4-amino-m-cresol

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From April 4, 1984 to Aug. 1, 1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, followed method comparable guideline, GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1984

Report date:

1984

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

51956-65-1

Cas Number:

51956-65-1

IUPAC Name:

51956-65-1

Constituent 2

Reference substance name:

1-Hydroxy-3-methyl-4-aminobenzol-sulfat

IUPAC Name:

1-Hydroxy-3-methyl-4-aminobenzol-sulfat

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material: 1-Hydroxy-3-methyl-4-aminobenzol-sulfat
- Molecular formula: Not reported
- Molecular weight: Not reported
- Substance type: Pure active substance
- Physical state: Solid powder (light-beige)
- Stability under test conditions: As salt unlimitedly stable
- Storage condition of test material: Not reported

Test animals

Species:

rat

Strain:

other: Wistar (BOR:WISW)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Winkelmann, Versuchstierzucht, 4791 Borchen 1, Germany
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: Body weight range of males and females was 111 to 149 g and 110 to 133 g, respectively.
- Fasting period before study: Not reported
- Housing: Animals were housed individually in Makrolon cages (type II). Animal cages were changed weekly.
- Diet: Pelleted rodent diet (Ssniff-R Alleindiat fur Ratten), ad libitum
- Water: Aqua fontana (as for human consumption), ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22±2°C
- Relative humidity: 50-85%
- Air changes: 16 times per hour (SPF quality air)
- Photoperiod: Artificial light (160 Lux) in a 12 hours day/night cycle per day

STUDY INITIATION DATE: April 4, 1984
STUDY COMPLETION DATE: Aug. 1, 1984

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Aqua deionized

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Test substance solution was prepared in water (Aqua deionized). Three different test solutions were prepared by dissolving 150/600/1200 mg of test substance in 100 mL of water (Aqua deionized).

VEHICLE
- Concentration in vehicle: 1.5, 6 and 12 mg/mL
- Amount of vehicle: 10 mL/kg bw

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:
15, 60 and 120 mg/kg bw
Basis:
nominal in water

No. of animals per sex per dose:

20/sex/group
For recovery observations, satellite groups of additional 5 animals/sex for control and high dose group were investigated after a 4-week treatment free period

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses were selected based on following criteria:
a) The appointed concentration, which would be possible under the conditions of hair coloring,
b) The possible penetration rate,
c) A sufficient high safe zone,
d) The results obtained by a preliminary dose-range-finding.
- Rationale for selecting satellite groups: To observe the signs of recovery (if any).
- Post-exposure recovery period in satellite groups: 4 weeks

TEST TREATMENT: The treatment doses were as follows:
Control: Water (10 mL/kg bw)
Group I (Low dose): 15 mg/kg bw
Group II (Mid dose): 60 mg/kg bw
Group III (High dose): 120 mg/kg bw

Examinations

Observations and examinations performed and frequency:

MORTALITY: Yes
- Time schedule: Twice daily

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations checked: For hair coat, body orifices, urine and fecal excretion and their general health status.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily (Sensory and motor behavior)

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Weekly

FOOD EFFICIENCY: Yes
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: Weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to beginning of treatment (Day 0) and after 6 and 13 weeks
- Dose groups that were examined: 5 males and 5 females of each group and from the remaining animals at the end of the recovery period.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Before the beginning of the test (0 value) and after 6 and 13 weeks and from the remaining animals at the end of the recovery period.
- Anaesthetic used for blood collection: No data. The blood was taken from the retrobulbar venous plexus by means of non heparinized hematocrit tubules.
- Animals fasted: No data
- How many animals: 20 males and 20 females
- Parameters checked: Erythrocytes, Hemoglobin, hematocrit, MCV, MCH, MCHC, reticulocytes, inclusion bodies, thrombocytes, leucocytes (total count, differential and ESR) and prothrombin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Before the beginning of the test (0 value) and after 6 and 13 weeks and from the remaining animals at the end of the recovery period.
- Anaesthetic used for blood collection: No data. The blood was taken from the retrobulbar venous plexus by means of non heparinized hematocrit tubules.
- Animals fasted: No data
- How many animals: 20 males and 20 females
- Parameters checked: Albumin, alkaline phosphatase, calcium, chloride, cholesterol, creatinine, CPK, glucose, GOT, GPT, inorg. phosphorus, potassium, serum electrophoresis, serum iron, sodium, total bilirubin, total protein, triglyceride and urea nitrogen.

URINALYSIS: Yes
- Time schedule for collection of urine: Prior to treatment (Day 0), Week 6 and 13 of the study.
- Metabolism cages used for collection of urine: Yes. For individual urine-sample production the animals were housed in metabolic cages for 18 hours after a 20 mL/kg intragastric administration of water.
- Animals fasted: No data
- How many animals: 5 males and 5 females
- Parameters checked: Specific gravity, protein, pH, glucose, bilirubin, urobilinogen, blood, nitrate, ketones and sediment.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to beginning of treatment (Day 0) and after 6 and 13 weeks
- Dose groups that were examined: 5 males and 5 females of each group and from the remaining animals at the end of the recovery period.
- Battery of functions tested: Sensory activity (Modified Irvin)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, the animals were sacrificed by CO2 asphyxiation. Moribund animals were sacrificed to avoid organ autolysis. The blood was removed by cutting the A. carotis. A complete autopsy with recording of macroscopic findings was performed in all animals. Hereby a final external examination of the animals and a registration of abnormal findings in the cranial, thoracal, abdominal and pelvic cavity under veterinary supervision was carried out.

HISTOPATHOLOGY: Yes, histological slides were prepared and evaluated from 10 males and 10 females of control and high dose groups. Histological sections of vertical body, epiphysis and diaphysis of bone were prepared and examined. The bone marrow smears were also prepared and examined. The details on preparation and analysis are provided in the study report. The details on tissues collected, fixation of organs, embedding, production of tissue–sections and staining for histology are provided in the study report.

Other examinations:

ORGAN WEIGHT (grams): Organ weight of all the animals of all groups was determined. The organs collected were cerebrum with cerebellum, pituitary, heart, liver, kidneys (l + r), adrenals (l + r), spleen, prostate gland, testes (l+ r), ovaries (l+ r) and uterus.

Statistics:

- For evaluation of weight changes, food consumption and, if indicated, water consumption a one- resp. two-factorial analysis of variance was performed. To compare the group mean values the method of "Scheffe" was employed.
For weight gain phase the ratio weight (food efficiency) = (weight changes per week)/(food consumption per week) x 100
- The organ weights were evaluated by the analysis of co-variance. Thereby the animal weight is the independent variable, the organ weight the dependent one. The comparison of the mean values was performed by the method of "Scheffe" for the analysis of co-variance.Values of clinical chemistry and hematology were analysed as follows:
a) Analysis of variance for dose-effect curves with the factors group and time and the interaction group/time. The degrees of freedom for the factor time and the interaction group/time were corrected according to Greenhouse and Geisser (Epsilon-correction).
b) Mean values were compared according to the method of Scheffe after a preceding analysis of co-variance. The comparison (of the mean values) was carried out by correction with analysis of co-variance in such a manner as if the curves originated from the same starting-point.
c) If there were available one point time values only, an analysis of variance with subsequent Scheffe test for analysis of variance was performed.
Significance levels in the tables are marked by asterisks:
* p < 0.05 slightly significant
**p < 0.01 significant
***P < 0.001 highly significant

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

MORTALITY:
- One female in the high dose group died in week 11, according to the necropsy this was due to a gavage error. No other mortality was observed during the study.

CLINICAL SIGNS
- Clinical observations did not reveal any test substance related differences between control and dose group animals.
- The animals of Group II and III showed dark discolored urines from Week 8 to the end of treatment. This was due to compound discoloration.

BODY WEIGHT AND WEIGHT GAIN
- The development of the bodyweight gain was without significant intergroup differences throughout the entire treatment phase.

FOOD CONSUMPTION
- Food intake was near to equal in all test groups during each investigation phase and over the entire test period.

FOOD EFFICIENCY
- The rate of food efficiency showed the weight gain in gram (factor 100) which was gained by the consumption of 1 g of food. Continuing and essential differences between control and dose groups were not found.

OPHTHALMOSCOPIC EXAMINATION
- Ophthalmological examinations did not reveal any important differences between control and test groups.

HAEMATOLOGY
- The evaluated parameters revealed no abnormal alterations in the hemograms of the treated animals.

CLINICAL CHEMISTRY
- Highly significant increase in creatinine values was observed in the females of Group III, after 13 weeks of treatment. No treatment related effects were observed in other animals.

URINALYSIS
- The analysis did not reveal any practically important differences between control and test groups.

ORGAN WEIGHTS
- Test substance related increase in absolute spleen weight was observed in high dose females (statistically significant increased) and in high dose males (not statistically significant). No treatment related effect was observed in other treated animals.

GROSS PATHOLOGY
- The macroscopic changes observed were commonly seen in rats. They were considered to be spontaneous alterations and not attributable to treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC
- No test substance related findings were observed.

NEUROBEHAVIOURAL EXAMINATION
- Hearing tests did not reveal any important differences between control and test groups.
- Neuro-pharmacological examinations (Irvin) did not reveal any test substance related differences between control and dose group animals.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

60 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Increase in absolute spleen weights of high dose group (120 mg/kg bw) females.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Results on recovery period: No treatment related effects were observed for clinical signs, body weight, food consumption, hematology, clinical chemistry, urinalysis, autopsy and organ weights.

Applicant's summary and conclusion

Conclusions:

Oral administration of 1-Hydroxy-3-methyl-4-aminobenzol-sulfat to male and female Wistar (BOR: WISW) rats at dose levels of 15, 60 and 120 mg/kg bw by oral gavage for a period of 90 days revealed a NOAEL of 60 mg/kg bw. Based on this result on the sulfat form, the converted NOAEL for the registered substance 4-hydroxy-3-methylphenol was defined at 42.6 mg/kg bw/day.

Executive summary:

The subchronic oral toxicity of 1-Hydroxy-3-methyl-4-aminobenzol-sulfat was determined by following the method similar to the OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents).

The study was designed to determine the toxicity of test substance when administered at three dose levels by oral gavage for 90 days.

A total of 192 (96 males and 96 females) Wistar (BOR:WISW) rats of 6 weeks age, weighing 119-149 g (males) and 110-133 g (females)) (source: Winkelmann, Versuchstierzucht, 4791 Borchen 1) were used in the study. Animals were housed individually in Makrolon cages (type II). Animals were maintained under standard laboratory conditions (temperature: 22±2°C, humidity: 50-85%; air changes: 16 times per hour and photoperiod: artificial light (160 Lux) in a 12 hours day/night cycle per day). Animals were acclimated for 6 d and had free access to food and water throughout the treatment period.

Test solutions were prepared in water. Post acclimation, animals were randomized into treatment/control group (each consisting of 20 animals/sex). For recovery observations, satellite groups of 5 additional animals/sex for control and high dose groups were investigated after a 4-week treatment free period. Animals treated with water served as controls. All animals were treated daily for 13 weeks with a dose volume of 10 mL/kg bw. The treatment doses were as follows:

Control: Water (10 mL/kg bw)

Group I (Low dose): 15 mg/kg bw

Group II (Mid dose): 60 mg/kg bw

Group III (High dose): 120 mg/kg bw

Animals were observed twice daily for mortality/morbidity and once daily for clinical abnormalities. Body weight and food consumption were recorded weekly. A detailed ophthalmological investigation as well as an evaluation of auditory function and reflexes, according to a modified Irwin screen test (FOB) were performed on 5 animals/sex/group with special regard to awareness, co-ordination and autonomous nervous system functions. The investigations were conducted prior to treatment, after week 6, at the end of the treatment period and at the end of week 17 (recovery groups). Haematology and clinical chemistry evaluations were performed in 20 animals/sex/dose at Day 0, and after 6 and 13 weeks in all dose groups and after 17 weeks in the recovery group. The urinalysis was performed in the 5 animals/sex/dose at Day 0, and after 6 and 13 weeks in all dose groups and after 17 weeks in the recovery group.

At the end of the treatment period, all animals were killed and subjected to a detailed necropsy. Recovery group animals were sacrificed after Week 17 of the study. Selected organs were weighed. A wide range of organs/tissues of the control and high dose animals was examined histopathologically. In addition, all gross lesions noted were examined microscopically.

One female in the high dose group died in Week 11, according to the necropsy this was due to a gavage error. Clinical observations and neuro-pharmacological examinations (Irvin) did not reveal any test substance related differences between control and dose group animals. The animals of Group II and III showed dark discolored urines from Week 8 to the end of treatment.

The only test substance related finding was an increase in absolute spleen weight in high dose females (statistically significant) and in high dose males (not statistically significant). In the high dose recovery group an increase in absolute spleen weight was not observed.

No treatment related effects were observed in body weight gain, food efficiency, urinalysis, hematology, clinical chemistry, gross pathology and histopathology parameters at any dose levels.

Upon ophthalmoscopic and hearing examination, no treatment related effects were observed.

No treatment related effects were observed during the recovery period.

Based on above, oral administration of 1-Hydroxy-3-methyl-4-aminobenzol-sulfat to male and female Wistar (BOR: WISW) rats at dose levels of 15, 60 and 120 mg/kg bw by oral gavage for a period of 90 days revealed a NOAEL of 60 mg/kg bw.