Diethyl [[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methyl]phosphonate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

31 October 1979 - 12 February 1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study was performed similar to OECD guideline 409 and under GLP-like quality controlled conditions with QAU statement provided. Study meets generally accepted scientific standards, and is described in sufficient detail. No purity of test substance available.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1980

Report date:

1980

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)

GLP compliance:

no

Remarks:

but QUA statement included

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Calcium diethyl bis[[[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methyl]phosphonate]

EC Number:

265-512-0

EC Name:

Calcium diethyl bis[[[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methyl]phosphonate]

Cas Number:

65140-91-2

Molecular formula:

C17 H29 O4 P. 1/2Ca

IUPAC Name:

calcium diethyl bis[[[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methyl]phosphonate]

Details on test material:

Physical appearance: white, solid powder

Test animals

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Balbeggie Kennels, Fife, Scotland
- Age at arrival: 20-22 weeks
- Weight at study initiation: 5.2-9.1 kg
- Identification: tattoo on the right hind limb
- Housing: individually housed in indoor kennels measuring 0.7 x 1.6 m and equipped with under-floor heating. Brief periods of supervised exercise were permitted daily and more extended periods of up to six hours were allowed approximately weekly, in covered outside runs.
- Diet: A moistened powdered dog diet (Laboratory Diet A from BP Nutrition Products Ltd., Witham, Essex, England) was offered as two 300 g meals given three hours apart at approximately the same times daily and any food uneaten was withdrawn the following morning. This procedure was modified before blood sampling or urine collection, when any food uneaten was withdrawn on the day preceding blood sampling or overnight urine collection.
- Water: Drinking water was supplied ad libitum to each pen via an automatic 'Lixit' valve system, except during urine collection.
- Acclimation period: All animals were held at these laboratories for a period of at least 19 weeks without treatment before assignment to this study, and for an additional 15 weeks after assignment before the commencement of treatment. With the exception of replacement animal, the dogs were 54 or 55 weeks of age at the commencement of treatment. The replacement animal was 82 weeks old and had been held without treatment for a total of 55 weeks at these laboratories at commencement of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): mean daily maximum = 19°± 2°C, mean daily minimum = 13° ± 4°C
- Humidity (%): 78±8
- Photoperiod: 12 hrs dark / 12 hrs light
- Air changes: 15 per hour
Dogs were held in a limited access facility. Personnel entering were required to wear protective clothing. Each room was kept at positive pressure with respect to the outside and had its own supply of filtered fresh air which was passed to the atmosphere and not re-circulated.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
On the first four occasions of formulation, the initial dispersal of the test substance into dry powdered diet was made by mixing the required quantity of the test substance with 100 g of diet in a Kenwood planetary mixer. Thereafter this was achieved by shaking the constituents of the admixture in a closed container and homogenising this product by passage through a grinder. This modification was adopted to minimise the generation of dust and hence possible loss of compound. The resulting pre-mix was further diluted with dry powdered diet in a Kenwood Chef planetary mixer for ten minutes followed by further dilution with diet in a Hobart A200 planetary mixer (15 minutes) to give a final pre-mix of 8800 ppm. The final admixtures for Group 3 (600 ppm) and Group 4 (2000 ppm) were achieved by diluting the required amount of pre-mix with diet in a Gardner 50L trough mixer with interrupted spiral agitator (15 minutes). The final admixture for Group 2 (200 ppm) was achieved by prediluting the required amount of pre-mix with diet in a Hobart A200 planetary mixer (ten minutes) with a final dilution with diet in the Gardner trough mixer (15 minutes). Each product, and untreated control diet, was wetted with tap water (200 mL/kg): and mixed in the Gardner trough mixer (15 minutes). At the request of the Sponsor, the mixing times were extended from 17 January 1980 (Week 12) by five minutes for each mixing procedure (excluding the wetting stage) involving the Kenwood planetary, Hobart A200 planetary and Gardner trough mixers. The final products were stored at 4°C for no more than four days and issued daily to the animal house.

DIET PREPARATION
- Rate of preparation of diet (frequency): at least every 4 days
- Storage temperature of food: 4°C

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the unwetted diet (200 g of the single common pre-mix and of each test group final mix and 500 g of control diets) prepared before the commencement of treatment and for use in weeks 1, 5, 8/9, 13 and the last week of treatment were supplied to the sponsor for analysis. In addition, a further 50 g sample of each test diet was retained for at least three months in sealed polythene sachets and stored at room temperature in these laboratories pending possible future analysis as required by Section 58.113 of the Good Laboratory Practice regulations. These samples were not analysed and were eventually discarded.

Duration of treatment / exposure:

7 days/week; 13 weeks (The treatment was intended to continue for 13 weeks. In fact the animals were held for at least one additional day before the terminal sacrifice commenced. Necropsies took up to 13 days to complete for all animals. Throughout this additional period, daily treatment continued)

Frequency of treatment:

Continuous dosing with feed.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
200, 600, 2000 ppm
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
8.5, 24.7, 84.1 mg/kg bw/day (males)
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
9.2, 28.3, 90.2 mg/kg bw/day (females)
Basis:
actual ingested

No. of animals per sex per dose:

6 animals per sex and dose

Control animals:

yes, plain diet

Examinations

Observations and examinations performed and frequency:

SIGNS
All dogs were inspected at least twice daily for any evidence of reaction to treatment or ill-health. A detailed record was maintained of any deviations from normal in respect of nature and severity, date and time of onset, duration and progress of the observed response.

PHYSICAL EXAMINATION
Each animal was subjected to a rigorous physical examination before treatment commenced and subsequently after three, nine and 12 weeks treatment, in which particular attention was paid to: Teeth and gums, mucous membranes and skin, ears (external auditory canal), superficial lymph nodes, abdomen - including palpation, external genitalia and mammary glands, chest - including auscultation of heart and lungs, gait and stance - including palpation of limbs, general behaviour and appearance. There were no abnormalities detected during physical examinations that required further investigation.

FOOD CONSUMPTION
Food residues, if any, were weighed and recorded daily for each pen. The total weekly intake per dog was calculated and, from these individual data, mean values for each group were derived.

BODYWEIGHT
The weight of each dog was recorded weekly for two weeks before commencement of treatment and throughout the study.

ACHIEVED CHEMICAL INTAKE
The achieved intake of test compound was calculated from the nominal dietary concentrations and the group mean values for bodyweight and food consumption.

WATER CONSUMPTION
In the absence of any treatment-related effects, no measurements were made.

OPHTHALMOSCOPY
Before commencement and after five, ten, and 12 weeks of treatment, both eyes of each dog were examined by means of a Fison's binocular indirect ophthalmoscope at least 20 minutes after the instillation of Mydriacil (1% tropicamide, Alcon Laboratories).

HAEMATOLOGY
Before commencement and after six and 12 weeks of treatment, blood samples were withdrawn from the jugular vein of each dog after overnight starvation. The following parameters were observed: packed cell volume (PCV), hemoglobine concentration (Hb), Erythrocyte count (RBC), Mean cell haemoglobin concentration (MCHC), Mean cell volume (MCV), leucocyte count (WBC; total and differential), Platelet count, prothrombin time (PT).

CLINICAL CHEMISTRY:
The following parameters were observed at the same schedule as for haematology investigations: Alkaline phosphatase (AP), Alanine amino-transferase activity (ALT:GPT), Aspartate amino-transferase activity (AST:GOT), Urea concentration, Glucose concentration, Direct bilirubin concentration, Total cholesterol concentration, Total protein concentration, Electrophoretic protein fractions, sodium (Na), potassium (K), chloride (Cl), calcium (Ca), lactate dehydrogenase (LDH). The majority of samples taken without anticoagulant before commencement of treatment were haemolysed. Repeat samples were obtained five days later and results for these repeat samples are reported only.

URINANALYSIS
Before commencement and after six and 12 weeks of treatment, each dog was placed in a metabolism cage for the collection of a urine sample. Drinking water was removed at 3 pm and urine collected from 5 pm to 9 am the following morning. The following parameters were observed: appearance, pH, specific gravity, protein, glucose, ketone, bile pigments, urobilin, blood pigments. The sediment from centrifugation at 3,400 rpm. for ten minutes was examined for epithelial cells (E), polymorph (P) and mononuclear (M) leucocytes, red :blood cells (R), casts (C), or other abnormalities (A).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All animals were subjected to a detailed necropsy involving the opening of the cranial, thoracic and abdominal cavities. During this, the appearance of any abnormality was recorded in detail. The external features of the dog were scrutinised and compared to any relevant comments on the history report. The first incision allowed rapid preparation of a costal bone marrow smear. The eyes complete with optic nerve and relevant adnexa were removed. The cranial cap was lifted and the brain dissected free of meninges. The pituitary was freed from the sella turcica and fixed separately. The ventral abdominal skin was reflected to allow observation of the subcutaneous structures, in particular, mammary glands and superficial lymph nodes. Abdominal and thoracic viscera were examined in situ and note made of any abnormal position, morphology or interaction. The urinary bladder was slightly inflated with fixative and the urethra ligated. The mucosa was examined after fixation at the time of embedding. The serosal surface of the entire intestinal tract was re-examined after removal. The tract was sectioned longitudinally, the mucosa examined and appropriate samples were retained for histology. After weighing, the major organs were scrutinised and, where appropriate, the cut surfaces examined. A section of thoraco-lumbar spinal cord was removed. Before disposal of the carcass, the senior prosector checked the retained tissue against the protocol and considered the necropsy report in detail.

ORGAN WEIGHTS
The following organs were dissected free of fat and other adjacent tissue before being weighed: Adrenals, Brain, Heart, Kidneys, Liver, Ovaries, Spleen, Testes, Thyroids. The ratio of organ weight to bodyweight was calculated in each case.

HISTOPATHOLOGY: Yes
Samples from each dog, together with any abnormality identified at necropsy, were preserved in buffered 4% formaldehyde except where otherwise specified. For processing, the tissues were dehydrated, embedded in paraffin wax, sectioned (at 5 µm thickness) and stained with haematoxylin and eosin. Four sections of the brain (to include cerebellum, cerebral cortex, thalamic nuclei, mid-brain and medulla) were prepared and examined. The spinal cord, in transverse and longitudinal sections, was prepared and examined. In addition a costal bone marrow smear was prepared, airdried, fixed in methanol and stained by the May-Gruenwald-Giemsa procedure.
The following tissues were examined microsopically: Adrenals, Aorta (thoracic), Brain, Colon, Duodenum, Epididymides, Eyes and optic nerves, Gall bladder, Heart (auricle, ventricle, ventricular septum, Ileum, Jejunum, Kidneys, Liver, Lungs (including mainstem bronchi), Lymph nodes (axillary, cervical, mesenteric), Mammary gland (caudal, cranial), Oesophagus, Ovaries, Pancreas, Parathyroids, Pituitary, Prostate, Salivary glands (left), Sciatic nerve (left), Skeletal muscle (thigh), Skin (dorsum), Spinal cord (cervical region, lumbar region), Spleen, Sternum, Stomach (cardia, fundus, pyloru), Testes, Thymus, Thyroids, Tongue, Trachea, Urinary bladder, Uterine cervix, Uterus

Statistics:

The significance of inter-group differences in food consumption, bodyweight, blood composition and organ weights was assessed by Students 't' test using a pooled variance.

Results and discussion

Results of examinations

Details on results:

SIGNS
Treated and control dogs remained similar in all aspects of behaviour and appearance.

CLINICAL SIGNS AND MORTALITY
No abnormalities attributable to treatment with the test substance were observed. No mortality occurred.

BODY WEIGHT AND WEIGHT GAIN:
The bodyweights of all animals, both treated and control, were within the normal range for beagle dogs. No biologically significant inter-group differences were observed in bodyweight changes over the treatment period.

FOOD CONSUMPTION AND COMPOUND INTAKE:
The superior food consumption of females receiving the test substance was also apparent before the treatment period and reflected the low appetite of two control animals. The food consumption of treated and control males was similar throughout the study.

OPHTHALMOSCOPIC EXAMINATION
Almost all animals suffered conjunctivitis of varying severity and isolated incidences of mild keratitis were observed before and during treatment. It is considered that these conditions resulted from the feeding of powdered diet and were not related to the administration of the test substance.

HAEMATOLOGY
The haematological characteristics of the blood were undisturbed by treatment with the test substance. Lower packed cell volume, haemoglobin concentration and erythrocyte count were found after six and 12 weeks of treatment in the treated males; these values were similar to those obtained before the commencement of the study and the deviations from control values are therefore attributed to chance.

CLINICAL CHEMISTRY
There were no inter-group differences attributable to treatment with the test substance.

URINALYSIS
On each occasion throughout the study, the composition of urine samples reflected the normal variation seen in beagle dogs.

ORGAN WEIGHTS
Comparison of organ weights, expressed either in absolute or bodyweight-relative terms gave no indication of any differences attributable to the administration of the test substance. Poor exsanguination at sacrifice of animals B522, B518 and B337 may have attributed to the high spleen weights of these animals.

GROSS PATHOLOGY
Unilateral cryptorchidism was present in two animals in Group 4. This confirmed similar observations before commencement of treatment. In one of these dogs the epididymis was also small. A small number of other changes was present and all are considered incidental and those commonly found in dogs.

HISTOPATHOLOGY:
No changes were detected that could be related to treatment with the test substance. There was a range of banal degenerative and inflammatory lesions present, similar in type and incidence to those considered usual in beagle dogs of this age at Life Science Research. These included hypercellularity of Peyer's patches in the ileum; craniopharyngeal cysts in the anterior pituitary gland, and mammary hyperplasia and secretion which can be correlated with the middle and late metoestral phase of the oestrous cycle. Two male dogs in the highest dosage group had a unilateral juvenile epididymis and testis due to cryptorchidism.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Highest dose tested.

2

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Highest dose tested.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

The calculated values for achieved dosage were consistent within each group throughout the treatment period; mean dosages were, for males and females respectively, 8.5 or 9.2, 24.7 or 28.3 and 84.1 or 90.2 mg/kg/day for those receiving 200, 600 or 2000 ppm.

The slightly higher values for female groups compared with the corresponding male groups reflected the similar appetites of the males and females combined with the lower group mean bodyweights of the females.

Applicant's summary and conclusion

Executive summary:

In a 90 day subchronic feeding study the test article was administered for at levels of 200, 600 or 2000 ppm in powdered diet to groups of six male and six female beagle dogs. A similar group received moistened diet without test article and acted as a control. The observed parameters were the following: Clinical signs, mortality, ophthalmologic examinations, body weight, food uptake, hematology, clinical chemistry, urine analysis, detailed gross pathology, organ weights and detailed histopathology. No deaths occurred during the study. Bodyweight and food consumption were unaffected by the administration of test material. No treatment-related signs were noted either during routine twice-daily observations or the scheduled physical and ophthalmic examinations. The results of haematology, blood chemistry and urine analysis after six and 12 weeks of treatment indicated no inter-group differences attributable to treatment. Macroscopic examination at necropsy, organ weight evaluation in absolute and bodyweight-relative terms, and .histopathological examination, did not reveal any toxicologically significant changes. Based on the results of this study, the NOAEL was set at the highest administered dose of 2000 ppm (84.1 and 90.2 mg/kg bod weight, for males and females respectively).