be partially caused by a high control value.

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Dark colouration of the urine samples from treated groups.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There was a dose related increase in body weight related liver wieghts. This effect was observed to a greater extent in males than females. There was a statistically significant increase in bodyweight related brain weight for male animals dosed at 600mg/kg/day.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic findings showing a treatlment related distribution were observed in the skin and urinary bladder. Orange staining of the skin was present in all treated animals. Orange or dark yellow discolouration of the contents of the urinary bladder was present in a proportion of male rats which received Imaxine FN. Some treated rats showed distension of the lumen.

Histopathological findings: non-neoplastic:

no effects observed

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

mortality

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Table 1 : Bodyweight

			Weeknumber
	Dose		1	2	3	4	5	Gain 1-5
Male	0	Mean	185	242	296	315	365	180
		SD	9,8	11,8	24	22,5	29	22,9
	100	Mean	186	246	302	317	371	185
		SD	8,9	13,4	22,8	34,7	41,4	36,4
	250	Mean	189	252	307	320	364	175
		SD	7,7	12,3	14,9	21,5	24,9	23,3
	600	Mean	188	241	292	303	338	151
		SD	6	12,5	12,9	14,3	24,1	24,9
Female	0	Mean	141	168	189	206	217	76
		SD	8,1	10,9	13,9	14,4	16,1	10,1
	100	Mean	146	176	189	215	224	78
		SD	8,6	11,3	13,3	17,1	15,8	11,4
	250	Mean	143	172	196	214	222	79
		SD	6,3	7,1	7,4	11,6	10,8	9,2
	600	Mean	144	172	194	209	217	74
		SD	5	6,1	7,1	8,7	7,6	7,1

 

Applicant's summary and conclusion

Conclusions:

Under the experimental condition of the study, the No Observe Adverse Effect Level of the test article 4-amino-3-nitrophenol was defined at 250 mg/kg/day.

Executive summary:

The purpose of this GLP compliant study was to assess the toxicity of Imexine FN when administered orally, by gavage for 4 weeks to rats.

The test article was administered, by gavage, once daily to 4 groups of Crl:CD-(SD)BR rats (10/sex) for 28 days. The test substance was administered at dosage levels of 100, 250 or 600 mg/kg bw. The control group received the vehicle (carboxymethylcellulose). All animals were sacrificed at the end of the study. All animals were observed daily for mortality and clinical signs. Water consumption was recorded before treatment and during week 3. Body weights and food consumption were recorded individually in weekly intervals. Ophthalmoscopic examination was performed. Blood samples were withdrawn from all survived animals for haematological and clinical chemistry investigations, during week 4. Organ weights were measured and macroscopy and histopathology were performed on all control and high dose animals.

Two animals (high dose group) died during the study (laboured respiration). All treated groups showed orange fur-staining, from day one. In the high dose group the following effects were observed: scabbing, perinasal staining, mild convulsions, significantly decreased body weight in the males. The dose level without adverse effects was 250 mg/kg bw.

The NOAEL was defined at 250 mg/kg bw per day.