Hexamethylene bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate]

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1975-04-03 to 1975-08-03

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non GLP but guideline compliant study. Well documented report.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1978

Report date:

1976

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)

Deviations:

no

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Hexamethylene bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate]

EC Number:

252-346-9

EC Name:

Hexamethylene bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate]

Cas Number:

35074-77-2

Molecular formula:

C40H62O6

IUPAC Name:

6-{[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propanoyl]oxy}hexyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propanoate

Details on test material:

- Physical state: white powder

Test animals

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Oloc (Western) Ltd.
- Age at study initiation: 29 weeks
- Weight at study initiation: 11922 g (9364 - 14480 g)
- Housing: individually in kennels
- Diet: 400 mg of complete dry diet (Spratts Dog Diet) were offered daily
- Water: ad libitum
- Acclimation period: 4 weeks

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Mixing appropriate amounts with: powdered diet

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Representative samples of dog diets containing the test substance were extracted with chloroform. The 500 ppm and 1500 ppm samples were then determined by TLC and visual comparison with standards. The 5000 ppm sample was determined by UV-spectrophotometry with the control extract in the reference beam and the test substance supplied with the samples as a reference standard. The estimated relative reproducibility of this method is ±10% or better. Within the limits of error of the sampling technique and the analytical method there was good correspondence between the values found and the nominal ones.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
500, 1500, 5000 ppm (eq. 14.7, 46.2, 148.7 mg/kg bw/d)
Basis:
nominal in diet

No. of animals per sex per dose:

4 - 5

Control animals:

yes, plain diet

Details on study design:

- Post-exposure recovery period in satellite groups: 4 weeks

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE:
- Time schedule for examinations: during week days only

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once before start of exposure and during week 4, 8 and 12
- Dose groups that were examined: all dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: once before start of exposure and during week 4, 8 and 12
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes, food was withdrawn for 16 hours
- How many animals: all animals
- Parameters examined: erythrocyte sedimentation rate (ESR), packed cell volume (PCV), haemoglobin (Hb), red cell count (RBC), reticulocyte count (Retics), mean corpuscular haemoglobin concentration (MCHC), mean cell volume (MCV), total white cell count (WBC), differential count (Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes), platelet count, prothrombin index (PTI)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: once before start of exposure and during week 4, 8 and 12
- Animals fasted: Yes, food was withdrawn for 16 hours
- How many animals: all animals
- Parameters examined: urea, glucose, total serum proteins, serum protein electrophoresis, alkaline phosphatase (AP), glutamic-pyruvic transaminase (GPT), bilirubin, sodium, potassium

URINALYSIS: Yes
- Time schedule for collection of urine: once before start of exposure and during week 4, 8 and 12
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes, food was withdrawn for 16 hours
- Parameters examined: pH, protein, reducing substances, glucose, ketones, bile pigments, urobilinogen, haemoglobin

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
The following organs were examined: brain, pituitary, heart, lungs, liver, spleen, pancreas, thymus, prostate/uterus, kidneys, thyroids, adrenals, gonads, aorta (arch and abdominal), trachea, lymph nodes (cervical and mesenteric), gall bladder, urinary bladder, salivary gland, tongue, oesophagus, stomach, duodenum, jejunum, ileum, colon, skin, mammary gland, skeletal muscle, bone marrow, peripheral nerve, eye and optic nerve,spinal cord

Other examinations:

ORGAN WEIGHTS: Yes
The following organs were weighed: brain, pituitary, heart, lungs, liver, spleen, pancreas, thymus, prostate/uterus, kidneys, thyroids, adrenals, gonads

Statistics:

Whenever it was necessary to determine whether significant differences existed between mean values relating to test and control animals, the method used was analysis of variance followed by Student's 't' test, the results being expressed as the 'least significant difference' (LSD), that is, the least difference that had to exist between mean values for dosed and control animals for significance at specified levels of probability. The only other statistical procedure adopted was that used to describe the range of results obtained during the pre-dosing investigation. These results have been described by the grand mean and the "95 % range", the latter phase being used to describe the mean ± ('t' x standard deviation).

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):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No clinical signs or mortality were observed in the course of the study.

BODY WEIGHT AND WEIGHT GAIN
From the figures illustrating bodyweight changes of individual animals, it can be seen that all dogs, with the exception of dog 566 (female) and 568 (female) (5000 ppm) showed an overall gain in weight during the pretreatment period. The latter animals were replacement dogs and lost weight during their first 2 weeks. However, both animals gained weight during the week prior to commencement of dietary administration. With the onset of dietary intake, no marked changes in bodyweight were seen. With the exception of dogs 539 (male), 541 (male) (Control) and 548 (female) (500 ppm) all dogs gained or maintained weight satisfactorily. These three animals all showed a slight weight loss over the 13 - week period of dietary intake. It should be noted, however, that minor fluctuations in bodyweight would be expected for dogs of this age. Examination of the group mean bodyweight graphs indicates that all groups gained weight at a similar rate. Statistical analysis of the data confirmed this observation, no significant group mean differences being demonstrated. During the 4 - week recovery period, all four animals showed an initial loss of bodyweight, with a subsequent return to normal.

FOOD CONSUMPTION AND COMPOUND INTAKE
Each dog was offered a basic ration of 2800 grams of powdered diet per week. Throughout the experimental period, appetite was good for all dogs, many animals consuming all of the food offered. During weeks 1 and 13 of treatment mean food consumption was 2625 (control), 2610 (500 ppm), 2694 (1500 ppm) and 2662 (5000 ppm) g per week for male and female animals.

WATER CONSUMPTION AND COMPOUND INTAKE
The quantities of water consumed by individual dogs during the 4 - week pre-treatment period, during weeks 1 - 4 and 9 - 12 of dietary intake, and during weeks 1 - 4 of the recovery period were measured on weekdays only. As the study progressed, the water intake increased, as would be expected for growing animals. Throughout the experimental period, the mean weekly quantities of water consumed were comparable, no significant group differences being demonstrated.

OPHTHALMOSCOPIC EXAMINATION
No abnormalities that could be associated with dietary intake were detected in male and female animals.

HAEMATOLOGY
All findings were within normal limits throughout the study period for male and female animals.

CLINICAL CHEMISTRY
Before dietary intake commenced plasma glucose levels in excess of our normal upper limit of 110 mg% were recorded for male dogs numbered 533, 537, 541 (Control); 543, 549 (500 ppm). Repeated investigations confirmed these findings.
After 4 weeks of dietary intake, male dog 541 (Control) again had a plasma glucose level in excess of 110 mg%, whilst the levels for male dogs 533, 537 (Control), 543 and 549 (500 ppm) were on this occasion within our normal limits. Female dog 542 (Control) had a serum potassium level in excess of our upper limit of 5.5 mEq/L and male dog 543 (500 ppm) had an GPT level that was above our normally accepted upper limit of 50 mU/mL. These findings were confirmed by repeated investigations. All other results were within normal limits.
Atfer 8 weeks of dietary intake male dogs 541 (Control) and 543 (500 ppm), as well as female dog 554 (1500 ppm) had GPT levels that were above our normally accepted upper limit of 50 mU/mL. Male dog 549 (500 ppm) had a plasma glucose level slightly in excess of normally accepted upper limit of 110 mg%. Repeated investigations confirmed these findings. All other findings were within normal limits.
After 12 weeks of dietary intake male dogs 541 (Control) and 565 (5000 ppm) had AP levels that were above our normally accepted upper limit of 35 KA units. Repeated investigations confirmed these findings. All other findings including GPT levels were considered to be within normal limits.

URINALYSIS
Before dietary intake commenced all specimens of urine were considered to be normal and all dogs were showing satisfactory urinary concentrations.
After 4 weeks of dietary intake the urine specimen from female dog 542 (Control) had a specific gravity of less than 1.035 which is our normally accepted lower limit. This was confirmed by a repeat investigation. All other specimens were considered to be normal and the remaining dogs were showing satisfactory urinary concentration.
After 8 and 12 weeks of dietary intake all specimens of urine were normal and all dogs were showing satisfactory urinary concentration.

ORGAN WEIGHTS
On completion of the macroscopic post mortem examination, the major organs were removed and weighed. These weights have been expressed as a percentage of the bodyweight and as a percentage of the brain weight. The organ weights for animals which were maintained for the 4 - week recovery period have been included in the study report, but excluded from the means and statistical analysis. The relative liver weights for male dogs numbered 537, 541 (Control), 565, 567 (5000 ppm) and 563 (5000 ppm - Recovery) were slightly in excess of our normally accepted upper limit of 4% of the bodyweight. The same has been observed in one female dog (No. 568) of the highest dosing group. Routine statistical analysis revealed that when expressed in grams, as a percentage of the bodyweight, and as a percentage of the brain weight, the mean liver weight for animals that had received 5000 ppm was significantly greater than that of the control mean, the differences being significant at the 1 % , 5% levels of probability respectively (LSD =99 grams, 0.63% and 88%). All other organ weights were considered to be within normal limits.

GROSS PATHOLOGY
The only findings which may possibly have been related to treatment were areas of congestion seen in the gastro-intestinal tract. Minimal congestion of the intestinal mucosa was seen in male dogs numbered 543, 547 (500 ppm); 555 (1500 ppm) and 559, 561, 565 (5000 ppm). This has also been observed in female dogs numbered 544, 546, 550 (500 ppm); 558 (1500 ppm) and 568 (5000 ppm). Similar observations were made for male dog 537 (Control - Recovery) and female dog 564 (5000 ppm - Recovery). This congestion was mainly confined to the jejunum and ileum.

HISTOPATHOLOGY: NON-NEOPLASTIC
Lungs: Minimal changes were encountered in the lungs in a proportion of control and treated animals. They included peribronchial, peribronchiolar, perivascular or interstitial foci of chronic inflammation and they were associated with several areas of pneumonitis in male dog 563 (5000 ppm, recovery). In addition, an area of bronchopneumonia was observed in female dog 558 (1500 ppm). These are common findings in the lungs of laboratory dogs and, as there was no evidence of a treatment-related effect, they were considered to have no toxicological significance.

Liver: Inflammatory cell foci, with or without eosinophils, were noted in occasional portal tracts in male dogs 539 (Control) and 547 (500 ppm). Similar effects have been observed in female dogs 536 (Control), 544, 546 (500 ppm) and 556 (1500 ppm). A focus of necrosis with acute inflammatory cell infiltration was identified in female dog 552 (1500 ppm). These changes were probably related to parasitic infestation and, thus, were considered to be of no importance.
Slight to moderate periportal glycogen depletion was observed in female dogs 540, 542 (Control), 550 (500 ppm), 556 (1500 ppm), 560, 562, 568 (5000 ppm). Since controls and animals of all test groups were similarly affected, this change was regarded as being without toxicological significance.
Varying degrees of bile duct hyperplasia with on associated inflammatory reaction were encountered in male dogs 551, 553, (1500 ppm) and 559 (5000 ppm) as well as in female dogs 560 and 568 of the highest dosing group. This change was not seen in any of the controls nor in animals receiving 500 ppm. In male dog 553 (1500 ppm) and female dog 560 (5000 ppm), the degree of bile duct hyperplasia was greater than that previously identified in untreated Beagle dogs. There was no evidence of bile duct hyperplasia in either of the two dogs receiving 5000 ppm after a recovery period of four weeks. These changes tended to be focal non-specific in nature, and probably related to parasitism. It is unlikely that they were related to treatment with the test substance. The significance of the intracytoplasmic, eosinophilic globules in hepatocytes in a single dog (female No. 552, 1500 ppm) was unknown.

Kidney: Treatment related changes were not detected. Minimal changes, commonly encountered in the kidneys of laboratory dogs, included the following:
fat deposition in the epithelium of a variable number of cortical tubules in the majority of control and treated dogs from all groups;
minimal calcification within a few medullary tubules in a proportion of control and treated dogs;
peripelvic chronic inflammation in female dogs 536 (Control), 544, 550 (500 ppm), 552 (1500 ppm) and 560, 562 (5000 ppm);
cortical foci of intestitial chronic inflammation in male dogs 543 (500 ppm) and 561 (5000 ppm) as well as in female dogs 540 (Control) and 566 (5000 ppm).

Intestine: There were no morphological findings corresponding to the mucosal congestion noted macroscopically at post mortem examination. Parasitic worms were seen within the lumen of the small intestine in male dog 539 (Control) and female dog 552 (1500 ppm) and minimal inflammatory changes, probably related to parasitic infestation, were identified in the mucosa or submucosa of the gastro-intestinal tract in female dogs 538 (Control, recovery), 544, 548, 550 (500 ppm).

No morphological change of toxicological significance was seen in any of the other tissues examined.

Effect levels

Dose descriptor:

NOAEL

Effect level:

148.7 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were seen in any of the animals at this dose.

Target system / organ toxicity

Critical effects observed:

not specified