3-amino-2-chlor-6-methylphenol

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24.06. - 21.10.2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

see field "Any information on materials and methods"

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EPA 712-C-98-199, 90-Day Oral Taxicity in Rodents, 1998

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3-amino-2-chlor-6-methylphenol

EC Number:

283-144-9

EC Name:

3-amino-2-chlor-6-methylphenol

Cas Number:

84540-50-1

Molecular formula:

C7H8ClNO

IUPAC Name:

3-amino-2-chloro-6-methylphenol

Test material form:

other: beige powder

Details on test material:

molecular weight: 157.5

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Recognised by international guidelines as the recommended test system.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Age: Approx. 6 weeks
Females were nulliparous and non-pregnant.
Conditions:
Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21.0 ± 3.0°C (actual range: 17.3 - 24.9°C), a relative humidity of 30-70% (actual range: 30 - 100%) and 12 hours artificial fluorescent light and 12 hours darkness per day. Temporary fluctuations from the light/dark cycle (with a maximum of 1 hour) occurred due to performance of functional observations in the room. Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.
Accommodation:
Group housing of 5 animals (recovery group allocation) or 4 animals (main group allocation) per sex per cage in stainless steel suspended cages with wire mesh floors (55 x 34 x 21.5 cm height). Acclimatisation period was at least 5 days before start of treatment under laboratory conditions. During activity monitoring, animals were individually housed overnight in Macrolon plastic cages (type 111, height 15 cm.) with sterilised sawdust (Woody-Clean type 3/4, Tecnilab­BMI BV, Someren, The Netherlands) provided as bedding. Results of bedding analyses for contaminants are examined and archived.
Diet:
Free access to standard pelleted laboratory animal diet (from Altrom in code VRF-1, Lage, Germany).
Animals were dosed up to the day prior to necropsy.
Water:
Free access to tap water.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Dose volume: 10 ml/kg body weight. Actual dose volumes were calculated weekly according to the latest body weight.

Vehicle:

propylene glycol

Duration of treatment / exposure:

90 days

Frequency of treatment:

Once daily for at least 90 days, 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

group 1 (vehicle control)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

group 2

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

group 3

Doses / concentrations 4

Dose / conc.:

600 mg/kg bw/day (nominal)

Remarks:

group 4

No. of animals per sex per dose:

12

Control animals:

yes

Details on study design:

Repeated dose 90-Day oral toxicity study with A 094 by daily gavage in the rat, followed by a 28-day recovery period.
Based on a 28-day range finding study, the dose levels for this 90-day oral gavage study were selected to be 0, 100, 300 and 600 mg/kg/day.

The test substance was administered daily for at least 90 days by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested,each consisting of 12 males and 12 females. An additional 5 animals per sex in the control and high dose group were allowed 28 days of recovery. The following parameters were evaluated: clinical signs daily; functional observation tests; body weight and food consumption weekly; ophthalmoscopy at pretest and in
week 13; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

Examinations

Observations and examinations performed and frequency:

Mortality/Viability:
At least twice daily. The time of death was recorded as precisely as possible.

Clinical signs:
At least once daily, detailed clinical observations were made in all animals. Once prior to start of treatment and at weekly intervals, this was also performed outside the home cage in a standard arena. The time of onset, degree and duration were recorded.
All symptoms were recorded and graded according to fixed scales: Maximum grade 1: grade O = absent, grade 1 = present
Maximum grade 3 or 4: grade 1 = slight, grade 2 = moderate, grade 3 = severe, grade 4 = very severe

Functional Observations:
During week 12-13 of treatment, the following tests were performed on all animals of the recovery groups and on all animals of the first cage of each main
group allocation (animal 26 replaced the decedent animal no. 22):
- hearing ability (HEARING), pupillary reflex (PUPIL L/R), static righting reflex (STATIC R) and grip strength (GRIP) (Score O = normal/present, score 1 = abnormal/absent).
- motor activity test (recording period: 12 hours during overnight for individual animals, using a computerised monitoring system, Pearson Technical Services, Debenham, Stowmarket, England). Based on the initial motor activity results, an additional motor activity measurement was performed in week 13 for animal nos. 11-14, 61-64, 81-84 and 131-134.

Ophthalmoscopic Examination ( direct):
Following instillation of tropicamide solution (5 mg/ml), both eyes were examined by means of an ophthalmoscope (Heine Beta 200): at pretest : All animals
at week 13 : Groups 1 and 4.

Body weights: Weekly.

Food consumption: Weekly

Water consumtion: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

Results and discussion

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

clinical signs

haematology

organ weights and organ / body weight ratios

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

kidney

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

Wistar rats were treated with A 094 for 90 consecutive days by oral gavage, followed by a 28-day treatment-free recovery period.
One male at 100 mg/kg/day and one female at 600 mg/kg/day were found dead in week 7/8 of treatment. A cause of death could not be established microscopically. Since there were no apparent signs of toxicity during treatment and the mortality incidence was unrelated to the dose, these deaths were not regarded to be related to treatment.

Histopathological examination of the liver revealed centrilobular hypertrophy at 300 and 600 mg/kg/day, the effect being more marked in males than in females. At 600 mg/kg/day, these changes correlated with increased absolute and relative liver weights and significant deviations in clinical biochemistry parameters (increased cholesterol levels in males and females, reduced urea level in males, and increased alanine aminotransferase activity in females), and are suggestive of perturbations in liver function. Although there was no clear morphological evidence of hepatocellular necrosis, the increased ALAT levels do suggest that hepatocellular damage may have occurred at 600 mg/kg/day. At the end of the recovery period, centrilobular hepatocytic hypertrophy remained in evidence in all high dose males at slight severity. Hepatocytic hypertrophy was also noted at a minimal degree in one male only at 100 mg/kg/day, and occurred in the absence of any other supportive functional or morphological changes. Centrilobular hypertrophy of hepatocytes is frequently an adaptive change in response to treatment with xenobiotics and at low incidence and severity considered not to be adverse.

lncidental histologic alterations in males consisted of hepatocellular necrosis (ranging from focal to organising nature) and chronic inflammation. The incidence of these observations did not show a relation to the dose. In addition, various animals (primarily males) distributed among the dose groups displayed increased plasma alanine and/or aspartate aminotransferase activity levels at the end of treatment or recovery phase. There was no clear dose-related incidence and there was no clear relation to the necrotic lesions seen in the liver. Therefore, these changes were considered to be of no toxicological relevance.

An increased incidence of cortical tubular basophilia was observed in the kidneys of most high dose males. Furthermore, forestomach limiting ridge hyperplasia (with two cases of squamous hyperplasia of the forestomach) was noted in all high dose males at the end of treatment. These histological lesions were not apparent in females and had resolved at the end of the recovery phase. The limiting ridge hyperplasia probably represented a response to local irritation to test material residing in the forestomach, and is a commonly observed typical adaptive phenomenon in rodent studies. This finding is regarded to be of no relevance to man.

The brown discolouration of the urine observed among all groups of treated animals during treatment was probably caused by the test substance and/or a test substance metabolite and was not regarded as adverse.

The increased mean corpuscular volume values noted in males and females at 100 and 600 mg/kg/day and the decreased mean corpuscular haemoglobin concentration values noted in males at 100 and 600 mg/kg/day and in all groups of treated females are suggestive of macrocytic hypochromic erythrocytes in treated animals. However, there was little evidence for an anaemic response; only males at 600 mg/kg/day showed marginally reduced erythrocyte counts, while haemoglobin levels and red cell distribution width were essentially similar in all groups. These deviations were therefore regarded not indicative of an adverse effect on red blood cells.

The increased bilirubin and plasma potassium levels in high dose males may relate to the changes seen in red blood cell parameters, but may also relate to the effects noted in the liver and kidneys, respectively.

There were no effects on body weight gain, food intake, ophthalmoscopy and macroscopy findings.

From the results presented in this report a No Observed Adverse Effect Level (NOAEL) for A 094 was established at 100 mg/kg/day.

Executive summary:

Repeated dose 90·-Day oral toxicity study with A 094 by daily gavage in the rat, followed by a 28-day recovery period.

Based on a 28-day range finding study, the dose levels for this 90-day oral gavage study were selected to be 0, 100, 300 and 600 mg/kg/day.

The study was based on the following guidelines.

-       EG Directive 67/548/EEC, B Repeated Dose (90 days) Toxicity (oral), 2001.

-       OECD 408, Repeated Dose 90-day Oral Toxicity Study in Rodents, 1998.

-       EPA 712-C-98-199, 90-Day Oral Toxicity in Rodents, 1998.

The test substance was administered daily for at least 90 days by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 12 males and 12 females. An additional 5 animals per sex in the control and high dose group were allowed 28 days of recovery.

The following parameters were evaluated: clinical signs daily; functional observation tests; body weight and food consumption weekly;

ophthalmoscopy at pretest and in week 13; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.