1-[[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Sep 1983 to 18 Oct 1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

May 1982

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

Version / remarks:

May 1982

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA OPP 80-1-4

Version / remarks:

Nov 1992

Test guideline 4

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPP 82-1 (90-Day Oral Toxicity)

Version / remarks:

Nov 1982

Test guideline 5

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPP 83-1 (Chronic Toxicity)

Version / remarks:

Nov 1982

Principles of method if other than guideline:

The purpose of this study was to determine the potential cumulative toxicity and dose-response relationships of test substance in Beagle dogs upon continuous daily administration in the feed for at least 12 months, and to establish a no-observable-effect level (NOEL).

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1-[[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole

EC Number:

262-104-4

EC Name:

1-[[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole

Cas Number:

60207-90-1

Molecular formula:

C15H17Cl2N3O2

IUPAC Name:

1-{[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl}-1H-1,2,4-triazole

Test animals

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Approximately 4 to 6 months of age
- Weight at study initiation: About 9.7 - 10.1 and 7.7 - 7.9 kg for males and females, respectively
- Housing: Animals were individually housed in pens with hardwood chip bedding "Beca Chips".
- Diet: Animals received daily portions of 400 g/dog of pulverised Purina Certified Canine Diet #5007. Food was presented for approximately 2 hours daily.
- Water: Tap water ad libitum.
- Acclimation period: Approximately 6 weeks.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 2
- Humidity (%): 40 - 70
- Air changes (/hour): 12 - 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 15 Sep 1983 To: 18 Oct 1984

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet: Diets were prepared approximately every 10 days.
- Mixing appropriate amounts with type of feed: The test article was administered orally in the diet admixed to the food (pulverised) at the levels 5, 50 and 250 ppm. The high (250 ppm) and mid (50 ppm) level test diets were prepared by first preparing a premix and then diluting aliquots of the premix with blank feed, to achieve the desired concentration of test diet. The 5 ppm diet was prepared by diluting on aliquot of the 50 ppm diet with blank feed. All premixes were mixed using an 1 kg capacity blender containing a high speed intensifier bar. All test diets were mixed using a 70 kg capacity blender. Prior to the start and twice during the study, diets were prepared for the purpose of determining homogeneity of mix of the test substance in the diet. Batches of test diet at each of the selected dietary concentrations were mixed.
- Storage temperature of food: Diets were stored in stainless steel containers at room temperature prior to and during use.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Diets were prepared approximately every 10 days. Samples from each dose level were taken from three locations in the blender and analysed for the concentration of test substance. Samples were also obtained from each dietary level for the first four mixes and every third mix thereafter and analysed for the test article concentration. Samples from each of the dietary levels of one mix were stored at room temperature for 21 days and analysed to determine the stability of test substance in the diet.

Duration of treatment / exposure:

53 consecutive weeks

Frequency of treatment:

Continuously

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

5 ppm

Remarks:

Group T-I: Low dose. Dietary equivalent to 0.17 and 0.19 mg/kg bw/day for males and females, respectively

Doses / concentrations 2

Dose / conc.:

50 ppm

Remarks:

Group T-II: Mid dose. Dietary equivalent to 1.9 and 1.9 mg/kg bw/day for males and females, respectively

Doses / concentrations 3

Dose / conc.:

250 ppm

Remarks:

Group T-III: High dose. Dietary equivalent to 8.4 and 8.9 mg/kg bw/day for males and females, respectively

No. of animals per sex per dose:

7 dogs/sex in the control and high dose groups, and 5 dogs/sex in the low and mid dose groups

Control animals:

yes, plain diet

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: all animals were observed daily for external signs of toxicity. Two morality checks, at last 5 hours apart (a.m. and pm), were conducted each day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Palpation for masses and detailed clinical examinations were conducted weekly.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights and food consumption were measured prior to initiation of feeding (week -1), weekly for the first 13 weeks, and monthly thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; Individual food consumption were measured prior to initiation of feeding (week -1), weekly for the first 13 weeks, and monthly thereafter.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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: Yes; Food conversion (body weight gain per 100 g of food consumed) was calculated weekly for the first 13 weeks from body weight and food consumption data (Food Conversion = Δ Body Weight/Food Consumption 100).


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ocular examinations were performed by a veterinary ophthalmologist on all dogs prior to the initiation of the study and after 52 weeks of feeding, and on 8 dogs at the conclusion of the 4 week recovery period. Eyes were examined by indirect ophthalmoscopy after pupil dilation with 1 % Hydriacyl (tropicamide).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples for haematology determinations were collected prior to the start of the study and after 3, 6 and 12 months of dosing from all animals. The same determinations were made on the recovery animals at the time of their necropsy. Blood was drawn from the jugular vein.
- Anaesthetic used for blood collection: No
- Animals fasted: Animals were fasted 19 - 20 hours prior to blood collection.
- Parameters checked: haemoglobin, haematocrit, erythrocyte count, total and differential leucocyte counts, reticulocyte count, lotting time, prothrombin time, Heinz bodies, erythrocyte indices (CV, MICH, MCHC), platelet count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples clinical chemistry determinations were collected prior to the start of the study and after 3, 6 and 12 months of dosing from all animals. The same determinations were made on the recovery animals at the time of their necropsy. Blood was drawn from the jugular vein.
- Animals fasted: Animals were fasted 19 - 20 hours prior to blood collection.
- Parameters checked: albumin, inorganic phosphorus, sodium, serum alanine aminotransferase (serum glutamic-pyruvic transaminase), potassium, globulin calcium chloride, serum aspartate aminotransferase (serum glutamic-oxaloacetic glucose transaminase), gamma glutamyl transpeptidase, total cholesterol, blood urea nitrogen, serum alkaline phosphatase, creatinine, creatinine phosphokinase, total protein, total bilirubin.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine samples for urine analysis were collected prior to the start of the study and after 3, 6 and 12 months of dosing from all animals. The same determinations were made on the recovery animals at the time of their necropsy.
- Metabolism cages used for collection of urine: Yes
- Parameters checked: pH, specific gravity, glucose, urobilinogen, bilirubin, occult blood, ketones, albumin, microscopic examination of sediment.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- After 53 weeks of feeding, 5 dogs per sex per group (40 total) were sacrificed and subjected to gross necropsies. The remaining 8 dogs (2 par sex from the control and high dose groups) were sacrificed and subjected to gross necropsies following the 4 week recovery period.
- Animals were euthanised with an intravenous injection (sodium pentobarbital) and exsanguinated. The gross necropsy included examination of the carcass, the external surface and all orifices, the cranial cavity, the nasal cavity and paranasal sinuses, the external and cut surfaces of the brain and spinal cord, the thoracic, abdominal, and pelvic cavities and their viscera, as well as the cervical issues and organs.
- Fresh organ weights were obtained and organ-to-body weight and organ-to-brain weight ratios were determined for the following organs from all dogs: ovaries, adrenal glands, pituitary, brain (including brain- stem), spleen, testes with epididymides, heart, kidneys, thyroid (with para- thyroids), liver.

HISTOPATHOLOGY: Yes
- For each animal, a complete set of tissues and organs was saved in 10 % oral offered formalin with the exception of the eyes which were fixed in Zenker's solution.
- Microscopic examination of paraffin embedded haematoxylin and eosin stained tissue sections was performed on the following organs and tissues from all animals: adrenal glands, parathyroid glands, aorta, pituitary, brain (cerebrum, cerebellum, brainstem), prostate, rectum, salivary gland, cecum, colon, sciatic nerve, skeletal muscle, duodenum, epididymides, oesophagus skin spinal cord (three levels cervical, mid-thoracic, lumbar), eyes, femur, gall bladder, spleen, heart, ileum, sternum (marrow), stomach (cardia, fundus, pylorus), kidneys, thymus, liver (2 lobes), lungs (which mainstem bronchi), thyroid, trachea, lymph nodes (cervical, mesenteric), urinary bladder, uterus horns, corpus, cervix), vagina, mammary glands, ovaries, all gross lesions (including tissue and draining lymph pancreas nodes if tumours).

Statistics:

Continuous data (body weight, body weight gain, food consumption, food conversion, haematological and clinical laboratory values, relevant urinalysis values, absolute organ weights) were analysed using one-way analysis of variance (Snedecor and Cochran, 1967). Differences among groups were identified using the Least Significant Difference Test.
Non-parametric data (relative organ weights) were analysed using the Kruskal-Wallis and Mann-Whitney U-tests (Siegel, 1956).
Score data (urinalysis scores) were analysed using a modification of the Mantel-Haenszel chi-square test (Mantel, 1963).
Pathology and daily observation incidence data were analysed using Fisher's Exact Test (Siegel, 1956). For all statistical analyses, significance was judged at the level of p ≤ 0.05.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The most prevalent and persistent observations noted involved the skin. Alopecia, thinning of the hair, loose hair, sores, reddening of the skin and thick skin were observed in both male and female animals of all groups. The etiology of the skin findings was most likely related to a mange (Demodex) infestation. With the exception one animal whose body weight decreased from 11.9 to 9.0 kg from week 13 through 53, the skin changes did not appear to compromise the health of the animals or to be associated with any toxicological abnormalities. The skin findings were present in control animals and the incidence of any one lesion in the high dose group was not significantly increased from the incidence in the respective control group, thus indicating that the skin lesions were not related to administration of test substance. None of the other observations noted during the course of the study were considered to be related to administration of test substance.

Mortality:

no mortality observed

Description (incidence):

All animals survived through the 53 week dosing period.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No statistically significant differences in mean body weight, food consumption or food conversion ratios were noted among groups during the study. Group mean body weight gain in females at the 50 ppm dose level was significantly decreased in comparison to the control value at week 33. Since group mean body weight gain data for this group was comparable to control values during the remainder of the study, the decrease in body weight gain at week 33 was not considered toxicologically significant. No other statistically significant differences in group mean body weight gain data were noted among groups. Examination of the individual body weight gain data showed a loss of body weight in the majority of male dogs, and to a lesser extent, female dogs throughout all groups at week 17 of the study. To insure adequate nutritional intake, the daily food allotment for all male dogs was increased from 400 to 500 g on day 128 of the study. The daily food allotment for females remained at 400 g per dog. Intermittent slight body weighs decreases were seen in several animals during the study, however, the decreases were transient in nature and unrelated to administration of test substance. The weight loss noted previously for one animal as most likely related to demodectic mange present in this animal. No other animal showed a sustained decrease in body weight during the study.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The data indicate that compound consumption in a particular group was consistent during the feeding period. Male animals at the 5, 50 and 250 ppm levels received an average of 1.2 ± 0.2, 13 ± 2 and 59 ± 8 mg/kg bw of test substance per week, respectively, while females at the 5, 50 and 250 ppm levels received an average of 1.3 ± 0.2, 13 ± 2 and 62 ± 10 mg/kg bw of test substance per week, respectively.

Food efficiency:

no effects observed

Description (incidence and severity):

No statistically differences in food conversion ratios were noted among groups during the study.

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Ocular examinations revealed the presence of a number of lesions in both and female animals at the termination of the 52 week feeding period, and lesions in two recovery animals. However, the nature and distribution of these lesions did not suggest a toxic origin. Thus, no ocular manifestations of a toxic nature were attributed to feeding of test substance at the dose levels used in this study.

Haematological findings:

no effects observed

Description (incidence and severity):

No statistically or toxicologically significant differences were noted between any group treated with test substance and the respective control group for any of the parameters examined after 3, 6, or 12 months of feeding.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Pre-test serum calcium levels in female animals at the 50 ppm level were significantly increased when compared to the control value, however, the value was within the normal range for beagle dogs of that particular age (FDRL historical data; calcium, 8.7-13.6 meq/L). Calcium levels in the 50 ppm females were comparable to control values after 3, 6 and 12 months of feeding. No statistically or toxicologically significant differences were noted between any group treated with test substance and the respective control group for any of the other parameters examined after 3, 6 or 12 months of feeding.

Endocrine findings:

not examined

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

The specific gravity of the urine in male animals at the 50 and 250 ppm dose levels was significantly increased, in comparison to the control value, after 3 months of feeding. Since urine specific gravity levels in male animals at the 50 and 250 ppm dose levels were comparable to control values after 6 and 12 months of feeding, the increase after 3 months of feeding was not considered toxicologically significant. No affects related to administration of test substance were noted on any of the other parameters examined for either sex after 3, 6 or 12 months of feeding.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The following statistically significant differences were noted between the groups fed test substance and the respective control group for the animals sacrificed at the completion of the 53 week dosing period: 1) decreased absolute mean brain weight in female dogs at the 50 ppm level; 2) increased mean organ-to-body and organ-to-brain weight ratio for the adrenal glands in female dogs at the 50 ppm level; 3) increased mean organ-to-body weight ratio for the adrenal glands in female dogs at the 250 ppm level; 4) decreased mean organ-to-body weight ratio for the pituitary gland in male dogs at the 250 ppm level; 5) increased mean organ-to-brain weight ratio for the pituitary gland in female dogs at the 50 ppm level.
Since no gross or microscopic abnormalities were observed in the brain, adrenal glands or pituitary gland of the above animals which could account for changes in organ weight, these changes were not considered to be due to administration of test substance increase in the organ-to-brain weight ratio for the adrenal glands and the pituitary gland in the 50 ppm females was most likely due to the decreased mean absolute brain weight noted for this group. Mean absolute brain weight and mean organ-to-brain weight ratio for the adrenals and pituitary in females at the high dose level (250 ppm) were not significantly different from controls, thus, indicating that the organ weight changes in mid-dose females were not test article-related. No clear dose response relationship was evident for the increased organ-to-body weight ratio for the adrenal glands in the 50 and 250 ppm females. No other significant differences between treatment and respective control groups occurred with regard to absolute organ weights, organ-to-body weight ratios, or organ-to-brain weight ratios after 53 weeks of feeding.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No gross or microscopic abnormalities related to feeding of test substance were noted in any organ (including the stomach) or tissue examined in either sex.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No abnormalities were observed.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No abnormalities were observed.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

>= 250 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Dietary equivalent to 8.4 and 8.9 mg/kg bw/day for males and females, respectively

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

DIET ANALYSIS
Results of the homogeneity analysis prior to initiation of feeding showed no apparent Location effects. The relative standard deviation of diets ranged from 1.7 (250 ppm) to 16.7 (5 ppm) %. The stability analysis showed test substance to be stable in the feed at room temperature for at least 21 days.
Diet analyses during the first 13 weeks of the study indicated that the diets were prepared accurately and homogeneously during this period. However, due to a gradual change in the physical condition. Crystallisation of the test article during refrigerated storage, non-homogeneous diets at the 50 and 250 pp levels pace fed to the animals for a period of 58 days from weeks 14 - 21. Following removal of the stock test article from the refrigerator and gentle heating of the test article prior to mixing, all test diets were prepared accurately and homogeneously for the remainder of the study. Over the entire course of the study, animals received the following time-weighted average dietary concentration of test substance: 4.93 ± 0.8 ppm (T-I), 47.6 ± 5.0 ppm (T-II), and 253.8 ± 67.2 ppm (T-III).

 

RECOVERY ANIMALS
Comparison of the data (daily observation, body weight, food consumption, haematology, clinical chemistry, urine analysis, ophthalmology, organ weight, pathology) from the 2 animals per sex from the high dose group which were held for an additional 28 days as a recovery group with the control recovery dog date and with the data collected from the high dose dogs during and at termination of the feeding period, further indicated that administration of test substance resulted in no toxicological or pathological effects.

 

Table 1. Summary of Daily Observations in Males

Observation	Control	Test substance
		5 ppm	50 ppm	250 ppm
Alopecia	5/7a	0/5	0/5	3/7
Diarrhoea	1/7	0/5	0/5	0/7
Pustules (Mouth region)	1/7	0/5	0/5	0/7
Red Skin	2/7	1/5	1/5	3/7
Soft Stools	1/7	0/5	0/5	0/7
Sore(s)	3/7	0/5	0/5	2/7
Swelling,  Right Axillary  Region	0/7	0/5	0/5	1/7
Thin Skin	1/7	1/5	0/5	2/7
Thin Hair	5/7	1/5	1/5	2/7
Vomiting	1/7	0/5	0/5	0/7

a = Number of animals with observations/total number of animals

 

Table 2. Summary of Daily Observations in Females

Observation	Control	5 ppm	50 ppm	250 ppm
Alopecia	3/7a	3/5	3/5	2/7
Diarrhea	0/7	0/5	0/5	1/7
Laceration	0/7	1/5	0/5	1/7
Loose Hair	0/7	2/5	0/5	0/7
Red Skin	2/7	4/5	3/5	3/7
Rough Coat	0/7	2/5	0/5	0/7
Soft Stools	0/7	0/5	1/5	0/7
Sore(s)	3/7	3/5	2/5	4/7
Thick Skin	1/7	4/5	3/5	2/7
Thin Hair	3/7	4/5	3/5	2/7
Wart-Like Growth (Left Ear)	0/7	0/5	1/5	0/7

 

Table 3. Summary of Organ Weight Data(a

	Sex and Treatment Level (ppm)	Body Weight (kg)	Brain Weight (g)	Brain %BWt	Liver (g)	Liver %BWt	Liver %BrWt	Spleen (g)	Spleen %BWt	Spleen %BrWt	Kidneys (g)	Kidneys %BWt	Kidneys %BrWt
Males	Control	12.3	83.7	0.7	416.9	3.4	493.3	66.0	0.6	79.8	56.2	0.5	66.8
	(±)	2.6	5.9	0.1	109.6	0.5	99.1	26.2	0.2	32.5	13.6	0.1	12.5
	5 ppm substance	12.7	90.9	0.7	395.6	3.1	421.0	46.6	0.4	51.3	61.1	0.5	67.0
	(±)	0.9	3.7	0.0	57.6	0.4	46.5	22.4	0.2	24.3	11.0	0.1	9.6
	50 ppm substance	12.4	83.4	0.6	419.9	3.4	502.7	42.8	0.4	51.4	62.3	0.5	74.5
	(±)	1.3	3.5	0.1	64.6	0.2	70.6	15.4	0.1	18.4	10.1	0.0	10.6
	250 ppm substance	13.4	80.8	0.6	457.3	3.4	561.5	53.3	0.4	64.6	68.5	0.5	83.7
	(±)	2.8	7.5	0.1	124.8	0.7	130.9	23.1	0.1	22.7	16.7	0.1	13.9
Females	Control	11.5	84.5	0.7	351.7	3	414.5	55.4	0.5	66.0	49.0	0.5	56.0
	(±)	1.5	3.5	0.1	61.5	0.3	57.0	16.0	0.2	21.0	8.3	0.1	9.3
	5 ppm substance	10.7	82.6	0.8	359.4	3.3	433.4	52.6	0.5	63.8	51.1	0.5	61.4
	(±)	1.3	6.4	0.1	70.8	0.4	72.2	16.2	0.1	20.0	11.5	0.1	11.2
	50 ppm substance	9.9	75.3b	0.8	360.5	3.7	478.7	47.7	0.5	63.5	49.1	0.5	65.1
	(±)	0.9	3.4	0.1	31.2	0.5	32.3	23.3	0.3	30.9	7.2	0.1	18.5
	250 ppm substance	10.5	81.8	0.8	338.6	3.2	413.3	71.7	0.7	88.8	45.7	0.4	55.9
	(±)	1.1	5.6	0	37	0.4	29.7	36.2	0.4	47.5	4.0	0.0	4.3

a) Values are group means ± SD for 5 observations

b) Significantly different from control, p ≤ 0.05

 

Table 4. Summary of Organ Weight Data)a

	Sex and Treatment Level (ppm)	Body Weight (kg)	Brain Weight (g)	Heart (g)	Heart %BWt	Heart %BrWt	Adrenal (g)	Adrenal %BWt  *1000	Adrenal %BrWt	Thyroid/ Parathyroid (g)	Thyroid/ Parathyroid %BWt  *1000	Thyroid/ Parathyroid %BrWt
Males	Control	12.3	83.7	103.2	0.8	122.7	1.4	11.9	1.7	0.9	7.7	1.1
	(±)	2.6	5.9	18.9	0.1	16.2	0.2	1.6	0.2	0.2	1.0	0.2
	5 ppm substance	12.7	90.9	107.9	0.9	133.1	1.5	11.5	1.6	1.1	9.0	1.3
	(±)	0.9	3.7	13.0	0.1	28.5	0.2	1.9	0.3	0.1	1.1	0.1
	50 ppm substance	12.4	83.4	114.2	0.9	136.7	1.6	12.9	1.9	1.0	8.2	1.2
	(±)	1.3	3.5	9.6	0.1	7.4	0.4	1.9	0.4	0.1	1.5	0.2
	250 ppm substance	13.4	80.8	113.2	0.8	139.0	1.5	11.4	1.9	1.1	7.9	1.3
	(±)	2.8	7.5	23.8	0.0	20.5	0.5	1.1	0.4	0.5	2.2	0.5
Females	Control	11.5	84.5	98.6	0.9	116.3	1.2	10.8	1.5	0.9	7.9	1.1
	(±)	1.5	3.5	22.9	0.2	24.0	0.2	1.6	0.3	0.1	1.2	0.1
	5 ppm substance	10.7	82.6	87.9	0.8	106.1	1.3	11.9	1.6	0.8	7.4	1.0
	(±)	1.3	6.4	11.4	0.1	7.6	0.4	2.1	0.4	0.2	1.8	0.3
	50 ppm substance	9.9	75.3b	92.0	0.9	122.5	1.5	15.4	2.0	0.9	9.4	1.2
	(±)	0.9	3.4	9.7	0.1	15.4	0.2	2.9	0.3	0.2	2.5	0.2
	250 ppm substance	10.5	81.8	88.0	0.9	107.9	1.4	12.9	1.6	0.9	8.4	1.1
	(±)	1.1	5.6	2.8	0.1	6.9	0.2	0.9	0.2	0.2	1.2	0.2

a) Values are group means ± SD for 5 observations
b) Significantly different from control, p ≤ 0.05

 

Table 5. Summary of Organ Weight Data)a

	Sex and Treatment Level (ppm)	Body Weight (kg)	Brain Weight (g)	Pituitary (g)	Pituitary %BWt	Pituitary %BrWt	Testes/Epididymides (g)	Testes/Epididymides %BWt	Testes/Epididymides %BrWt
Males	Control	12.3	83.7	75	6.3	9.0	23.9	0.2	28.3
	(±)	2.6	5.9	12	1.5	1.6	6.0	0.0	5.5
	5 ppm substance	12.7	90.9	80	6.3	8.8	26.4	0.2	29.1
	(±)	0.9	3.7	16	1.0	1.7	5.1	0.0	5.1
	50 ppm substance	12.4	83.4	59	4.9	7.1	21.6	0.2	25.8
	(±)	1.3	3.5	12	1.3	1.4	4.2	0.0	4.5
	250 ppm substance	13.4	80.8	56	4.1b	6.9	23.6	0.2	28.8
	(±)	2.8	7.5	25	1.1	2.5	7.3	0.0	6.6

a) Values are group means ± SD for 5 observations
b) Significantly different from control, p ≤ 0.05

 

Table 6. Summary of Organ Weight Data)a

	Sex and Treatment Level (ppm)	Body Weight (kg)	Brain Weight (g)	Pituitary (g)	Pituitary %BWt	Pituitary %BrWt	Ovaries (g)	Ovaries %BWt	Ovaries %BrWt
Females	Control	11.5	84.5	54	4.8	6.4	1.2	10.6	1.4
	(±)	1.5	3.5	22 (4)	2.0 (4)	2.5 (4)	0.2	1.3	0.2 (4)
	5 ppm substance	10.7	82.6	60	5.5	7.2	1.1	10.6	1.4
	(±)	1.3	6.4	22	1.8	2.7	0.2	1.6	0.2
	50 ppm substance	9.9	75.3b	73	7.4	9.7b	1.2	11.6	1.6
	(±)	0.9	3.4	10	1.3	1.2	0.3	2.9	0.5
	250 ppm substance	10.5	81.8	56	5.4	6.8	1.2	11.2	1.4
	(±)	1.1	5.6	6	0.9	1.0	0.2	2.0	0.2

a) Values are group means ± SD for 5 observations, unless otherwise indicated in ( ).
b) Significantly different from control, p ≤ 0.05.

 

Table 7. Summary of Organ Weight Data (Recovery))a

	Sex and Treatment Level (ppm)	Body Weight (kg)	Brain Weight (g)	Brain %BWt	Liver (g)	Liver %BWt	Liver %BrWt	Spleen (g)	Spleen %BWt	Spleen %BrWt	Kidneys (g)	Kidneys %BWt	Kidneys %BrWt
Males	Control	16.6_b	92.1	0.6	518.5	3.2	562.8	93.1	0.5	100.4	79.4	0.5	86.2
		(2)	(2)	(2)	(2)	(2)	(2)	(2)	(2)	(2)	(2)	(2)	(2)
	250 ppm substance	13.0_b	82.9	0.7	406.5	3.2	493.6	49.3	0.4	58.7	55.1	0.4	71.3
		(2)	(2)	(2)	(2)	(2)	(2)	(2)	(2)	(2)	(2)	(2)	(2)
Females	Control	12.9	88.9	0.7	362.4	2.9	411.5	89.9	0.7	105.0	49.2	0.4	55.8
		(2)	(2)	(2)	(2)	(2)	(2)	(2)	(2)	(2)	(2)	(2)	(2)
	250 ppm substance	12.9	83.0	0.7	371.7	2.9	448.4	53.1	0.5	64.1	57.2	0.5	69.0
		(2)	(2)	(2)	(2)	(2)	(2)	(2)	(2)	(2)	(2)	(2)	(2)

a) Values are group means for number of observations in ( ).
b) Insufficient number of observations for calculations of standard deviation and statistical analysis.

 

Table 8. Summary of Organ Weight Data (Recovery))a

	Sex and Treatment Level (ppm)	Body Weight (kg)	Brain Weight (g)	Heart (g)	Heart %BWt	Heart %BrWt	Adrenal (g)	Adrenal %BWt  *1000	Adrenal %BrWt	Thyroid/ Parathyroid (g)	Thyroid/ Parathyroid %BWt  *1000	Thyroid/ Parathyroid %BrWt
Males	Control	16.6_b	92.1	139.2	0.9	151.0	1.7	9.9	1.8	1.3	7.8	1.4
		(2)	(2)	(2)	(2)	(2)	(2)	(2)	(2)	(2)	(2)	(2)
	250 ppm substance	13.0_b	82.9	106.6	0.9	130.4	1.3	9.7	1.6	1.1	8.5	1.4
		(2)	(2)	(2)	(2)	(2)	(2)	(2)	(2)	(2)	(2)	(2)
Females	Control	12.9	88.9	94.9	0.8	107.8	1.5	11.3	1.7	0.9	7.0	1.0
		(2)	(2)	(2)	(2)	(2)	(2)	(2)	(2)	(2)	(2)	(2)
	250 ppm substance	12.9	83.0	91.1	0.7	109.9	1.6	12.4	2.0	1.3	10.0	1.6
		(2)	(2)	(2)	(2)	(2)	(2)	(2)	(2)	(2)	(2)	(2)

a) Values are group means for number of observations in ( ).
b) Insufficient number of observations for calculations of standard deviation and statistical analysis.

 

Table 9. Summary of Organ Weight Data (Recovery))a

	Sex and Treatment Level (ppm)	Body Weight (kg)	Brain Weight (g)	Pituitary (g)	Pituitary %BWt	Pituitary %BrWt	Testes/Epididymides (g)	Testes/Epididymides %BWt	Testes/Epididymides %BrWt
Males	Control	16.6_b	92.1	61	3.6	6.6	28.9	0.2	31.4
		(2)	(2)	(2)	(2)	(2)	(2)	(2)	(2)
	250 ppm substance	13.0_b	82.9	79	5.9	9.1	24.3	0.2	29.4
		(2)	(2)	(2)	(2)	(2)	(2)	(2)	(2)

a) Values are group means for number of observations in ( ).
b) Insufficient number of observations for calculations of standard deviation and statistical analysis.

 

Table 10. Summary of Organ Weight Data (Recovery))a

	Sex and Treatment Level (ppm)	Body Weight (kg)	Brain Weight (g)	Pituitary (g)	Pituitary %BWt	Pituitary %BrWt	Ovaries (g)	Ovaries %BWt	Ovaries %BrWt
Females	Control	12.9	88.9	71	5.5	8.0	1.7	12.9	19.2
		(2)	(2)	(2)	(2)	(2)	(2)	(2)	(2)
	250 ppm substance	12.9	83.0	59	4.5	7.2	1.2	9.5	13.9
		(2)	(2)	(2)	(2)	(2)	(2)	(2)	(2)

a) Values are group means for number of observations in ( ).
b) Insufficient number of observations for calculations of standard deviation and statistical analysis.

Applicant's summary and conclusion

Conclusions:

Oral administration (via the feed) of test substance for 53 consecutive weeks to beagle dogs at levels of 5, 50 and 250 ppm resulted in no toxicological or pathological effects which were related to administration of the test material. Thus, the no observed adverse effect level for this study is 250 ppm.

Executive summary:

In a GLP-compliant study the potential cumulative toxicity effects and dose-response relationships of the test substance were evaluated in Beagle dogs following administration of the test article in the diet for 53 consecutive weeks. The maximum tolerated dose was established at 1200 ppm in a previously conducted 90 day feeding study in dogs. Groups of 7 dogs per sex in the control and high dose groups, and 5 dogs per sex in the low and mid dose groups were fed either a control diet or diet supplemented with test substance at levels of 5, 50 or 250 ppm. Two dogs par sex in the control and high dose groups were held for an additional 28 days as a recovery group, during which time they were fed basal diet containing no test material. Animals were observed daily; body weights and food consumption were measured weekly for the first 13 weeks, and monthly thereafter. Food conversion was calculated weekly from body weight and food consumption data for the first 13 weeks of the study. Blood and urine were collected for haematology, clinical chemistry, and urine analysis prior to the initiation of feeding and at months 3, 6 and 12, as well as on the recovery animals at 13 months. At the termination of the study, complete gross necropsy and histo-pathologic examinations were performed on all animals.
No deaths occurred during the course of the study, nor were there any daily observations which indicated toxicity related to administration of the test substance. There were no changes in body weight, body weight gain, food consumption, food conversion, organs weights, or histological, clinical chemistry of urine parameters which indicated any test article-related toxicity. No gross necropsy or histopathological effects were observed in any organ or tissue examined which were related to administration of the test substance.

In conclusion, oral administration of the test substance for 53 weeks at levels of up to 250 ppm resulted in no toxicological or histopathological effects which were related to administration of the test material, thus, establishing a no observed adverse effect level of 250 ppm.