4,4',4''-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol]

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 December 1985 to 27 March 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study compliant to GLP and reviewed by US EPA

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Principles of method if other than guideline:

Four groups, each of 20 male and 20 female sprague dawley rats, exposed orally via diet up to a maximum dose of 5000ppm mg/kg daily over a period of 13 weeks.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4,4',4''-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol]

EC Number:

217-420-7

EC Name:

4,4',4''-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol]

Cas Number:

1843-03-4

Molecular formula:

C37H52O3

IUPAC Name:

4-[4,4-bis(5-tert-butyl-4-hydroxy-2-methylphenyl)butan-2-yl]-2-tert-butyl-5-methylphenol

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

The test article, a fine white powder, was received in clear plastic bags packed in 4 cardboard drums (circa 3.6 to 4.1 kg each) from ICI America Inc., Wilmington, Delaware 19897, USA, on October 3, 1985.
The test article was stored in the original containers at room temperature away from direct light when not in use.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

One hundred and four (104) male and 104 female Sprague-Dawley rats (Rattus norvegicus, Crl :CDR[SD]BR), 28 + 1 day old, were received from Charles River Canada Inc., St. Constant, Quebec, on December 10, 1985.
Subsequent to arrival, all animals were subjected to a complete physical examination by a qualified veterinarian and/or veterinary aide to ensure "normal" health status. Ten (10) animals/sex were used to establish the health of the animals used in this study. Blood samples were obtained from the aorta of rats under ether anesthesia for evaluation of hematology and blood biochemistry parameters. Gross pathological examination was then carried out on all health screen animals. Such examination revealed no abnormal findings.
The body weight range prior to randomization was 86 to 127 g for males and 76 to 112 g for the females.
Each animal was housed individually in a stainless-steel cage of conventional design equipped with an automatic watering system. Each cage was labeled with a color-coded card indicating the study number, animal number, group, sex and dose level.

All animals were housed in a single room with controlled temperature and humidity (target temperature 21 ± 3°C and target humidity 50 ± 20%). Although some of the values were occasionally outside the prescribed range, these deviations were not considered to have influenced the outcome of the study. A 12-hour light/12-hour dark photoperiod and 12 air changes/hour were maintained throughout. An 11-day acclimation period preceded treatment.

A commercially available certified rodent diet (Purina Laboratory Chow #5002) and fresh municipal tap water were available ad libitum except when laboratory investigations were carried out. Routine water analyses were performed every 3 months at Bio-Research Laboratories Ltd. for microbiological counts, while chemical analyses were performed by Eco-Research Inc., 121 Hymus, Pointe Claire, Quebec.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

not specified

Details on oral exposure:

Dietary Preparation
Fresh treated diets of the appropriate concentration (mg/kg diet -ppm) for each group receiving the test article were prepared weekly. New diet was offered to the animals once weekly within 3 days of preparation.

The high-dose dietary preparation (5000 ppm) was prepared first by adding 50-100 g of preweighed basal diet to the appropriate amount of preweighed Topanol CA and mixing it in a glass beaker with a rubber spatula to form a premix. This premix was then added to the remaining required amount of basal diet and mixed in a V-blender for approximately 15 minutes.

Following the high-dose preparation, the V-blender was cleaned with an air jet gun and 5 kg of basal diet was mixed in the V-blender for approximately 5 minutes to prevent carry-over of the test article. The basal diet was then discarded. Thereafter, the low-dose diet (100 ppm) was prepared by dilution of the required amount of 5000 ppm diet preparation with the appropriate quantity of basal diet arid mixed in the V-blender for 15 minutes. The intermediate-dose diet (500 ppm) was prepared last, using the same procedure as the low-dose diet preparation.

Each diet was stored in prelabeled stainless-steel buckets (away from direct light), in order to eliminate any possibility of toluene (present as an impurity due to the manufacturing process) leaching material (s) from plastic buckets. Buckets were stored at room temperature, under a fume hood.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

METHOD
The method of analyses is described in Bio-Research SOP No. C.20.65, developed and validated by Bio-Research Laboratories. The method is by HPLC and is summarized as follows:

Chromatography was carried out on a DuPont Zorbax ODS column, 4.6 nm internal diameter by 25 cm in length. Detection was by UV absorption at 280 nm. The mobile phase consisted of acetonitrile and distilled water mixed in the ratio 90:10 (v:v).

Standard curves consisted of Topanol CA prepared in mobile phase. A high and a low concentration Quality Control (QC) sample were processed with each standard curve. The actual concentration of each QC sample was calculated from the standard curve, and was considered to be acceptable if between 90% and 110% of nominal.

Diet (approximately 10 g) was extracted by shaking with a mixture of acetronitrile and distilled water (9:1). The internal standard, 2-methylanthraquinone, was mixed with each extract to yield a final injected concentration of approximately 2 mcg/mL. Diet particles were allowed to settle, and a portion of the clear extract was diluted to between approximately 2.5 and 51 mcg/mL Topanol CA for injection.

CALCULATION AND LINEARITY
Standard concentrations for method validation ranged from approximately 2.5 to 51 mcg/mL. Standard curves were constructed from the data pairs of standard concentration and peak area or peak height ratio using an Apple computer linear regression program. The data pairs are weighted by l/concentration and the equation is of the form: y = mx + b.

Where:
y = peak area or peak height ratio of test article to internal standard.
m = slope of the regressed line.
x = concentration of Topanol CA standard
b = y-intercept of the regressed line.

QC sample concentrations ranged from 91.7% to 106% of claim. Table 2 summarizes the back-calculated standard concentrations from standard curves, illustrating linearity of Topanol CA in the
range of 2.5 to 51 mcg/mL when calculated both by peak area ratio and by peak height ratio.

RECOVERY FROM 100 PPM DIET SAMPLES
During initial attempts at proving diet homogeneity, persisting problems with the 100 ppm level diets were encountered. Details of these assay results are not reported. (See reason under Diet Homogeneity below.) At the time, however, it was not possible to unequivocally determine whether the problem was with the assay or due to the mixing procedure.

In order to provide a check on the recovery of Topanol CA from diet, "Spiked" diet samples were prepared in Analytical Chemistry. These samples were designed to be independent of diet homogeneity. An exact volume of Topanol solution was added to approximately 10 g of blank diet to render it equivalent to approximately 100 ppm. The Topanol/diet mixture was "dried" under a gentle stream of nitrogen in order to mimic the actual condition of compound in the diet. The entire 10 g was then extracted by the usual extraction procedure and chromatographed.

STABILITY OF DIET
Diet samples at concentrations of 10000 and 5000 ppm were prepared in the pharmacy and submitted to the Analytical Chemistry department for stability evaluation. Samples were stored at room temperature protected from light.

Top, Middle, and Bottom samples from 10000 and 5000 ppm diets showed no appreciable differences from the original assay when re-assayed 16 days following preparation. Thus stability over this period of time is indicated. In addition, one sample (bottom) of the 5000 ppm diet was assayed 33 days following preparation, and again, no appreciable concentration change was observed.

Stability testing was attempted on the 100 ppm samples. Results of the day 0 and day 7 assays are not reported due to a suspected problem with the assay which resulted in low recovery. Assays of 100 ppm diet 14 days post preparation (KX-21) did not indicate a problem with stability over this time period.

HOMOGENEITY OF DIET
Prior to commencement of animal dosing, Top, Middle and Bottom samples of 100, 1000 and 5000 ppm diet preparations were submitted for evaluation of homogeneity. Homogeneity at all three dose levels was shown to be good based upon consistency between Top, Middle, and Bottom sample assay results.

In the 1000 and 5000 ppm levels, percent nominals ranged from 104.7% to 112.3% and from 101.9% to 103.4% respectively. Although no result was obtained for the Bottom 5000 ppm sample, it was not deemed necessary to repeat this sample in view of the overall consistency seen in the 2 homogeneity assays examined.
Although the homogeneity of the 100 ppm sample was good, its average percent nominal was 151.6%. Upon investigation of the mixing procedure, a problem was discovered.

Duration of treatment / exposure:

Duration of treatment: 13 weeks

Frequency of treatment:

Feed was available ad libitum execpt when laboratory investigations were carried out.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 500, 5000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

4 groups of Sprague-Dawley rats, each consisting of 20 males and 20 females, were treated with appropriate concentrations of test article added to the diet to obtain dose levels of 0, 100, 500 or 5000 ppm.

Control animals:

yes, plain diet

Details on study design:

The dose levels used in this study were selected by the Sponsor and were based on the results of a preliminary pilot study performed at Bio-Research Laboratories Ltd.

In the pilot study administration of diets containing 10000, 5000 or 2500 ppm to groups of 5 male rats for up to 3 weeks revealed severe palatability problems leading to death in some animals at 10000 ppm, an initial reduction in food intake and body weight gain in rats receiving 5000 ppm and no apparent effects in rats receiving 2500 ppm.

Randomization/Identification: One week before treatment commenced, the rats were weighed and randomly assigned to each of the four groups using a computer-based pseudorandom number generator so that each group had a similar distribution of animals according to body weight. Male and female animals were randomized separately and were subsequently permanently identified by individual ear notch. Rats in poor health or at the extremes of the body weight range were not assigned to the study. Animals showing Ophthalmological findings at the pre-treatment examination were replaced by spare animals prior to the start of treatment.

Positive control:

No data

Examinations

Observations and examinations performed and frequency:

Clinical Examinations: During the pre-treatment and the treatment period, all animals were examined twice daily for mortality and morbidity. In addition, during the treatment period, all animals were examined daily (excluding days 0.2 and 0.3) for signs of ill health or reaction to treatment and rats showing clinical signs were examined once weekly by a veterinary aide.

Body Weights: During the week preceding the start of treatment and during the treatment period, individual body weights were recorded weekly. Starved body weights were also recorded prior to necropsy.

Food Consumption: Individual weekly food consumption was measured during the last week pre-treatment and throughout the treatment period. From these data, group mean intakes and total food consumption were calculated.

Food Conversion Ratios
Food conversion ratios over successive 4-weekly time periods were calculated using group mean body weight and food consumption data according to the following formula:

Food conversion ratio = Total mean food consumption for period (g)/Mean body weight gain for period (g)

Mean body weight gains were calculated using the group mean body weight data.

Ophthalmoscopy: Funduscopic (indirect ophthalmoscopy) and biomicroscopic (slit lamp) examinations were performed on all animals during the pretreatment period and again during week 13 of treatment. Following the administration of atropine sulphate (0.5% solution) as a mydriatic, the conjunctivae, sclera, cornea, pupil, lens, lacrimal apparatus, limbus, anterior chamber, membrana nictitans, globe, lid margins, iris and fundus (optic disk, retinal vessels and choroid) were examined by a qualified ophthalmologist.

Laboratory Investigations: Prior to commencement of treatment, laboratory investigations (hematology and clinical biochemistry) were performed on the 10 male and 10 female rats used for health screen assessment. In this instance, blood was obtained from the abdominal aorta immediately following ether anesthesia. Animals were starved overnight prior to the blood collection.
During week 13, the same laboratory investigations were performed on 10 males and 10 females randomly selected from each study group. Blood samples were obtained on this occasion from the orbital sinus using light ether anesthesia. Rats were also starved overnight prior to the blood collection.

Sacrifice and pathology:

Gross Pathology: A complete gross pathological examination was performed on all animals sacrificed pretreatment (health screen animals) and at treatment termination. Animals killed for health screen purposes were anesthetized with ether and then exsanguinated via the abdominal aorta. At completion of the treatment period, all animals were killed by intraperitoneal injection of sodium pentobarbital followed by exsanguination from the abdominal aorta. All animals were fasted before scheduled sacrifice, and a similar proportion of animals from each group and sex were killed on each day.

Organ Weight Assessment: For all animals killed at termination, organs were dissected free of fat and weighed.

Other examinations:

Not specified

Statistics:

Group mean values and standard deviations were calculated for all quanti¬tative data (body weight, food consumption, hematology and clinical biochemistry parameters, and absolute and relative organ weights). Following Bartlett's test, the statistical significance of differences from the control group was assessed using analysis of variance (Anova) followed by Dunnett's "t" test for homogeneous data or analysis of variance (Kruskall-Wallis) followed by Dunn's test for heterogeneous data.
Statistical significance was assessed at the 0.05 and 0.01 levels using the Dunnetts 't* test for body weight and food intake data and at the 0.05 level for other data, while significance was assessed at levels of 0.05, 0.01 or 0.001 using Dunn's test for all data types.
The incidence of hepatocyte vacuolate on was analyzed for stati stical significance using the minimum logit Chi-square procedure. In addition the incidence of this finding in each treated group was compared to the control incidence using Fisher exact Chi-square analysis.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

at high dose

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

at high dose

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

Description (incidence and severity):

at high dose

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

Mortality: No deaths occurred during the 13-week treatment period.
Clinical Signs: During the 13-week treatment period, the clinical signs that were noted were those commonly observed in rats of this age and strain, and none of these findings were considered to be attributable to treatment with Topanol CA.
Evidence of a mild sialodacryoadenitis (SDAV) virus infection, characterized by swelling of the ventral cervical region and ocular discharge, redness and/or swelling of the periorbital regions, associated with "sneezing" or nasal discharge or abnormal respiratory sounds in isolated individuals, was first noted towards the end of week 2 of treatment.
The majority of animals in all groups showed such signs. There was no evidence of any treatment-related difference in distribution, type or severity of signs observed, and all signs of SDAV had virtually subsided by the beginning of week 6.
During weeks 2 and/or 3 of treatment, this transient infection was associated in some individuals with a small reduction in food intake, which was not clearly reflected in the weekly group mean values. Subsequently, food intake values returned to expected ranges.

Body Heights: Among animals receiving 5000 ppm, a statistically significantly lower group mean body weight (P < 0.001 on occasion), was noted, resulting in significantly lower overall body weight gains when compared to those of the controls. This lower mean body weight was observed from the end of the first week of treatment and was up to 18% (males and females) lower than controls over the 13-week treatment period.
Males and females receiving 500 ppm showed a marginal but consistent trend to lower mean body weights, although such differences did not attain a level of statistical significance (up to 6% lower for males and females).
Body weight gains for rats receiving 100 ppm are considered to be essentially comparable to those of the controls.

Food Consumption: Rats receiving 5000 ppm showed a consistent slight reduction in weekly food intake values when compared to controls, these differences being statistically significant on several occasions, particularly among males. Total food intakes for the 12-week treatment period for rats receiving 5000 ppm were 88% (males) and 94% (females) of the control intake.
Food intake of other treated rats was considered essentially similar to that of the controls.

Food Conversion Ratios: A slight reduction in the efficiency of food utilization was noted for males and females receiving 5000 ppm as indicated by slightly higher food conversion ratios (weeks 1-4, 9-12 and 1-12 males; weeks 1-4, 5-8, 9-12 and 1-12 females) when compared to controls.
This reduction in the efficiency of food utilization was more evident among females receiving this dose level.
Rats receiving 100 or 500 ppm showed food conversion ratios which were considered to be comparable with those of the controls.

Test Article Intake: The average daily intakes of Topanol CA in mg/kg body weight/day were calculated using the weekly group mean body weight and food consumption values and are presented in table form.

Ophthalmoscopy: Ophthalmoscopic examination (funduscopy and biomicroscopy) performed on all animals during week 13 revealed the following findings:

1502 (control), left eye - corneal edema and neovascularization,
2508 (100 ppm), left eye - mild conjunctivitis, 1+ mucoid discharge,
4001 (5000 ppm), right eye - corneal edema and neovascularization,
4019 (5000 ppm), right eye - cortical cataract,
4519 (5000 ppm), both eyes - mucoid discharge 1+.

The low incidence of ocular findings was not considered to indicate any changes attributable to Topanol CA treatment.

Laboratory Investigations

Hematology: Group mean values for hematological parameters assessed prior to the start of treatment on 10 rats/sex selected for health screen, and again during week 13 on 10 randomly selected rats/sex/group.
The pre-treatment data did not demonstrate any unusual or unexpected clinical pathological findings, thereby assuring the health status of the animals for the study.
The evaluation of hematology data for parameters assessed during week 13 of treatment revealed a slight increase in RBC count among males and females receiving 5000 ppm, such increase being statistically significant for females when compared to controls. Rats receiving 5000 ppm also showed a reduction in MCV and MCH (statistically significant for males), which was considered to reflect the change observed, in the RBC count.
The values obtained for total WBC and differential count (neutrophil [segmented] and lymphocyte), although in some instances showing small differences between controls and rats receiving 5000 ppm, were within the normally expected range for rats of this age and strain.

Blood Biochemistry: The blood biochemistry values obtained prior to the start of treat¬ment were all within the normally expected range for rats of this age and strain.
The evaluation of the clinical biochemical investigations conducted during week 13 of treatment showed a statistically significant increase in serum GPT, GOT and a tendency to slightly higher alkaline phosphatase levels among males receiving 5000 ppm when compared to controls. A tendency to a marginal but statistically significant increase in serum GPT levels was also noted among the high-dose females when compared to control animals.
One female receiving 100 ppm (#2520) also showed a marked increase in serum GPT and GOT levels when compared to controls.
Among rats receiving 5000 ppm, marginal reductions, in some instances attaining a level of statistical significance when compared to control values, were noted in glucose and total protein for males and females, total bilirubin for males and albumin and A/G ratio for females. However, since values obtained were at the lower end of the normal range, the toxicological significance of these findings is uncertain at this time.

Terminal Studies

Gross Pathology: Gross pathological examination of rats killed after 13 weeks of treatment revealed a low incidence of pathologies commonly seen in rats of the age and strain used. The number, distribution and type of such findings showed no indication of any disturbance attributable to Topanol CA treatment.

Organ Weight Analysis: Males and females receiving 5000 ppm showed absolute liver weights which were comparable to those of the controls. However, these high-dose animals showed a marked reduction in fasted body weight, and thus relative liver weights were significantly higher than those of the controls.
Absolute and relative liver weights for other treated rats were comparable to those of the controls.

A marginal but statistically significant increase in absolute spleen and adrenal weights was noted for females receiving 5000 ppm com¬pared to controls, with an associated increase in relative weights (statistically significant). Males receiving this dose level showed comparable absolute spleen and adrenal weights, but relative weights for these organs were slightly but significantly increased. Since no histological changes were observed in these organs which could be related to treatment with Topanol CA, the increase in absolute and/or relative spleen and adrenal weights observed among high dose animals was considered of doubtful toxicological significance and to most likely be related to the low body weights.

Similarly, other small differences between values obtained for animals receiving 5000 ppm and the controls, although on occasions attaining a level of statistical significance, were considered to be of doubtful toxicological significance. Such differences were considered to most probably be related to the lower terminal body weights recorded for these treated rats.

Histopathology: Histopathological examination of the livers from all control rats and animals receiving 5000 ppm revealed fine and/or coarse cytoplasmic vacuolation of the hepatocytes generally located periportally in 11/ 20 males and 4/20 females receiving 5000 ppm. In view of this finding the livers of all rats receiving 100 or 500 ppm were examined. Vacuolation of hepatocytes was observed in 2 males and 2 females receiving 100 ppm and 1 male and 5 females receiving 500 ppm. The severity of this change showed no clear evidence of any dose-related difference and was not associated with any inflammatory or degenerative changes.
Statistical analysis of the overal incidence of hepatocyte vacuolation revealed a significant linear trend with dose for males only and a significantly higher incidences for males receiving 5000 ppm and females receiving 500 ppm when compared with controls (Fishers exact Chi square analysis).
In order to further evaluate the hepatocyte vacuolation, fixed frozen sections of liver from all rats were stained for fat with 0il-Red-0.
Variable amounts of fat were noted in both control and treated rats with the Oil-Red-0 stain.
The subjective assessment of the total fat content of liver sections indicated no appreciable difference in total fat for all treated females and for males receiving 100 or 500 ppm when compared to controls. A proportion of males receiving 5000 ppm showed a slightly increased amount of total fat when compared to controls or other treated rats.
However, among treated rats where hepatocyte vacuolation was noted using hematoxylin and eosin staining there appeared to be an increase in the number of large fat droplets in the hepatocytes when compared to controls.
Examination of all other tissues from controls and rats receiving 5000 ppm and tissues showing gross lesions from rats receiving 500 or 100 ppm revealed a low incidence of changes commonly seen in rats of this age and strain. The distribution and type of such findings showed no evidence of changes attributable to Topanol CA treatment.

Effect levels

Dose descriptor:

NOAEL

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Group mean body weight gains over the first 12 weeks+ of treatment were as follows:

Group/Treatment	Mean (±S.D.) Body Weight Gains (g)
	Males	% of control	Females	% of control
	Weeks 0-12+		Weeks 0-12+
1. Control	361.3 ±54.14	-	162.2 ±26.84	-
2. Topanol CA, 100 ppm	348.4 ±38.37	96	167.4 ±24.91	103
3. Topanol CA, 500 ppm	341.5 ±50.23	95	150.8 ±19.46	93
4. Topanol CA, 5000 ppm	266.8** ±23.48	74	105.8** ±15.08	65

Statistically significant differences from control group 1 ** P < 0.01, Dunnett’s. +Gain calculated to week 12 since a proportion of animals from each group were starved prior to necropsy on the day of week 13 body weight measurements.

 

The average intakes of Topanol CA in mg/kg body weight/day over the 13-week treatment period for each dietary concentration were as follows:

Group/Treatment	Average Intake* (mg/kg body weight/day)
	Males	Females
2. Topanol CA, 100 ppm	7.6 (5-13)	8.8 (6-13)
3. Topanol CA, 500 ppm	38.1 (25-62)	45.5 (31-68)
4. Topanol CA, 5000 ppm	391.5 (270-644)	490.2 (397-720)

*Figures in paratheses indicate minimum and maximum intake.

Applicant's summary and conclusion

Conclusions:

From the results of this study, it is concluded that administration of Topanol CA by dietary admixture at concentrations of 100, 500 or 5000 ppm to rats for a period of 13 weeks produced no mortality or clinical signs of reaction to treatment or any effect on ophthalmoscopic or gross pathology findings.

Executive summary:

The study was conducted with four dose groups of 20 male and 20 female sprague dawley rats over a period of 13 weeks with test sample exposed to animals by dietary exposure at concentrations of 0, 100, 500 and 5000ppm.

Exposure at the highest exposure group resulted in statistically significant reduction in growth rate associated with reduced food intake, plus reduced relative liver weights and small increases in ralative spleen and adrenal weights.

A slight reduction in body weights was observed at 500 ppm but these were so slight compared to control animalks to be considered not statistically or toxicologically significant and no effect on animals at 100 ppm. The NOAEL is therefore considered to be 500ppm.