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Repeated dose toxicity: oral

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Administrative data

sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Material tested according to OECD guideline and according to GLP

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes (incl. certificate)
Limit test:

Test material

Details on test material:
Material was from a single batch of 4-aminophenol with a certificate of analysis provided; stability confirmed through analytical verification during last phase of the study
- Analytical purity: 99.3%
- Purity test date: 25 Feb 1994
- Lot/batch No. 2070155

Test animals

Details on test animals and environmental conditions:
- Source: Charles River France
- Age at study initiation: Approx. 6 weeks old
- Weight at study initiation: 206 g for the males and 176 g for the females
- Fasting period before study:- Housing:
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 7 days

- Temperature (°C): 21 +- 2 degrees C
- Humidity (%): 50 +-20 percent
- Air changes (per hr): 12 cycles/hour
- Photoperiod (hrs dark / hrs light):12 hours/ 12 hours light dark (7:00-19:00)

First day of treatment 29-Sept-1994
Last day of treatment: 28-Dec-1994

Administration / exposure

Route of administration:
oral: gavage
CMC (carboxymethyl cellulose)
0.5% aqueous colloid emulsion
Details on oral exposure:
0.5% aqueous carboxymethylcellulose solution
- water for injections, batch 9799, 0419 and 0675 provided by BioSedra (92240 Malakoff, France); and
- carboxymethylcellulose, batch Nos. 71H0397 and 33H0576, provided by Sigma (38297 Saint-Quentin-Fallavier, France)
The test substance was ground to a fine powder using a mortar and pestle, suspended in the vehicle in order to achieve the concentration of 6 mg/ml, and then homogenized using a magnetic stirrer. The 2.4 mg/ml preparation was prepated by direct dilution of the 6 mg/ml preparation.

- Justification for use and choice of vehicle (if other than water): solubility
- Concentration in vehicle: 0.5%
- Lot/batch no. (if required): No. 71H0397 provided by Sigma
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Before the begining of the study the homogeneity of test substance suspended in 0.5% aqueous colloid emulsion of carboxymethylcellulose was checked. A preparation containing 2 mg/ml of 4-aminophenol was sampled at three different levels (top, middle and bottom) and analyzed in duplicate.
Method of analysis: HPLC with UV detector validated method
Stability: The same preparation was sampled in duplicate after 5 hours of storage at room temperature to check the stability over a mock treatment period, It was tested on the day of preparation and after 2,4 and 7 days of storage at +4 degrees C. Samplings of day 0, day 2, and day 4 were diluted with degazed solvent containing a solvent containing a stabilizer (ascorbic acid at 0.01% in water) and kept frozen at -20 degrees C until analysis on day 7.
On weeks 1, 4, 8 and 12, each preparation (control group included) was checked for achieved concentration of the test substance. Two additional anlysis were achieved on weeks 8 and 12 for preparations performed at a new time for groups 2 and 3 respectively.
Except for the low concentration for which the preparation was made daily during the first 8 days of dosing, the test substance preparations were made up for 7 days of use and were delivered to the animal room protected from light.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Doses / concentrationsopen allclose all
Doses / Concentrations:
10 mg/kg/day
other: analytically verified
Doses / Concentrations:
30 mg/kg/day
other: analytically verified
Doses / Concentrations:
100 mg/kg/day
other: analytically verified
No. of animals per sex per dose:
10 male and 10 female
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were determined in agreement with the sponsor following the results of a preliminary 4-week oral toxicity study performed at 15, 50, 200 and 1000/500 mg/kg/day. In this preliminary study tubular nephrosis was noted at 200 mg/kg/day after 4-weeks of treatment only; therefore 100 mg/kg/day was selected as the high dose and 10 mg/kg/day as a potential NOEL and the intermediate dose chosen as the approximate geometric mean between the 10 and 100 mg/kg/day treatment groups.


Observations and examinations performed and frequency:
- Time schedule: Once/day at approximately the same time.

- Time schedule:Once/day at approximately the same time

- Time schedule for examinations: Once before allocation of the animals into groups, on the first day of treatment, then once a week until the end of the study.

The quantity of food consumed by the animals of each cage was recorded once a week (over a 7-day period) until the end of the study.
Food intake per animal and per day was calculated using the amount of food given and left in each cage, divided by 2 (# animals/cage).

The food conversion ratio ws calculated on a weekly basis for each sex and each group, using body weight and food consumption means.
Food Conversion Ration = mean food consumption in g/animal/week/ mean body weight gain in g/animal/week

- Time schedule for examinations:Performed on all animals of each of the control and the high dose groups before the beginning of the treatment period and on week 13.

- Time schedule for collection of blood: approximately 24 hours after final treament, blood samples were taken from the orbital sinus of the fasted animals under light ether anesthesia
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- How many animals: All
- Parameters checked as per OECD protocol
Full CBC with differntial and platelet count, differential
Full Coagulation Panel including PT, APTT, Fibrinogen
Metabolic pannel includeing Na, K, Cl, Inorg Phosphorus, Glucose, Urea, Creatinine, Bilirubin, Total Protein, Albumin, Albumin/blobulin ratio, Cholesterol, Triglycerides, Alk Phosphatase, ASAT, ALAT,

- Time schedule for collection of urine: Collected into a tube containing thymol crystals during an overnight period of about 18 hours
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes
- Parameters checked : Volume, pH, Specific gravity, proteins, glucose, ketones, Bilirubin, Nitrites, Blood, Urobilinogen, microscopic cytology for leucocytes, erythorcytes, cylincers, mag ammonium phosphate crystals, calcium oxalate crystals and cells; Qualitative parameters - appearance


Sacrifice and pathology:
A complete macroscopic examinations was performed on all animals. Any gross observations were recorded individually

For all animals, the body weight was recorded before necropsy and the following organs were weighted wet as soon as possible after dissection:
adrenals heart kidney liver ovaries spleen testes thymus

All the macroscopic lesions and the following tissues were preserved in 10% buffered formalin (except for the eyes and pituitary gland which were fixed in formol-sublimate). The tissues marked with an asterisk * were preserved in fixative for possible future microscopic examination.
Adrenals aorta brain including medulla/pons, cerebellar and cerebral cortex caecum duodenum eyes with Harderian glands* heart ileum jejunum kidneys liver lungs with bronchi lymph nodes (mandibular and mesenteric) mammary glands oesophagus ovaries pancreas pituitary gland prostate rectum salivary glands* (sublingual and submaxillary) sciatic nerve seminal vesicles* skeletal muscle* skin* spinal cord* (cervical, thoracic and lumbar) spleen sternum with bone marrow stomach testes and epididymides thymus thyroids with parathyroids tongue* trachea urinary bladder uterus (horns and cervix) vagina*
All tissues for microscopic examination were embedded in paraffin wax and exctions and stained with hematoxylin eosin. Microscopic examinations were performed on: all macroscopic lesions and tissues listed above (except those marked with an asterisk*) in animals of the control and high dose group; and all macroscopic lesions and lungs, liver and kidneys of all animals of the low and intermediate dose groups.
The following sequence was used for the statistical analysis of the body weight, food consumption, hematology, blood biochemistry, urinalysis and organ weight data: Normality of distributions of the values in each group checked by Komolgorov-Smirnov test. If the distributions was normal, the homogeneity of variances between the groups was assessed by Bartlett's test (more than 2 groups) or Fisher's test (2 groups). If no significnat heterogenity of the variances was established, the comparison between treated and controls groups was performed by Dunnett's test. If the variances were heterogeneous, the comparison between treated and control groups was performed by Dunn's test (more than 2 groups) or by Mann Whitney's test (2 groups). If the distribution of values in the groups was not normal, the anlaysis was repeated after logarithmic transformation of the values (except for the organ weights). If this logarithmic transformation failed to normalize the distribution of the values, comparion of treated and control groups was performed by Dunn's test using original values.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No mortality occurred during the treatment period. Dose-related cloration of urine (yellow or orange until week 5, then brown until the end of the study) was observed in almost all animals given 30 or 100 mg/kg/day. Brown colored tail was noted in some f
mortality observed, treatment-related
Description (incidence):
No mortality occurred during the treatment period. Dose-related cloration of urine (yellow or orange until week 5, then brown until the end of the study) was observed in almost all animals given 30 or 100 mg/kg/day. Brown colored tail was noted in some f
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A slightly lower body weight gain was recorded in all treated females when compared to the controls (-16%, -12%, -19% at 10, 30, and 100 mg/kg/day, respectivelyent
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Slightly lowere mean sodium and chloride levels were minimal (< or = 4%) and not dose-related and were not considered to be of toxicological significance.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Higher mean absolute and/or relative to body weight adrenal gland, heart, kidney and lver weights were noted in the treated females given 100 mg/kg/day. As these differences were minor and were found in animals with a decreased body weight, they were not
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Body extremities or hair were brown in 3/10 females given 100 mg/kg/day; this was considered to be due to the physical properties of the test substance, which has a use as a dyeing substance
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See details below
Histopathological findings: neoplastic:
no effects observed
Details on results:
See above for areas where no effect were observed.
The principal clincal signs observed were attributed to the elination of the test substance or its metabolites in urine as follows:
1) yellow colored urine in2/10 males given 30 mg/kg/day (weeks 1 to 5)
2) orange colored urine in alltreated males and females given 100 mg/kg/day (weeks 1 to 5)
3) brown colored urine in all treated males and feamles given 30 or 100 mg/kg/day (from week 5)
4) brown coloration of the tail in 4/10 females given 100 mg/kg/day from week 13

Ptyalism was observed in 3/10 females at 100 mg/kg/day on a few occasions from week 5.

Minimal to marked tubular nephrosis was recorded in 5/10 males and 2/10 females given 30 mg/kg/day and in 9/10 males and 10/10 females given 100 mg/kg/day versus none in the controls. This was characterized by the degeration /necrosis of the tubular epithelium together with exfoliation of the tubular cells in the tubular lumen, dilated tubules and/or denuded basement membrane. This was associated in almost all animals with slight to marked tubular basophilia.
Miniaml to slight (often unilateral) tubular basophilia was noted in 9/10 males and 3/10 females given 10 mg/kg/day versus 6/10 males nd 2/10 females from the control group. As the severity of this tubular basophilia was comparatively similar between the two groups, the slightly higher incidence observed in the animls given 10 mg/kg/day was considered to bek irrelevant to the treatment with the test substance.

Effect levels

open allclose all
Dose descriptor:
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: see 'Remark'
Dose descriptor:
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: See discussion under LOEL

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

The administration of the test substance 4-aminophenol, daily by gavage at 10, 30, or 100 mg/kg/day for 13 weeks induced at all doses a not dose-related lower body weight fain in females only and ptyalism in females only at 100 mg/kg/day. The major finding consisted of tubular nephrosis, with a dose-related incidence at the two highest doses tested in both sexes. As the dose of 10 mg/kg/day induced a lower body weight gain in females, this does was considered to be or at the NO Observed Effect Level. The doses for further oncogenicity studies based on these results were determined to be between 2 and 30 mg/kg/day.