5-amino-o-cresol

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 21st, 1978 to September 21st 1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The method was equivalent to OECD 408 guideline method with deviations : animals were treated only days per week instead of 7 ; the study was not GLP-compliant ; However this study is not up to modern standard but was considered reliable for assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1979

Report date:

2005

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:

yes

Remarks:

see "rationale for reliability included deficiences" remarks above

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

5-amino-o-cresol

EC Number:

220-618-6

EC Name:

5-amino-o-cresol

Cas Number:

2835-95-2

Molecular formula:

C7H9NO

IUPAC Name:

5-amino-2-methylphenol

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by Henkel Inc., batch No. 851 from the lot of 13/06/1977
- 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:stored at room temperature
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/vehicle: stable suspension
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was ground in an agate planetary mill in order to obtain a reasonably stable suspension and to avoid a displacement of the gavage tube by too large particles during administration. The test substance was freshly suspended eachday prior to the administration in 0.1% gelatin-2500 swelling
- Final preparation of a solid: grounded

FORM AS APPLIED IN THE TEST (if different from that of starting material)
in suspension in 0.1% gelatin-25000 swelling

Test animals

Species:

rat

Strain:

other: Him:OFA (SD) SPF

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:Research Institute for Experimental animal breeding
- Age at study initiation: not specified
- Weight at study initiation: 114g (male) ; 108 g(female)
- Fasting period before study: not specified
- Housing:Makrolon type III cages
- Diet (e.g. ad libitum): pelleted Altromin breeding diet No. 1314 "fortified", sterilised by CO60 irradiation (2Mrad) ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 8 days

DETAILS OF FOOD AND WATER QUALITY:sterilised by CO60 irradiation (2Mrad)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21.5°C
- Humidity (%): 55-60%
- Air changes (per hr): air conditionned rooms, no more details
- Photoperiod (hrs dark / hrs light): 10/14 hours

From June 13rd, 1978 to September 21st 1978

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The substance was administered by a metal gavage tube once daily between 9 a.m. and noon from Monday to Friday (inclusive).

Vehicle:

other: 0.1% gelatin 250000 swelling

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was ground in an agate planetary mill in order to obtain a reasonably stable suspension and to avoid a displacement of the gavage tube by too large particles during administration. The test substance was freshly suspended eachday prior to the administration in 0.1% gelatin-2500 swelling

VEHICLE
- Justification for use and choice of vehicle (if other than water): not specified
- Concentration in vehicle: not specified
- Amount of vehicle (if gavage): 10 mL/kg

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

once daily (5 days per week)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

900 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

2 700 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 animals per sex per dose were used

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses were selected according to LD50 value of 3600 in an acute toxicity oral test and a dose finding study with oral administration during 14 days. This last study revealed, at a dose of 2700 mg/kg, a lower feed consumption and apathetic behaviour after the administration as the major changes in the animals.
- Rationale for animal assignment (if not random):
- Rationale for selecting satellite groups:
- Post-exposure recovery period in satellite groups:
- Section schedule rationale (if not random):

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily from Monday to Friday

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a week

BODY WEIGHT: Yes
- Time schedule for examinations: once a week

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Feed consumption was determined once a week, for 2 animals together. An amount of 500 mg feed provided at a time.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water consumption was determined once a week from Monday to Thursday

OPHTHALMOSCOPIC EXAMINATION: Yes
An ophtalmoscopy of all animals was caried out before the start of administration and at the day 87.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: one day before the start of administration, on the day 30 and on the day 84 and 86
- Anaesthetic used for blood collection: Yes (light ether)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked : Number of red blood cells, Haematocrit, Haemoglobin content, Mean Corpuscular volume, Number of white blood cells, differential blood count

CLINICAL CHEMISTRY:Yes
- Time schedule for collection of blood: one day before the start of administration, on the day 30 and on the day 84 and 86
- Anaesthetic used for blood collection: Yes (light ether)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked :Enzymatic determination of urea in serum ; determination of D glucose in serum, total protein, alkaline phosphatase activity, GPT activity, Na+ and K+

URINALYSIS: Yes
- Time schedule for collection of urine: 6 days before the start of the administration, on the 34th day (male) or 36th day (females), and on the day 83 (males) and 84 (females)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: External evaluation (colour, odour, turbidity,...) ; volume ; density using a weighed pipette; pH ; Iron ; glucose ; ketone bodies ; urobilinogen ; bilirubin and blood ; urinary sediment

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Following organs were weighed : kidneys, adrenal glands, spleen, testes, heart, liver, brain, pituitary gland

HISTOPATHOLOGY: Yes
Following organs were taken and fixed : kidneys, adrenals glands, spleen, stomach, duodenum, pancreas, ileum, caecum, lymphocentrum mesentericum, urinary bladder, ovary, uterus, testes, muscle (m. biceps fem.), peripheral nerve (n. ischiadus), mamma, skin, trachea, thyroid gland, thymus, lung, aortic arch, heart, esophagus, liver, eyes, brain, pituitary gland, spinal cord (from cervical and cranial thoracic area), lymphocentrum mandibulare

Statistics:

Either the mean ± standard deviation or the median together with the minimum and maximum values is given for each results. Measured values and frequencies above approximately 10 (e.g. the number of neutrophils) were tested for equality of the group means with the simple analysis of variance and subsequent Scheffé test. The level of significance was set at P= 0.05. Only the significant differences compared to control group 1 are identified in the tables. The applicability of the analysis of variance was tested with the Bartlett test (P=0.001). The H-test was used if the conditions for the analysis of variance were not fullfilled. The t-test was used if only two groups were available (P=0.05). Frequencies below approximately 10 (E.G. the number of monocytes) and ordinal data (e.g. the analyses in the urinary examination) were compared with the H-test and subsequent test according to Nemenyi or the median-test in the case of unequal sample sizes (P=0.05).

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

generally, the symptoms observed (somnolence to unconsciousness, tremor, convulsions, bradypnea) were more pronounced and persisted longer during the first days of administration than later on. After approximately one month, the animals of the group 4 in particular were only somnolent for a few minutes.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two animals died before the end of experiment. In both cases, necropsy showed that a faulty administration by gavage tube was causative for the premature death.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Male animals of group 4 showed a significantly lower body weight than control group 1 from the day 36 until the end of the experiment. Male animals of the group 2 and 3 did not differ to the controls. For female animals, no group lagged behind controls in the development of the body weight. Group 3 gained more weight than the other groups, but the difference to controls was only significant on two days (36 and 34)

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Feed consumption of treated male animals did not substantially differ from controls. In female animals, a significantly higher consumption occured twice, namely once in group 3 (day 35 to 42) and once in group 4 (day 63 to 70).

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

no effects observed

Description (incidence and severity):

The water consumption of group 4 was significantly higher compared to controls for nearly the entire duration of the experiment in females and until the day 58 in males.

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

One positive finding was obtained on the day 87 for one animal from each groups 3 and 4. There was considered as biologically not relevant.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The number of white blood cells was lower in group 4 than in control group 1 on the day 30 (significant only in females). No specific shift was discernible in the differential blood count, with only the number of eosinophils being lower in group4 (significant only in males). The other parameters were without noticeable differences. A dose-dependent decrease of the number of red blood cells, together with a dose-dependent decrease of the hematocrit and the haemoglobin concentration, occured in females and males in the last experimental week. A significant difference to controls for the haematocrit was observed even in the lowest dose group 2, the mean corpuscular volume was considerably higher in group 4 males than in controls.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The examinations before the start of the treatment showed a significant, but not explicable, lower urea concentration for group 4 males and 4 females compared to the controls. This difference was no longer observed in the subsequent analyses. No significant differences occured in the other parameters measured. A dose increase of GPT in the last experimental week occured as the only finding after the start of the administration. The difference between group 4 males and controls was statistically significant. The protein content in serum also increased with the dose in the last experimental week (and ths behaved different than the number of erythrocytes); however, there were no statistically significant differences.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

The urine of treated animals was coloured, up to dark brown in the group 4, during the entire period of administration. A increased quantity of urine was obsrved in the high dose group at the day 28, consistent with the increased water consumption. The significantly increased density of urines from group 4 is ascribed to the considerable amount of test item to be excreted. There was no difference observed in urinary sediment.
The urine from high dose animals was more acidic than that from controls and reached significantly lower pH values in some cases of group 4. The urine volume was significantly higher in group 4 female than in control during the first collection period. The density was lower, although not significantly.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

As with the absolute organ weights, a dose dependent increases of the organ weights related to body weight occured for the kidney, adrenal gland and, in particular, liver. Significant differences compared to controls existed for males and females. Even the lowest dose group 2 males showed significantly increased values in the case of the liver. A increased relative weight of the spleen (group 4 males) and a decreased relative weight of the pituitary gland (group 2 males) were observed as individual findings that were not clearly dose dependant.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Substance specific alteration and lesions of the stomach were observed. The following were observed in group 3 and 4 and were described : brown red deposit were more frequently found on the glandular mucous membrane, and preferentially in the crypts, of the stomachs. These deposites were in part also observed spread. A thickening, hardening and increased plication of the upper layer of the mucous membrane was often seen at the cutaneous mucous membrane. In group 4, approximately half of the animals were affected by such lesions.
The alterations described as emphysematous focus was in no case high-grade lung emphysema but rather reduced retractation of the lung. The differences between the groups in relation to the occurence of this funding was statistically not significant.

Description (incidence and severity):

Isolated cases of pathological changes were found, which also did not allow any reference to the treatment with the substance (complete atrophy of the germinal epitheliuem of testis, cystitis, involution of thymus). The emphysemous foci in lungs found at necropsy could be confirmed histologically. These were not very clearly pronounced alterations, rather being only locally deficient retractions of the lung. Moreover, the first evaluation of the routine sections elicited a suspicion alterations and lesions of the liver, kidney, stomach and pancreas. He-stained sections from all animals of all groups were therefore prepared from these organs and evaluated. A yellow brown pigment appeared more frequently in the spleen of the animals from the high dose group.
An increased frequency of optically empty intraplasmic vacuoles of liver cells and sporadic necrosis were found in the liver , findings that were significant in the highest dose group 4. Although no statistically validated, a dose-dependent accumulation of infiltrates with mononuclear, lymphocytic elements was also observed. Amorphous, eosinophilic hyaline (cylinders) with secondary dilation of the tubules were seen dose-dependantly increased in the examination of the kidneys. Degenerative changes of the kidneys tubules were also dose-dependently increased, which ranged from squamous degeneration to tubulonephrosis - although always restricted to individual nephrons only. As with the cass in he tubules, hyaline casts in the collecting tubule system were dose-dependently increased. The increased frequency of the changes in the treated groups described could partly be statistically validated down to group 3. Hyperkeratoses of the cutaneous mucous membrane and erosions of the glandular mucous membrane were more frequently found in increasing dose in the stomachs, with the group 3 and 4 substantially differing from controls.
Sudan stain frozen sections analyses : For the kidneys, female animals of groups 3 and 4 showed significantly more fat inclusion in the tubular epithelia than the other animals. A fatty degeneration, although only to a slight degree, has thus to be assumed in addition to the lesions on the renal tubules.
Berlin blue-stained sections analysis: Significantly more haemosiderin than in controls was detected in male and female animals of group 4 in male animals of group 3.

Effect levels

Key result

Dose descriptor:

LOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

gross pathology

organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

The main toxic effects in this study were local irritative effects on the stomach mucosa, hepatotoxicity and toxic effects on the red blood cells, apart from transient effects on the general behaviour immediately after application in the beginning of the study. Although of less severity, some of these effects were also observed at the lowest dose level. Hence, particularly due to effect observed at the low dose level (organ weight changes), no NOAEL can be derived. The LOAEL was defined as 300 mg/kg bw/day.

Executive summary:

The study was performed in order to investigate the potential toxicity of the registered substance 4 -amino-2 -hydroxytoluene when administered orally by gavage during 13 weeks (5days per week).

Doses of 0, 300, 900 and 2700 mg/kg bw/day of 4-amino-2-hydroxytoluene were administered 5 days/week to Him:OFA (SPF) rats orally by gavage over a period of 13 consecutive weeks. A control group was treated similarly with the vehicle only. The dose volume in treated and control groups was 10 ml/kg body weight.

The groups comprised 20 animals per sex which were sacrificed after 13 weeks of treatment. Mortality and clinical signs were recorded at least once daily; body weights, water and food consumption were recorded weekly during the treatment period. Clinical laboratory investigations (haematology, blood/clinical biochemistry) as well as urinalysis were carried out before the start of administration and twice during the treatment period. All animals were subjected to a detailed necropsy and a number of organs (kidneys, adrenals, spleen, testes, heart, liver, brain and pituitary gland) were weighed; numerous tissues and organs were fixed and stored for further examination if required.

The main toxic effects in this study were local irritative effects on the stomach mucosa, hepatotoxicity and toxic effects on the red blood cells, apart from transient effects on the general behaviour immediately after application in the beginning of the study. Although of less severity, some of these effects were also observed at the lowest dose level. Hence, particularly due to effect observed at the low dose level (organ weight changes), no NOAEL can be derived. The LOAEL was defined as 300 mg/kg bw/day.