3,3',5,5'-tetra-tert-butylbiphenyl-2,2'-diol

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (GLP; no urinalysis, no neurobehavioural examination, histopathological examinations in limited nr. of tissues/organs [as required by the actual guideline])

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1989

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

May 12, 1981 (feeding)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Requirements of the Japanese Government under the revised Chemical Substance Law (1987) according to the notification of Dec. 9, 1986 by EA (No. 700), MHW (No. 1039) and MITI (No. 1014).

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3,3',5,5'-tetra-tert-butylbiphenyl-2,2'-diol

EC Number:

407-920-5

EC Name:

3,3',5,5'-tetra-tert-butylbiphenyl-2,2'-diol

Cas Number:

6390-69-8

Molecular formula:

C28 H42 O2

IUPAC Name:

3,3',5,5'-tetra-tert-butyl-[1,1'-biphenyl]-2,2'-diol

Details on test material:

- Name of test material (as cited in study report): generic name CA 16-178
- Physical state: yellowish powder
- Analytical purity: 97.7%
- Lot/batch No.: A 88103 / Z 1572R
- Storage condition of test material: room temperature

Test animals

Species:

rat

Strain:

other: albino rats

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Tif: RAIf (SPF), hybrids of RII/1 x RII/2 fom Animal Production, CIBA-GEIGY Limited, 4332 Stein / Switzerland
- Age at study initiation: approx. 4-5 weeks at delivery
- Weight at study initiation: 105.6 - 124.3 g in males; 98.8 - 116.7 g in females; at week -1
- Housing: in groups of 5 in macrolon cages type 4
- Diet (e.g. ad libitum): pelleted, certified standard diet (Nafag No. 890 Tox) containing the appropriate concentrations of the test article when necessary; ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2°C
- Humidity (%): 55±10%
- Air changes (per hr): 16-20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Mixing appropriate amounts with food: the test substance was weighed on a calibrated Mettler balance. The pulverized food was then homogeneously mixed with the appropriate concentrations of the test article and about 25% water was added before pelleting to ensure the necessary pellet quality. The pellets were subsequently air dried.
- Storage temperature of food: room temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Control analyses (analysis of content, homogeneity and stability) of the test article content were undertaken with diet used from treatment day 1-28 (GC). The contents of the test substance in the diet were in agreement with the nominal concentrations. The distribution of the active ingredient in the samples was considered homogeneous. All food samples were stable for 35 days at room temperature (concentrations of 82.3 - 97.1% of the nominal values were revealed).

Duration of treatment / exposure:

28 days treatment followed by 15 days recovery

Frequency of treatment:

continuously in diet

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
150, 650, 3000 and 12000 ppm
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
14.4, 61.0, 291 and 1150 mg/kg bw
Basis:
other: nominal dose ingested, calculated based on the food consumption and body weight

Doses / concentrations 3

Remarks:

Doses / Concentrations:
129/118, 572/577, 2750/2690 and 11700/11600 ppm (mean from study day 1-28; in males/females)
Basis:
other: analytical

Doses / concentrations 4

Remarks:

Doses / Concentrations:
11.8/11.7, 53.9/52.0, 264/253 and 1110/1120 mg/kg bw (in males/females)
Basis:
other: analytical dose ingested, calculated based on the food consumption and body weight

No. of animals per sex per dose:

5 (for main test), and 5 (for recovery)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: the dose levels were fixed based on the results of the acute toxicity study (study report number 884614).
The following doses were selected:
(1) 150 ppm expected to result in a daily intake of about 13 mg/kg and to cause no observable adverse effects,
(2) 650 ppm expected to result in a daily intake of about 55 mg/kg and to cause no or minimal adverse effects,
(3) 3000 ppm expected to result in a daily intake of about 250 mg/kg and to cause slight adverse effects, if any, and
(4) 12000 ppm expected to result in a daily intake of about 1000 mg/kg and to cause observable adverse effects, but no fatalities which would prevent a meaningful evaluation of the study.
- Rationale for selecting satellite groups: recovery evaluation
- Post-exposure recovery period in satellite groups: 15 days

Positive control:

not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: all animals were checked daily (a.m. and p.m. on working days, a.m. on weekends and holidays), in order to record mortalities, and to allow dead or moribund animals to be submitted to necropsy as soon as possible.
In order to detect changes in state of health or behaviour, or, in the case of dosed animals, any reaction to treatment, examination was carried out daily, and observations were recorded at least weekly.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly (midweek); the first weighing was done during the acclimatisation period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- 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: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: weekly (cagewise)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period, and at the end of the recovery period on surviving scheduled animals (each in the morning)
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes, overnight
- How many animals: all
- Parameters examined: erythrocyte count, hemoglobin, haematocrit, mean corpuscular volume, mean corpuscular hemoglobin, reticulocytes, leucocyte count, differential leucocyte count, thrombocyte count, prothrombin time, methemoglobin.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: as for heamatology
- Animals fasted: Yes
- How many animals: all
- Parameters examined: glucose, urea, creatinine, total bilirubin, total protein, albumin, globulins, albumin/globulins ratio, cholesterol, triglycerides, sodium, potassium, calcium, chloride, phosphorous inorganic, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma-glutamyl transpeptidase.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes; all animals were bled under ether anesthesia at the end of the treatment/recovery period and subjected to detailed autopsy. Besides the weight of the exsanguinated body, the following organs were weighed from all animals sacrificed at scheduled sacrifices: brain, liver, kidneys, adrenals, ovaries/testes. The following organs and tissues were preserved in neutral buffered 4% formalin: skin; mammary area; spleen; mesenteric lymph node; axillary lymph node; popliteal lymph node; sternum with bone marrow; femur with joint; skeletal muscle; trachea; lung; heart; aorta; submandibular salivary gland, both; liver; pancreas; esophagus; stomach; small intestine; large intestine; kidney, both; urinary bladder; prostate; seminal vesicle; testis, both; epididymis, both; uterus; vagina; ovary, both; pituitary gland; adrenal gland, both; thyroid with parathyroid gland; thymus; peripheral nerve; brain; spinal cord; eye with optic nerve, both; orbital gland, both; extraorbital lacrimal gland, both; Zymbal gland, both; muzzle; tongue; any tissue with gross lesions.
HISTOPATHOLOGY: Yes; after fixation, the organ samples listed below were embedded in paraplast, sectioned at 3-5 microns, stained with hematoxylin and eosin and subjected to microscopical examination: spleen; heart; liver; kidney, both; adrenal gland, both; any organ with gross lesions. These organs were examined from all animals of experimental main groups and consequent upon the findings in these animals, the livers of animals from experimental recovery groups were also examined.

Other examinations:

None

Statistics:

For each time point and parameter an univariate statistical analysis is performed. Nonparametric methods are applied, to allow for non normal as well as normal data distribution. Each treated group is compared to the control group by Lepage's two-sample test (a combination of Wilcoxon and Ansari-Bradley statistics. Increasing or decreasing trends in location from control to the highest dose group are tested by Jonckheere's test for ordered alternatives. A statistically significant difference between two values does not necessarily imply biological relevance of that deviation and is not conclusive for a treatment related effect. Hence, comment may not be made on statistically significant values lying within the physiological range and, or on statistically not significant values, which differ substantially from the expected normal values.

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY
- No death occurred during this study
- No relevant clinical symptoms and no signs of toxicity were observed.

BODY WEIGHT AND WEIGHT GAIN
The mean bodyweight gain of all treated groups was comparable to the respective controls. The marginally increased (males) and reduced (females) mean bodyweights were considered due to minimally imbalanced mean group weights at the beginning of the study and therefore unrelated to the treatment.

FOOD CONSUMPTION
The mean food consumption values of all treated groups were comparable to the respective controls. The observed differences in food consumption between treated and control groups were present from the beginning of the study and therefore considered unrelated to the treatment.

FOOD EFFICIENCY
The food consumption ratios of all treated groups were comparable to the respective controls.

WATER CONSUMPTION
The mean water consumption values of both untreated and treated groups showed some variations in both sexes, but were considered within the limits of our determination method and therefore unrelated to the treatment

HAEMATOLOGY
A dose-related decrease of red blood cell parameters (erythrocyte count, hemoglobin, packed cell volume) associated with higher values of reticulocytes was observed in animals of groups 650, 3000 and 12000 ppm. In addition, a mild leucocytosis occurred in the males of the high dose group (12000 ppm). Within the recovery period of two weeks partial reversibility of these effects was noted in the male groups only.

CLINICAL CHEMISTRY
A minor hyperglycemia and a dose-dependent increase of plasma protein levels, due to higher albumin and globulin levels, occurred in males and females of all treatment groups. Increased activities of aspartate aminotransferase and alanine aminotransferase were noted in both sexes of group 3000 and 12000 ppm, and higher alanine aminotransferase activities were also recorded in animals of group 650 ppm. In the absence of a dose-relationship, no biological relevance was attributed to the higher triglyceride levels noted in the males of some treatment groups. The alterations in clinical chemistry were not reversible within the recovery period of two weeks.

ORGAN WEIGHTS
At the end of the treatment period, absolute and relative mean liver weights showed a trend to dose-related increase. In males of groups 650, 3000 and 12000 ppm the absolute and relative mean liver weights were increased by 17-19%, 30-36% and 21-23%, respectively. In females of groups 3000 and 12000 ppm the absolute and relative mean liver weights were increased by 21-24% and 12-16%, respectively.
After the 2 weeks recovery period, absolute and relative mean liver weights in male groups 650, 3000 and 12000 ppm were still increased by 9-17%, 4-7% and 10-14%, respectively. In female groups 650, 3000 and 12000 ppm, the relative mean values were still increased by 12, 22 and 13%.
All other absolute and relative organ weights were considered within the historical control values.

GROSS PATHOLOGY
Very few macroscopical findings were noted. The only finding considered treatment-related was enlargement of the liver in 3/5, 4/5 and 4/5 males treated with 650, 3000 and 12000 ppm and 2/5, 1/5 females treated with 3000 and 12000 ppm respectively. This phenomenon was only observed in one male of the recovery group treated with 12000 ppm.

HISTOPATHOLOGY:
Histological examination revealed a treatment-related fatty change in the liver in all males and females treated with 650 ppm and above. This phenomenon appeared to be dose-related and was still present in animals of the same dose groups allowed a period of recovery but to a slightly lesser degree. Extramedullary haematopoiesis in the liver was increased above background levels in the same dose groups as above, and to a slightly lower extent in animals of the recovery group, particularly in females.
The other changes observed were related to commonly-occurring background diseases in laboratory rats. They were not dose-related and are considered to be incidental in nature and to have no experimental significance.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

11.7 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: 150 ppm (nominal) corresponding to 11.8 mg/kg bodyweight in males and 11.7 mg/kg bodyweight in females (analytical; calculated based on body weight and food consumption).

2

Dose descriptor:

LOAEL

Effect level:

52 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: 650 ppm (nominal) corresponding to 53.9 mg/kg bodyweight in males and 52.0 mg/kg bodyweight in females (analytical; calculated based on body weight and food consumption). Affected organs/tissues: liver, blood

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Mean body weight

	Mean body weight (g)
Dose level	0 ppm		150 ppm		650 ppm		3000 ppm		12000 ppm
Time point	Male	Female	Male	Female	Male	Female	Male	Female	Male	Female
Main test
Week -1	113.2	109.2	115.2	107.3	115.5	108.6	113.8	107.5	116.8	105.2
Week 1	184.9	153.3	188.7	152.4	189.2	151.8	185.1	154.0	188.2	147.5
Week 2	239.4	183.0	242.9	177.4	244.9	175.0	242.1	177.9	244.6	171.4
Week 3	280.3	202.4	289.0	198.3	291.9	191.3	288.6	197.3	290.8	189.0
Week 4	311.5	219.9	323.8	209.8	327.0	207.8	324.0	209.1	323.8	201.7
Recovery
Week 5	331.3	237.5	347.5	225.6	352.3	211.3	337.8	217.1	346.0	207.4
Week 6	351.9	251.0	371.1	234.6	376.1	226.6	362.9	229.6	365.4	217.0

Table 2: Hematology parameters showing influenced by treatment

Time period	Blood parameters	Mean values per dose group and per sex
		0 ppm		150 ppm		650 ppm		3000 ppm		12000 ppm
		Males	Females	Males	Females	Males	Females	Males	Females	Males	Females
Week 5	RBC (count/l)	7.477	7.731	7.519	7.738	7.113	7.407	7.127	7.248	7.049	7.196
	Hb (mmol/l)	9.596	9.796	9.597	9.856	9.407	9.421	9.188	9.356	9.073	9.321
	Reticulocytes (l)	0.034	0.028	0.037	0.026	0.050	0.040	0.052	0.042	0.055	0.038
	Hematocrit (l)	0.458	0.465	0.455	0.465	0.441	0.442	0.435	0.434	0.432	0.429
Week 7	RBC (count/l)	7.536	7.505	7.680	7.229	7.307	6.936	7.287	7.001	7227	6.856
	Hb (mmol/l)	9.523	9.718	9.695	9.476	9.611	9.286	9.606	9.277	9.499	9.164
	Reticulocytes (l)	0.035	0.029	0.046	0.038	0.054	0.036	0.050	0.043	0.053	0.042
	Hematocrit (l)	0.446	0.444	0.453	0.446	0.449	0.434	0.443	0.437	0.444	0.425

 

Table 3: blood chemistry parameters showing influenced by treatment

Time period	Blood parameters	Mean values per dose group and per sex
		0 ppm		150 ppm		650 ppm		3000 ppm		12000 ppm
		Males	Females	Males	Females	Males	Females	Males	Females	Males	Females
Week 5	Glucose (mmol/l)	7.364	6.019	9.206	6.972	8.811	6.331	8.615	7.128	9.578	7.079
	Plasma proteine (g/l)	65.62	64.50	69.71	66.32	71.17	68.57	71.47	69.56	72.48	69.63
	ASAT (U/l)	61.66	62.59	63.65	61.04	71.19	68.13	97.90	80.03	79.48	85.73
	ALAT (U/l)	46.10	36.03	47.16	39.99	71.21	53.23	109.1	77.66	91.49	93.20
Week 7	Glucose (mmol/l)	8.166	6.082	9.920	6.650	9.254	7.022	10.00	7.626	10.70	8.018
	Plasma proteine (g/l)	63.52	62.94	64.16	64.85	68.94	64.99	69.29	66.84	69.62	68.74
	ASAT (U/l)	65.78	67.48	65.06	56.08	59.58	67.80	64.70	86.94	82.34	110.5
	ALAT (U/l)	56.98	38.56	43.00	40.46	64.10	65.54	71.92	87.12	101.2	136.9

ASAT: Aspartate aminotransferase; ALAT: alanine aminotransferase. Bold format indicates significant difference to control values.