Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02/07/1991-12/08/1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline Study (OECD 407)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2,6-bis(1,1-dimethylethyl)-phenol
- Substance type: dialkylphenol
- Physical state: solid at 20°C (colourless solid)
- Analytical purity: 98.7%
- Lot/batch No.: 10/6
- Expiration date of the lot/batch: July 01, 1992
- Instructions for test substance storage: at room temperature in the dark
- Stability in vehicle: stable in polyethylene glycol for at least 48 h

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL Ltd. Basel, Switzerland
- Age at study initiation: approximately 6 weeks
- Housing: animals were housed 5 to a cage (same sex) in stainless steel suspended cages with wire mesh floors.
- Diet (e.g. ad libitum): free access to standard pelleted laboratory animal diet (Kliba, Klingeltalnuehle AG, 4303, Kaiseraugst, Switzerland). each batch was analysed for contaminants and results were examined and archived.
- Water (e.g. ad libitum): tap water, ad libitum. Results of chemical and contaminants analyses are examined and archived quarterly.
- Acclimation period: at least 7 days. Veterinary examination was performed prior to commencement of treatment to ensure that the animals were in a good state of health.
- Identification: earmark and tattoo
- Randomisation: computer-generated random algorithm according to body weight with all animals ± 20% of the sex mean



ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C
- Humidity (%): 55%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light


IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on oral exposure:
- Method: the test substance was heated to approximately 50°C and subsequently weighed into a glass flask on an analytical balance and the vehicle (w/w) added. Adjustment was made for specific gravity of vehicle. If necessary test substance formulations were re-heated to approximately 50°C in order to form a suitable suspension.
- Frequency of test substance formulation: daily immediately prior dosing
- Homogeneity of test substance in vehicle: by stirring. Homogeneity during treatment was maintained using a magnetic stirrer.
- Storage instructions for test substance formulation: at ambient temperature
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of formulations: samples of formulations prepared during week 1 were analysed to check accuracy of preparation. based on changes in appearance of previously heated and dispensed test substance, possible instability was suspected. Therefore samples were taken from visually normal or different appearing test substance and analysed for the actual contents, in order to check stability of test substance under storage conditions.

Analysis of dose preparations: Analytical report (appendix 2) Subacute 28-day oral toxicity with 2,6-bis(1,1-dimethylethyl)-phenol by daily gavage in the rat. Determination of the accuracy of preparation of 2,6-bis(1,1-dimethylethyl)-phenol in polyethylene glycol and the stability of test substance under storage conditions. The concentrations were determined using a HPLC method. Test substance formulations in polyethylene glycol formed a solution at all test concentrations. Analysis of the accuracy of dose preparations revealed values within the range -4 to -2% of nominal, which was considered for this type of formulation.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily, approximately the same time each day, 7 days per week.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg b.w./day
Basis:
other: oral gavage, 5 ml/kg body weight
Remarks:
Doses / Concentrations:
15 mg/kg b.w./day
Basis:
other: oral gavage, 5 ml/kg body weight
Remarks:
Doses / Concentrations:
100 mg/kg b.w./day
Basis:
other: oral gavage, 5 ml/kg body weight
Remarks:
Doses / Concentrations:
600 mg/kg b.w./day
Basis:
other: oral gavage, 5 ml/kg body weight
No. of animals per sex per dose:
5
Control animals:
yes
Details on study design:
- Dose selection rationale: a 5-day range finding study was performed (with 3 rats/sex/group at dose levels of 50, 200 and 100 mg/kg/day) to provide basis for selection of dose levels for a study of longer duration.
Animals receiving 1000 mg/kg/day showed hunched posture, piloerection, diarrhoea, bradypnoea and laboured respiration. After 5 days of oral treatment, males receiving 1000 mg/kg/day showed low body weight gain and females receiving 1000 mg/kg/day showed body weight loss, which were both associated with decreased food consumption. An irregular grey-white appearance of the forestomach was noted in 2/3 males and 3/3 females receiving 1000 mg/kg/day, in 1 female accompanied with thickening of the limiting ridge (junction between glandular stomach and forestomach). A small increase of absolute and relative liver weights were noted in males and females receiving 1000 mg/kg/day. Among animals receiving 200 or 50 mg(kg/day, changes occurred incidentally and were within normal background variation for rats of this age and strain.
Based on these observations and following consultation of the sponsor, treatment levels for a study of 28 days duration were selected
- 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: No
- Time schedule:
- Cage side observations checked in table [No.?] were included.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily. Severity of observations were graded.


BODY WEIGHT: Yes
- Time schedule for examinations: weekly and on the day preceding termination, prior to overnight fasting.
- body weight (g) and body weight gain (%)


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): weekly
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data:No


FOOD EFFICIENCY:
- 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: No


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): subjective appraisal was maintained during the study, but not quantitative investigation introduced as no effect was suspected.


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: both eyes were examined following instillation of tropicamide solution (5mg/ml) during the last week of treatment.
- Dose groups that were examined: all animals


HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of termination, immediately prior to post mortem examination, between 8.00 and 10.00 a.m.
- Anaesthetic used for blood collection: Yes (identity) light ether anaesthesia
- Animals fasted: Yes (overnight), but water was provided
- How many animals: all rats/sex/group
- Parameters checked in table were examined.


CLINICAL (BIO)CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of termination, immediately prior to post mortem examination, between 8.00 and 10.00 a.m.
- Animals fasted: Yes (overnight), but water was provided
- How many animals: all rats/sex/group
- Parameters checked in table were examined.


URINALYSIS: No
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / No data
- Animals fasted: Yes / No / No data
- Parameters checked in table [No.?] were examined.


NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:


OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see information below)
HISTOPATHOLOGY: Yes (see information below)
Statistics:
The following statistical methods were used to analyse the body weight, organ weights and clinical laboratory data:
Univariate one-way analysis of variance was used to assess the significance of intergroup differences.
If the variables could be assumed to follow a normal distribution, the Dunnett-test (many to one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups.
The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
All tests were two-sided and in all cases p<0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.
Individual values, means, standard deviations and statistics were rounded off before printing. For example, test statistics were calculated on the basis of exact values for means and pooled variances and then rounded off to two decimal places. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
The exact Fisher-test was applied to the ophthalmoscopic examination data.

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:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no clinical signs of toxicity or behavioural changes noted over the 29 day observation period that were considered to be related to treatment with the test substance.
No mortality occurred during the study period.

BODY WEIGHT AND WEIGHT GAIN
Body weights and body weight gain of treated animals remained in the same range as controls over the 4 week study period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
There were no differences in food consumption before or after allowance for body weight between treated and control animals.

FOOD EFFICIENCY


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)


OPHTHALMOSCOPIC EXAMINATION
There were no differences between control and treated animals, upon opthalmoscopic examination at week 4, that could be attributed to the treatment with the test substance.

HAEMATOLOGY
haematological parameters of treated rats were considered not to have been affected by treatment.

CLINICAL CHEMISTRY
Changes in clinical biochemistry parameters of the serum were noted in total protein, albumin, inorganic phosphate, calcium and urea.

URINALYSIS


NEUROBEHAVIOUR


ORGAN WEIGHTS
Toxicological significant differences between organ weights of treated and control animals were noted in the liver and kidney. At 600 mg/kg/day male and females had around 30 to 40% heavier livers than control animals whilst a 20% increase in relative (% of bodyweight) liver weight was noted in males dosed at 100 mg/kg/day.

GROSS PATHOLOGY
The principal change noted at macroscopic examination was enlarged caecum. Other changes, including enlarged liver, enlarged kidneys and swollen limiting ridge in the stomach, were incidentally occurring and considered to be of possible toxicological significance.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopic alterations that were considered to be treatment-related were noted in the liver and kidneys.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)


HISTORICAL CONTROL DATA (if applicable)


OTHER FINDINGS

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
15 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: clinical signs; mortality; body weight; food consumption; ophthalmoscopic examination; haematology; clinical chemistry; gross pathology; organ weights; histopathology.
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: Increased relative liver weight in males with no accompanying microscopic changes. Reduced serum urea in females. Macroscopically enlarged caecum in males. These changes were not of toxicological significance.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Clinical signs

Excessive salivation was noted in control animals and treated animals. Among control animals and animal receiving 15 mg/kg/day excessive salivation occurred incidentally in a few animals. All animals receiving 100 or 600 mg/kg/day showed intermittent periods of excessive salivation over the study period. However, this finding is frequently noted in rats of this age and strain following oral treatment via a stomach tube or may be associated with a bad taste or possible irritant effect of the test substance. Therefore no toxicological significance was attached to this finding.

Alopecia was noted in 1 control female (shoulder region) and in 4 out of 5 females receiving 100 mg/kg/day (cheek region). In 3 females receiving 100 mg/kg/day incidental wounds or scabs were also noted on the cheek. Alopecia is a common phenomenon in rats of this age and strain. Furthermore, given the increased incidence in 1 cage and the incidental occurrence of wounds and scabs it is very likely that this finding occurred at a higher incidence as a result of increased grooming and scratching activity rather than as a result of treatment.

Clinical Biochemistry

Statistically significantly increased total protein was noted in males and females receiving 600 mg/kg/day when compared with control animals. In males receiving 600 mg/kg/day, albumin levels were also noted to be statistically significantly higher than controls. No differences were noted between control animals and animals receiving 100 or 15 mg/kg/day, in total protein albumin levels.

Inorganic phosphate was noted to be statistically significantly decreased compared to controls in females receiving 600 mg/kg/day, but not in males receiving 600 mg/kg/day. Inorganic phosphate levels in animals receiving 100 or 15 mg/kg/day remained in the same range as control animals.

Urea values were noted to be statistically significantly decreased, in comparison with control females, in females receiving 600 or 100 mg/kg/day. Urea values in females receiving 15 mg(kg/day or treated males remained in the same range as control animals.

Among females receiving 600 mg/kg/day, statistically significantly increased calcium levels were noted when compared to control females. Females receiving 100 or 15 and treated males were not thus affected.

Macroscopic examination

Macroscopically observed enlarged caecum was noted in 4/5 males and all females receiving 600 mg/kg/day. Enlargement of the caecum was also noted in 2/5 males receiving 100 mg/kg/day. females receiving 100 mg/kg(day or animals receiving 15 mg/kg/day did not show changes of the caecum.

Enlarged liver and enlarged kidneys (accompanied with green appearance of the kidneys) was noted in 1 male receiving 600 mg/kg/day. Another male receiving 600 mg/kg/day had a thickened limiting ridge (junction between glandular stomach and forestomach).

A cyst, noted in the left uterus left horn of 1 female receiving 15 mg/kg/day, was considered within normal background variation for rats of this age and strain and not to be of biological significance.

Organ weights

Liver weights of males and females receiving 600 mg/kg/day were statistically significantly increased in comparison with controls before and after allowance for body weight. in males receiving 100 mg/kg/day, relative liver weights were also statistically significantly higher than controls. There were no differences noted between liver weights of control animals and those of females receiving 100 mg/kg/day or animals receiving 15 mg/kg/day.

Relative kidney weights were statistically significantly increased in males receiving 600 mg/kg/day when compared to control males. Realtive kidney weights of females receiving 600 mg/kg/days or animals receiving 100 or 15 mg/kg/day remained similar to kidney weights of control animals.

Microscopic examination

Among 4/5 and 5/5 females receiving 600 mg/kg/days, very slight hepatocellular hypertrophy was noted in the centrilobular of the liver.

In the renal cortex of males receiving 600 mg/kg/day, slight accumulations of tubular eosinophilic inclusion bodies were encountered in more rats and at a high degree than among control males.

There were no treatment-related microscopic changes noted in animals receiving 100 or 15 mg/kg/day. The small number and the degree of changes recorded, remained within the normal incidence of background variation.

There were no histopathological correlates noted in the caeca, which were notice to be macroscopically enlarged.

Analysis of the test substance and preparations

The stability of the test substance under the storage conditions applied in this study (room temperature and in the dark) was confirmed as satisfactory. All test substance formulations were solutions and the actual concentrations remained within -4 to -2% of nominal concentrations, which was considered to be acceptable for this type of formulation.

Discussion

In this 28 -day oral toxicity study in rats, organs that appear to be affected by treatment were the liver and kidneys. The liver of animals receiving 600 mg/kg/day and of males receiving 100 mg/kg/day were noted to be heavier than controls. Upon microscopic examination, these livers showed slight hepatocellular hypertrophy, which may be responsible for the increase in liver weights. There were no changes in liver enzymes noted in the serum, therefore, the increased liver weights and related increase in the number of liver cells may be attributed to an increased work hypertrophy. As this is a common phenomenon in rats, in order to metabolise a xenobiotic agent, these findings were considered not to be a change of toxicological significance.

Increased kidney weights were only noted in males receiving 600 mg/kg/day and may be associated to the histophatologically observed increase in tubular accumulations of eosinophilic inclusion bodies in the kidneys. There were no changes in serum biochemical parameters of males that may be possibly related to kidney damage. Changes in serum electrolyte levels(i.e. decreased phosphate and increased calcium) and decreased urea levels in females receiving 600 mg/kg/day did not show any corroborative pathological changes. These changes were not considered to be of toxicological significance.

Increased total protein values, and in male also increased albumin values, were noted in animals receiving 600 mg/kg/day. As there were no associated disturbances in nutritional status or water balance noted during the study, these increased serum protein values were considered not to represent a clear sign of toxicity.

At termination of the study animals receiving 600 mg/kg/day and a few males receiving 100 mg/kg/day showed and enlarged caecum at macroscopic examination. there were however, no microscopic changes noted that could be related to the macroscopically enlarged caecum. The toxicological significance therefore must remain unclear.

From the results presented in this study, a definitive No Observed Adverse Effect Level of 100 mg/kg/day was established.

Applicant's summary and conclusion

Conclusions:
Dose levels were 0, 15, 100 and 600 mg/kg/day. Treatment related changes were observed in the blood, liver and kidney. At 600 mg/kg/day males and females had around 30 to 40% heavier livers than control animals and these weight increases were accompanied by very slight centrilobular hepatocellular hypertrophy. Although relative liver weight (% of bodyweight) in males at 100 mg/kg/day was approximately 20% heavier than controls this was not accompanied by microscopic changes and liver weight in females was comparable to controls. The increased liver weight and centrilobular hypertrophy noted at 100 and 600 mg/kg/day was considered to be an adaptive change and of no toxicological significance. Reduced serum urea (17%) was present in females at 100 mg/kg/day and also noted in females at 600 mg/kg/day (33%); this is not considered to be a toxicologically relevant finding as a pathological change is normally reflected by an increase in serum urea levels. At necropsy examination enlarged caecum was noted in 100 and 600 mg/kg/day males. No microscopic changes were noted that could be related to the enlarged caecum noted macroscopically and therefore this finding was not considered to be of toxicological significance.
The NOEL in this study was determined to be 15 mg/kg/day. However, it may be considered that the findings at 100 mg/kg/day were not adverse in that they would not be considered to affect the performance of the whole organism or the ability of the organism to respond to an additional environmental challenge. The NOAEL in this study was therefore, 100 mg/kg/day.
Executive summary:
A subacute 28 -day toxicity study was performed to assess the repeated dose toxicity of the test material, 2,6 -bis(1,1 -dimethylethyl), in the SPF-bred Wistar rat. The method used followed that described in the OECD Guidelines for Testing of Chemicals, Section 4, Health Effects, No. 407, "Repeated Dose Oral Toxicity - Rodent: 28 or 14 day study", Paris Cedex, May 12, 1981 and EEC Directive 84/449/EEC, Annex V of the EEC Directive 67/548/EEC, Part B: Methods for the Determination of Toxicity; B.7: "Sub-acute Toxicity - Oral". Official Journal of the European Communities No. L251, September 1984.

The results may be used as a basis for classification and labelling.

Following a range-finding study, the test substance was administered daily by gavage to 4 groups of ten animals (five males and five females) at concentrations of 0 (control), 15, 100 and 600 mg/kg/day.

All animals were subjected to daily clinical observation. Body weight and food consumption were measured weekly and on the day before necropsy. During week 4 of treatment, both eyes of all animals were examined. On the day of termination blood was collected from each animal for clinical laboratory investigations. Subsequently a necropsy examination was performed, macroscopic observations and organ weights were recorded. A histopathological examination was performed on adrenals, heart, kidneys, liver, spleen and stomach.

At 15 mg/kg/day no treatment-related changes were detected.

At 100 mg/kg/day decreased serum urea was noted in females only. Increased relative liver weights were noted in males only. Enlarged caecum was noted in 2/5 males at macroscopic examination.

At 600 mg/kg/day an increased serum total protein level was noted in males and females and increased serum albumin level was noted in males only. Decreased serum urea was noted in females only. Decreased inorganic phosphate and increased calcium was noted in the serum of females only. Enlarged caecum was noted in 4/5 males and 5/5 females at macroscopic examination. Incidentally occurring findings included enlarged liver, enlarged kidneys and swollen limiting ridge in the stomach. Increased liver weights were noted in males and females and increased kidney weights were noted in males only. A microscopically observed very slight increase of hepatocellular hypertrophy in the centrilobular area of the liver was noted in males and females and increased slight accumulations of eosinophilic inclusion bodies were noted in the renal cotex of males only.

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