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

Endpoint summary

Description of key information

- Oral: NOAEL = 240 ppm (dietary equivalent to 15.9 and 16.8 mg/kg bw/day for males and females, respectively), rats, sub-chronic, 90 days, dietary, Sachsse 1979.

- Oral: NOAEL ≥ 250 ppm (8.4 and 8.9 mg/kg bw/day for males and females, respectively), dogs, sub-chronic, 1 year, dietary, Johnson 1985

- Oral: NOAEL = 600 ppm (dietary equivalent to 38 mg/kg bw/day) in males and 1500 ppm (dietary intake of 111 mg/kg bw/day) in females, rats, sub-chronic, 13 weeks, dietary, OECD TG 424, Herberth 2013a

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

May 1982

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

Version / remarks:

May 1982

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA OPP 80-1-4

Version / remarks:

Nov 1992

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

Nov 1982

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

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

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

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

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

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

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.

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.

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

Key result

Critical effects observed:

no

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.

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. 

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 Jul 2012 to 21 Dec 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 424 (Neurotoxicity Study in Rodents)

Version / remarks:

1997

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.6200

Version / remarks:

1998

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Approximately 6 weeks old
- Weight at study initiation: Individual body weights ranged from 190 - 243 g and 146 - 186 g for males and females, respectively.
- Housing: Upon arrival, all animals were housed 2 - 3 per cage by sex for approximately 6 days. Thereafter, all animals were housed individually in clean, stainless steel, wire-mesh cages suspended above cage-board. The cage-board was changed at least 3 times each week. Animals were housed by group within each column, and groups were intermixed to avoid housing all animals of the same group together. The columns were rotated weekly to avoid cage location bias.
- Diet: Basal diet, LLC Certified Rodent, LabDiet 5002 (meal) available, ad libitum
- Water: Reverse osmosis-treated (on-site) drinking water delivered by an automatic watering system was provided, ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes: Minimum of 10 per hour
- Photoperiod (hrs dark / hrs light): Fluorescent lighting illumination for 12/12

IN-LIFE DATES: From: 31 Aug 2012 To: 15 Nov 2012

Route of administration:

oral: feed

Vehicle:

acetone

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Diets for test and control groups were prepared approximately weekly
- Mixing appropriate amounts with (Type of food): The dietary formulations were prepared with acetone as the vehicle using an appropriate blender. The acetone was evaporated off overnight prior to feeding the diets to the animals on study.
- Storage temperature of food: Room temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and stability (following 11 days of room temperature storage) of test substance formulations were established in a previous study at concentrations of 200 and 6500 ppm. Therefore, stability was not assessed in this study.
Duplicate samples for homogeneity and concentration analyses were collected from the top, middle, and bottom strata of each prepared diet (including the control group diet) to be used on study during the first (study week 0), fourth (study week 3), eighth (study week 7), thirteenth (study week 12), and fourteenth (study week 13) weeks of dosing. One set of samples was analysed for concentration, and the remaining samples were stored frozen at approximately -20 °C as backup.

Duration of treatment / exposure:

Approximately 13 weeks

Frequency of treatment:

Continuously

Dose / conc.:

200 ppm

Remarks:

Group 2, low dose.

Dose / conc.:

600 ppm

Remarks:

Group 3, mid-dose.

Dose / conc.:

1 500 ppm

Remarks:

Group 4, high dose. Females only.

Dose / conc.:

3 500 ppm

Remarks:

Group 4, high dose. Males only.

No. of animals per sex per dose:

12

Control animals:

yes, concurrent no treatment

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS
- Time schedule: All animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity. Detailed physical examinations were recorded weekly, beginning 1 week prior to test diet administration and continuing until the scheduled necropsy. The absence or presence of findings was recorded for individual animals.

BODY WEIGHTS
- Time schedule: Individual body weights were recorded weekly, beginning approximately 1 week prior to test diet administration. Mean body weights and mean body weight changes were calculated for the corresponding intervals. Mean body weight changes were also calculated for the overall exposure period (study days 0 - 91).

FOOD CONSUMPTION AND TEST SUBSTANCE CONSUMPTION
- Time schedule: Individual food consumption was recorded weekly, beginning approximately 1 week prior to test diet administration. Food intake was calculated as g/animal/day for the corresponding body weight change intervals. Mean test substance consumption (mg/kg/day) for each group was determined by multiplying the concentration of the test substance in the diet (mg/kg of diet) by the g/animal/day food consumption value and dividing by the body weight (in kg) for each interval. When food consumption could not be determined for an animal during a given interval (due to a weighing error, food spillage, etc.), group mean values were calculated for that interval using the available data.

OPHTHALMIC EXAMINATIONS
- Time schedule: Ophthalmic examinations were conducted on all animals prior to the initiation of test diet administration (05 August 2012; study week -2) and near the end of the treatment period (06 November 2012; study week 12). All ophthalmic examinations were conducted using an indirect ophthalmoscope and slit lamp biomicroscope preceded by pupillary dilation with an appropriate mydriatic agent.

FUNCTIONAL OBSERVATIONAL BATTERY ASSESSMENTS
- Time schedule: FOB findings were recorded for all animals 1 week prior to administration of test substance (study week -1) and during study weeks 1, 3, 7, and 12. The FOB used at WIL Research is based on previously developed protocols (Gad, 1982; Haggerty, 1989; Irwin, 1968; Moser et al., 1988; Moser et al., 1991 and O’Donoghue, 1989). Testing was performed by the same biologists, to the extent possible, without knowledge of group assignment for the animals. The FOB was performed in a sound-attenuated room equipped with a white-noise generator set to operate at 70 ± 10 dB. All animals were observed for the parameters as described below:

HOME CAGE OBSERVATIONS
Posture; Convulsions/tremors; Feces consistency; Biting; Palpebral (eyelid) closure

HANDLING OBSERVATIONS
Ease of removal from cage; Lacrimation/chromodacryorrhea; Piloerection; Palpebral closure; Eye prominence; Red/crusty deposits; Ease of handling animal in hand; Salivation; Fur appearance; Respiratory rate/character; Mucous membranes/eye/skin color; Muscle tone

OPEN FIELD OBSERVATIONS
Mobility; Rearing; Convulsions/tremors; Grooming; Bizarre/stereotypic behaviour; Time to first step (seconds); Gait; Arousal; Urination/defecation; Gait score; Backing; Note: Open field observations were evaluated over a 2-minute observation period.

SENSORY OBSERVATIONS
Approach response; Startle response; Pupil response; Forelimb extension; Air righting reflex; Touch response; Tail pinch response; Eyeblink response; Hindlimb extension; Olfactory orientation

NEUROMUSCULAR OBSERVATIONS
Hindlimb foot splay; Grip strength-hind and forelimb

PHYSIOLOGICAL OBSERVATIONS
Catalepsy; Body temperature; Body weight

LOCOMOTOR ACTIVITY
- Time schedule: Locomotor activity (LMA) was assessed for all animals during pre-test (study week -1) and during study weeks 1, 3, 7, and 12.
- LMA, recorded after the completion of the FOB, was measured automatically using a personal computer-controlled system that utilises a series of infrared photobeams surrounding an amber plastic, rectangular cage to quantify the motor activity of each animal. Four-sided black plastic enclosures were used to surround the transparent plastic boxes and decrease the potential for distraction from extraneous environmental stimuli or activity by biologists or adjacent animals. The black enclosures rested on top of the photobeam frame and did not interfere with the path of the beams. The LMA assessment was performed in a sound-attenuated room equipped with a white-noise generator set to operate at 70 ± 10 dB. The testing of treatment groups was conducted according to replicate sequence. Each animal was tested separately.
- Data were collected in 5-minute periods, and the test session duration was 60 minutes. These data were compiled as six 10-minute subintervals for tabulation. Data for total and ambulatory LMA were tabulated. Total motor activity was defined as a combination of fine motor skills (i.e., grooming, interruption of 1 photobeam) and ambulatory motor activity (interruption of 2 or more consecutive photobeams).

Sacrifice and pathology:

NEUROPATHOLOGY - MACROSCOPIC EXAMINATION
At the termination of the study (study week 13), 5 randomly selected rats/sex/group were deeply anesthetised by an intraperitoneal injection of sodium pentobarbital and then perfused in situ with a 4.0 % paraformaldehyde/0.1M phosphate buffer solution. The central and peripheral nervous system tissues were dissected and preserved. Fixed brain weight and brain dimensions (length [excluding olfactory bulbs] and width) were recorded. Any observable gross changes, abnormal coloration, or lesions of the brain and spinal cord were recorded. After successful perfusion of the selected rats, all remaining (non-selected) rats were euthanised by carbon dioxide inhalation and discarded without macroscopic examination.

NEUROPATHOLOGY - MICROSCOPIC EXAMINATION
The following nerve tissues were collected from all rats subjected to in situ perfusion (5 rats/sex/group): Brain - olfactory bulbs, cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus, midbrain, cerebellum, pons, and medulla oblongata; Spinal cord - at cervical swellings C3-C7 and at lumbar swellings T13-L4, Trigeminal ganglia/nervesa; Lumbar dorsal root ganglia at T13-L4(b); Lumbar dorsal root fibers at T13-L4(b); Lumbar ventral root fibers at T13-L4(b); Cervical dorsal root ganglia at C3-C7(b); Cervical dorsal root fibers at C3-C7(b); Cervical ventral root fibers at C3-C7(b); Cervical spinal nerve; Lumbar spinal nerve; Sciatic nerves (mid-thigh region) (2)(c); Sciatic nerves (at sciatic notch) (2)(c); Sural nerves (2)(c); Tibial nerves (2)(c); Peroneal nerves (2)(c); Optic nervesa; Eyes(a); Skeletal muscle (gastrocnemius); Other sites (if deemed necessary).

Legend:
(a) - Both processed and evaluated microscopically
(b) - Four to 6 tissues were collected at necropsy; 2 were evaluated microscopically.
(c) - One processed for microscopic examination
(2) - Two sections (1 transverse and 1 longitudinal) of the tissue were evaluated from the right hind leg. The tissues from the left hind leg were collected and preserved for possible future evaluation.

The tissues listed above from the 5 rats/sex perfused in situ in the control and high dose groups were prepared for a qualitative histopathological examination by embedding in paraffin (central nervous system tissues) or plastic (peripheral nervous system tissues), sectioning, and staining with haematoxylin and eosin for microscopic evaluation. The tissues collected from the perfused low- and mid-dose animals were preserved for possible future examination.

Statistics:

Each mean was presented with the standard deviation (S.D.) and number of animals (N) used to calculate the mean. In addition, standard error (S.E.) was presented for body weight, food consumption, brain measurement, and LMA data. Due to the use of significant figures and different rounding conventions inherent in the types of software used, the means and standard deviations in the summary and individual tables may differ slightly. Therefore, the use of reported individual values to calculate subsequent parameters or means will, in some instances, yield minor variations from those listed in the report data.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related clinical findings observed in any group. Clinical findings observed in the test substance-exposed groups, including hair loss and red material on various body surfaces, were observed in the control group with similar frequency or in a limited number of animals and therefore, not considered test substance-related.

Mortality:

no mortality observed

Description (incidence):

All males and females survived to the scheduled euthanasia during study week 13.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A statistically significantly lower mean body weight gain was noted for the 3500 ppm males during study days 0-7; this was considered test substance-related. Mean body weight gains in this group were generally similar to or slightly lower than the control group throughout the remainder of the treatment period; these slight reductions, coupled with the initial reduction in body weight gain, resulted in a lower (not statistically significant) mean body weight gain that was 10.9 % lower than the control group when the entire treatment period (study days 0-91) was evaluated and mean body weights that were up to 7.4 % lower (not statistically significant) than the control group. Mean body weights and body weight gains for the 200 and 600 ppm males and the 200, 600, and 1500 ppm females were unaffected by test substance administration. A statistically significantly higher mean body weight gain was noted for 600 ppm females during study days 14-21; however, this did not occur in a dose-related manner and was considered incidental to treatment. Other differences from the control group were slight and/or not statistically significant.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Statistically significantly lower mean food consumption was noted for the 3500 ppm males during study days 0-7. This corresponded to a reduced mean body weight gain noted during this period. Mean food consumption in this group was generally similar to the control group throughout the remainder of the treatment period. Mean food consumption for the 200 and 600 ppm males and the 200, 600, and 1500 ppm females was unaffected by test substance administration.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No ophthalmic lesions indicative of toxicity were observed in any of the test substance-treated groups. All findings observed were typical in prevalence and appearance for laboratory rats of this age and strain.

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Home cage parameters were unaffected by test diet consumption. There were no statistically significant differences between the control and test substance-treated groups (by sex) at the study week 1, 3, 7, and 12 evaluations.
Handling observations parameters were unaffected by test diet consumption. There were no statistically significant differences between the control and test substance-treated groups (by sex) at the study week 1, 3, 7, and 12 evaluations.
Open field parameters were unaffected by test diet consumption. There were no statistically significant differences between the control and test substance-treated groups (by sex) at the study week 1, 3, 7, and 12 evaluations, with the following exception. The mean rearing count for males for the 200 ppm males was statistically significantly lower than the control group at the study week 3 evaluation; however, this did not occur in a dose-related manner and in the absence of other related observations was not considered test substance-related.
Sensory parameters were unaffected by test diet consumption. There were no statistically significant differences between the control and test substance-treated groups (by sex) at the study week 1, 3, 7, and 12 evaluations.
Mean body weights for the 3500 ppm males were lower (not statistically significant) than the
control group during the study week 7 and 12 evaluations. The effects on mean body weight correlated with test substance-related effects during the weekly body weight evaluations. There were no test substance-related effects on mean catalepsy or body temperature for males and females at any test substance concentration.
Locomotor activity patterns (mean ambulatory and total motor activity counts) were unaffected by test diet consumption. There were no statistically significant differences between the control and test substance-exposed groups when values obtained from the 6 sub-intervals (0-10 min, 11-20 min, 21-30 min, 31-40 min, 41-50 min, and 51-60 min) and the overall 60-min test session were evaluated during study weeks 1, 3, 7, and 12. No remarkable shifts in the pattern of habituation occurred in any of the test substance-exposed groups when the animals were evaluated on study weeks 1, 3, 7, and 12. During the pretest period, significantly (p≤0.016) higher motor activity counts were noted for the 200, 600, and 3500 ppm males during the 21-30 min sub-interval; however, this was attributed to normal biological variability and was not expected to impact LMA assessments during the treatment period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related alterations in brain weights and measurements for males and females at any dose level. Differences from the control group were slight and not statistically significant.

Neuropathological findings:

no effects observed

Description (incidence and severity):

Neuromuscular parameters were unaffected by test diet consumption. There were no statistically significant differences between the control and test substance-treated groups (by sex) at the study week 1, 3, 7, and 12 evaluations, with the following exception. A statistically significantly higher mean hindlimb footsplay value was noted for 3500 ppm males at the week 1 evaluations; however, this difference was not attributed to treatment, but rather to a low control group mean value (50.1 mm), as the value in the 3500 ppm group (65.2 mm) was similar to the mean value in the WIL historical control data (68.2 mm).

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related macroscopic findings observed in the brain or spinal cord of males and females at any dose level. The only finding noted in the test substance-treated groups was depressed areas of the brain for a single 200 ppm male; however, this finding did not occur in a dose-related manner and therefore was not attributed to treatment. There were no test substance-related microscopic observations. All histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test substance. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations. There were instances of axonal degeneration in the peripheral nerves and in the spinal nerve roots in control and treated animals. This axonal degeneration was of minimal severity, typically with only a single ‘digestion chamber’, and consistent with incidental alterations. Minimal axonal degeneration in the peripheral nerves and spinal nerve roots is a common background lesion.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

There were no test substance-related observations.

Key result

Dose descriptor:

NOAEL

Effect level:

600 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

Remarks on result:

other: Corresponding to a dietary intake of 38 mg/kg bw/day.

Key result

Dose descriptor:

NOAEL

Effect level:

1 500 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: Corresponding to a dietary intake of 111 mg/kg bw/day.

Key result

Critical effects observed:

no

ANALYSES OF DIETARY FORMULATIONS
The analysed dietary preparations were within the performing laboratory range for dietary formulations (8 5% to 115 % of target), met the protocol-specified criteria for mean concentration (90 % to 110 % of nominal with RSD of ≤ 5%), and were homogeneous, with the following exceptions. The mean concentrations of the 200, 600, 1500, and 3500 ppm formulations prepared on 08 November 2012 (dispensed during study week 13) were 88.9 %, 88.9 %, 88.3 %, and 88.7 % of target, respectively. These values were below the protocol-specified range, but were within the range; therefore, this did not affect the outcome of the study. The test substance was not detected in the control diet that was administered to the control group (Group 1).

 

Table 1. Results of Homogeneity and Concentration Analyses

Group 2		Group 3	Group 4F	Group 4M
(200 ppm)		(600 ppm)	(1500 ppm)	(3500 ppm)
Homogeneity and Concentration Assessment of the 09 August 2012 Formulations
Mean Concentration (mg/mL)	187	575	1433	3285
RSD (%)	0.98	0.72	0.94	2.6
Mean % of Target	93.6	95.8	95.5	93.8
Homogeneity and Concentration Assessment of the 30 August 2012 Formulations
Mean Concentration (mg/mL)	187	570	1477	3412
RSD (%)	2.2	1.4	2.3	2.2
Mean % of Target	93.5	94.9	98.5	97.5
Homogeneity and Concentration Assessment of the 27 September 2012 Formulations
Mean Concentration (mg/mL)	196	593	1487	3461
RSD (%)	0.86	1.1	0.97	0.99
Mean % of Target	98.0	98.8	99.1	98.9
Homogeneity and Concentration Assessment of the 01 November 2012 Formulations
Mean Concentration (mg/mL)	188	553	1443	3440
RSD (%)	3.0	3.6	2.3	2.1
Mean % of Target	94.2	92.2	96.2	98.3
Homogeneity and Concentration Assessment of the 08 November 2012 Formulations
Mean Concentration (mg/mL)	178	533	1325	3106
RSD (%)	0.72	1.9	1.5	1.4
Mean % of Target	88.9	88.9	88.3	88.7
M = Males
F = Females

 

Table 2.Summary of Test Substance Consumption 

Target Dietary	Mean Calculated Test Substance Consumption (mg/kg/day)
Concentration (ppm)	Males	Female
200	13	15
600	38	45
1500	NA	111
3500	222	NA

 

Table 3. Summary of Body Weights [g]

		Males				Females
		0 PPM	200 PPM	600 PPM	3500 PPM	0 PPM	200 PPM	600 PPM	3500 PPM
DAY -7	MEAN	147	147	147	147	126	126	126	126
	% DIFFERENCE		0	0	0		0	0	0
	S.D.	11.1	11.4	9.3	10.5	7.6	7.6	7.8	7.8
	S.E.	3.2	3.3	2.7	3	2.2	2.2	2.2	2.3
	N	12	12	12	12	12	12	12	12
DAY 0	MEAN	214	214	214	213	161	165	162	165
	% DIFFERENCE		0	0	-0.5		2.5	0.6	2.5
	S.D.	14.4	14.5	12.5	10	9.9	6.3	10.5	9.1
	S.E.	4.2	4.2	3.6	2.9	2.8	1.8	3	2.6
	N	12	12	12	12	12	12	12	12
DAY 7	MEAN	273	269	270	261	182	185	187	187
	% DIFFERENCE		-1.5	-1.1	-4.4		1.6	2.7	2.7
	S.D.	18.1	16.8	16.5	13.5	14.3	11.2	13.9	12
	S.E.	5.2	4.9	4.8	3.9	4.1	3.2	4	3.5
	N	12	12	12	12	12	12	12	12
DAY 14	MEAN	312	310	313	304	198	206	204	205
	% DIFFERENCE		-0.6	0.3	-2.6		4	3	3.5
	S.D.	23.3	23	20.6	15.1	19.1	16	16.6	14.1
	S.E.	6.7	6.7	6	4.4	5.5	4.6	4.8	4.1
	N	12	12	12	12	12	12	12	12
DAY 21	MEAN	355	350	356	345	215	227	227	218
	% DIFFERENCE		-1.4	0.3	-2.8		5.6	5.6	1.4
	S.D.	27.8	29.3	24.3	19.4	19.1	18	16.5	15.8
	S.E.	8	8.5	7	5.6	5.5	5.2	4.8	4.5
	N	12	12	12	12	12	12	12	12
DAY 28	MEAN	386	379	388	374	225	238	238	228
	% DIFFERENCE			0.5	-3.1	5.8	5.8	1.3
	S.D.	30.5	32.2	31.5	21.4	24.4	19.6	20.7	13.3
	S.E.	8.8	9.3	9.1	6.2	7	5.7	6	3.9
	N	12	12	12	12	12	12	12	12
DAY 35	MEAN	35	411	420	403	237	248	248	238
	% DIFFERENCE		-2.1	0	-4	4.6	4.6	0.4
	S.D.	33.9	36	34	24.5	24.9	22.1	21.4	15.6
	S.E.	9.8	10.4	9.8	7.1	7.2	6.4	6.2	4.5
	N	12	12	12	12	12	12	12	12
DAY 42	MEAN	446	435	450	424	247	257	260	249
	% DIFFERENCE		-2.5	0.9	-4.9	4	5.3	0.8
	S.D.	37.3	36.3	39	25.6	27.1	24.6	20.9	14.7
	S.E.	10.8	10.5	11.3	7.4	7.8	7.1	6	4.2
	N	12	12	12	12	12	12	12	12
DAY 49	MEAN	471	460	475	446	257	268	268	255
	% DIFFERENCE		-2.3	0.8	-5.3	4.3	4.3	-0.8
	S.D.	40.8	38.5	43.1	25.7	25.2	25.5	22.6	16.3
	S.E.	11.8	11.1	12.5	7.4	7.3	7.4	6.5	4.7
	N	12	12	12	12	12	12	12	12
DAY 56	MEAN	489	475	492	461	259	271	271	260
	% DIFFERENCE		-2.9	0.6	-5.7	4.6	4.6	0.4
	S.D.	44.2	41.4	46.9	28.7	25.9	24.6	24.9	11
	S.E.	12.8	11.9	13.5	8.3	7.5	7.1	7.2	3.2
	N	12	12	12	12	12	12	12	12

 

Conclusions:

There was no evidence of neurotoxicity associated with exposure to test substance based on FOB evaluations, LMA assessments, mean brain weights and measurements, and macroscopic and microscopic findings. A lower mean body weight gain, with a corresponding reduction in food consumption, was noted for the 3500 ppm males during study days 0-7. As a result, mean absolute body weight for the 3500 ppm males at the end of treatment (study day 91) was 6.9 % lower than the control group. No effects on body weights or food consumption were noted for the 200 and 600 ppm males or the 200, 600, and 1500 ppm females. Based on these data, the NOAEL values for systemic toxicity in males and females were 600 ppm (38 mg/kg bw/day) and 1500 ppm (111 mg/kg bw/day), respectively.

Executive summary:

The test substance was administered on a continuous basis in the diet for approximately 13 weeks to Crl:CD(SD) rats in Groups 2 - 4 at concentrations of 200, 600, and 3500 ppm for males and 200, 600, and 1500 ppm for females. This study was conducted according to OECD TG 424 and followed GLP criteria. A concurrent control group (Group 1) was offered the basal diet on a comparable regimen. Animals were approximately 6 weeks old at the initiation of test diet administration. Each group consisted of 12 rats/sex. All animals were observed twice daily for mortality and moribundity. Detailed physical examinations, body weights, and food consumption were recorded weekly. Functional observational battery (FOB) and locomotor activity (LMA) data were recorded for all animals during the pre-test period and the second, fourth, eighth, and thirteenth weeks of test diet administration (study weeks 1, 3, 7, and 12, respectively). Ophthalmic examinations were performed prior to the start of test diet administration (study week -2) and study week 12 (near the end of test diet administration). Five rats/sex/group were deeply anesthetised and perfused in situ during study week 13; brain weights and brain dimensions (excluding olfactory bulbs) were recorded. Of the rats perfused in situ, those in the control and high dose groups (3500 ppm males; 1500 ppm females) were subjected to a neuropathological evaluation of selected tissues from the central and peripheral nervous systems. Animals not selected for in situ perfusion were euthanised and discarded without macroscopic examination.

Mean test substance consumption for the 200, 600, and 3500 ppm males was 13, 38, and 222 mg/kg bw/day, respectively, over the entire study (study days 0-91). Mean test substance consumption for the 200, 600, and 1500 ppm females was 15, 45, and 111 mg/kg bw/day, respectively, over the entire study. All animals survived to the scheduled necropsy. No test substance-related clinical findings were noted during the weekly observations. Findings attributed to treatment with  test substance were limited to the 3500 ppm males, and included a statistically significant decrease in mean body weight gain, with a corresponding reduction in mean food consumption, during study days 0-7. Overall (study days 0-91) mean body weight gain for the 3500 ppm males was 10.9% lower than the control group. At the end of treatment (study day 91), the mean absolute body weight for the 3500 ppm males was 6.9% lower than the control group. Mean absolute body weights, body weight gains, and food consumption for the 200 and 600 ppm males and the 200, 600, and 1500 ppm females were unaffected by  test substance exposure. FOB parameters, including home cage, handling, open field, sensory, neuromuscular, and physiological parameters, were unaffected by  test substance exposure. There were no test substance-related effects on mean total and ambulatory LMA counts for males and females at any dose level. No remarkable shifts in the pattern of habituation occurred in any of the test substance-treated groups during the evaluation periods of study weeks 1, 3, 7, and 12. There were no  test substance-related ophthalmologic changes or macroscopic/microscopic findings. There were no effects on brain weights or brain measurements at any  test substance concentration.

There was no evidence of neurotoxicity associated with exposure to test substance based on FOB evaluations, LMA assessments, mean brain weights and measurements, and macroscopic and microscopic findings. A lower mean body weight gain, with a corresponding reduction in food consumption, was noted for the 3500 ppm males during study days 0-7. As a result, mean absolute body weight for the 3500 ppm males at the end of treatment (study day 91) was 6.9% lower than the control group. No effects on body weights or food consumption were noted for the 200 and 600 ppm males or the 200, 600, and 1500 ppm females. Based on these data, the NOAEL values for systemic toxicity in males and females were 600 ppm (38 mg/kg bw/day) and 1500 ppm (111 mg/kg bw/day), respectively.

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Jan 1979 to 17 Apr 1979

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

other: Tif: RAf (SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: approx. 4 weeks
- Weight at study initiation: 123 - 125 g, males; 111 - 116 g, females
- Fasting period before study:
- Housing: The animals were housed in groups of 5 in Macrolon cages type 4 with standardized granulated soft wood bedding.
- Diet: Pelleted standard diet, ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): : 22 ± 1
- Humidity (%): 55 ± 5
- Air changes (per hr): At least 15-17
- Photoperiod (hrs dark / hrs light): 10 hours light / 14 hours dark

IN-LIFE DATES: From: 15 January 1979 To: 17 April 1979

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
Test substance. was weighed into an Erlenmeyer flask on a balance. The pulverised food was then homogeneously mixed with the appropriate concentrations of the compound and 12 % water was added before pelleting to ensure the necessary pellet quality. The pellets were subsequently airdried.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to the initiation of the study pretest feed samples were analysed for concentration and stability of the test material. The same was undertaken with
the food batches applicated during the test.

Duration of treatment / exposure:

3 months

Frequency of treatment:

Continuous

Dose / conc.:

240 ppm

Remarks:

Group 2 low dose: dietary equivalent to 15.9 and 16.8 mg/kg bw/day for males and females, respectively.

Dose / conc.:

1 200 ppm

Remarks:

Group 3 mid dose: dietary equivalent to 76.1 and 77.6 mg/kg bw/day for males and females, respectively.

Dose / conc.:

6 000 ppm

Remarks:

Group 4 high dose: dietary equivalent to 461.7 and 481 mg/kg bw/day for males and females, respectively.

No. of animals per sex per dose:

20

Control animals:

yes, concurrent no treatment

Details on study design:

- Fasting period before blood sampling for clinical biochemistry: yes, overnight fasting.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
- Time schedule: twice a day

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: daily

BODY WEIGHT:
- Time schedule for examinations: weekly

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

OPHTHALMOSCOPIC EXAMINATION:
- Time schedule for examinations: prior to dosing and at the end of the treatment
- Dose groups that were examined: all

HAEMATOLOGY:
- Time schedule for collection of blood: 4, 8 and 13 week
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table 2 were examined.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: 4, 8 and 13 weeks
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table 3 were examined.

URINALYSIS:
- Time schedule for collection of urine: 4, 8 and 13 weeks
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table 4 were examined.

OTHER: A hearing test was performed prior to dosing and at the end of the treatment period.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes: brain, heart, liver, kidneys, adrenals and gonads

HISTOPATHOLOGY: Yes: brain (cerebrum, cerebellum, brainstem), spinal cord, eye, pituitary, salivary gland, heart, thymus, thyroid, lungs, trachea, spleen, bone marrow, lymph nodes (axillary and mesenteric), sciatic nerve, oesophagus, stomach, small intestine, large intestine, adrenal glands, pancreas, liver, kidneys, urinary bladder, ovaries or testes, prostate or uterus, skin (mammary area) and skeletal muscle

Statistics:

For each time point and parameter a uni-variate statistical analysis was conducted. Due to the routine manner of the analysis system parameter free methods were applied. Each treated group was compared to the control group in respect of dispersion and displacement*. In addition a trend test** was applied considering all groups. For comparing some haematological, biochemical and urinalysis values, random samples from untreated male and female RAI (outbred) rats (Sisseln) were taken from different age periods for calculating the Grand Mean, its Standard Deviation and Tolerance Limits.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight gain of all male and female rats of the highest concentration (6000 ppm) and that of the females of the intermediate group (1200 ppm) were significantly decreased (weeks 9-13) when compared to the controls.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Due to the reduction of body weight gain the mean food conversion of the males and females of group 4 (6000 ppm) was slightly increased when compared with the controls. The values of all animals of groups 2 and 3 (240 and 1200 ppm) were comparable to the controls.

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The observed haematological findings beween treated rats and controls were generally unremarkable and considered within the confidence limits of the control values. Some statistically significant intergroup differences were observed in the study, however, these changes were minimal and the result of normal individual variations in these parameters.
Erythrocyte count, haematocrit and haemoglobin concentration were found to be somewhat lower in the female rats of the high-dose group at week 13. The toxicological significance if any, could not be assessed from the data.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No obvious changes related to the treatment were observed between treated rats and controls. The findings were considered within the confidence limits of the controls. The only change of note was a marginal increase in alkaline phosphatase activity in the female rats of group 4 at week 13 and the y-glutamyl transpeptidase activity in male and female rats at weeks 4, 8 and 13. In general the changes observed were small and inconclusive with little evidence of toxicological manifestations.

Urinalysis findings:

no effects observed

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 terminal body weights of all male and female rats of the highest concentration and that of the females of the intermediate concentration were significantly decreased when compared with those of the controls. The organ weights, organ to body weight and organ to brain weight ratios were changed accordingly.
Adrenal, brain, gonad, liver and/or heart weights, relative to body weight were increased in males and females fed 6000 ppm and females fed 1200 ppm when compared with the controls.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A small number of lesions were observed, none of which was related to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In the spleen of all female rats fed 6000 ppm, slightly increased haemosiderosis was observed. No other gross or microscopic changes which could be related to the administration of the test substance were found in the treated animals.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

AUDITORY PERCEPTION
Examinations made prior to dosing and after the treatment period revealed no evidence of loss of the hearing ability.

Key result

Dose descriptor:

NOAEL

Effect level:

240 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: Dietary equivalent to 15.9 and 16.8 mg/kg bw/day for males and females, respectively.

Key result

Critical effects observed:

no

Table 5. Intergroup comparison of mean body weight

	Dietary concentrations (ppm)
	Males				Females
week	0	240	1200	6000	0	240	1200	6000
1	125.5	123.0	125.4	124.5	115.1	111.3	115.0	116.1
2	193.7	188.6	189.1	168.5*	158.2	155.0	155.4	145.3*
3	248.7	240.1	238.7	210.7*	185.8	182.6	181.1	169.4*
4	293.4	284.1	279.6	244.1*	208.5	203.3*	198.2	186.4*
5	321.8	312.9	309.5	263.7*	223.7	217.2	211.7	195.7*
6	351.7	346.8	342.6	287.2*	235.1	231.3	223.8	207.6*
7	382.5	377.0	372.1	311.6*	249.5	245.1*	237.5	218.3*
8	399.9	397.0	393.4	324.4*	261.0	260.0	249.0	224.9*
9	411.8	407.8	404.7	328.0*	267.7	260.1	249.4*	220.9*
10	432.4	427.4	425.5	342.3*	281.0	273.9	26.1.5*	231.2*
11	451.2	445.1	443.0	355.6*	291.3	282.6*	271.7*	236.2*
12	466.6	456.4	455.0	365.4*	296.0	287.2*	274.1*	240.7*
13	469.9	463.1	458.9	371.7*	293.2	286.5	269.4*	235.0*
*Statistically significant difference from control group mean p<0.01 (Lepage test).

Table 6. Mean Dose Received (mg/kg/day)

test substance (ppm)	240	1200	6000
Males	15.9	76.1	461.7
Females	16.8	77.6	481.0

Table 7. Intergroup comparison of selected organ weights (g)

Dietary concentrations (ppm) Males Organ 0 240 1200 6000 Brain: Abs Relative 2.165 0.471 2.145 0.474 2.209 0.489 2.102 0.572* Heart: Abs Relative 1.296 0.280 1.186 0.262* 1.151* 0.253* 1.017* 0.276 Liver: Abs Relative 16.545 3.590 16.095 3.542 16.906 3.713 15.667 4.247* Kidneys: Abs Relative 3.028 0.657 2.834 0.624 2.813 0.618 2.520* 0.683 Adrenals: Abs Relative 0.061 0.013 0.060 0.013 0.057 0.012 0.049* 0.013 Gonads: Abs Relative 3.444 0.748 3.444 0.760 3.512 0.774 3.489 0.951*   Dietary concentrations (ppm) Females Organ 0 240 1200 6000 Brain: Abs Relative 1.981 0.668 1.953 0.680 1.984 0.734* 1.961 0.867* Heart: Abs Relative 0.829 0.279 0.815 0.283 0.811 0.299* 0.767* 0.338* Liver: Abs Relative 10.860 3.659 10.596 3.681 10.650 3.925* 10.201 4.500* Kidneys: Abs Relative 1.9990 0.670 1.997 0.693* 1.893* 0.699 1.670* 0.737* Adrenals: Abs Relative 0.085 0.029 0.090 0.031 0.097* 0.036* 0.081 0.036* Gonads: Abs Relative 0.134 0.045 0.140 0.049 0.140 0.052* 0.133 0.058* Abs – absolute weight           Relative – adjusted for final body weight * Statistically significant difference from control group mean p<0.05 (Lepage test).

Conclusions:

It can be inferred from the observations made during the 90-d sub-chronic toxicity study in rats, that 240 ppm of test substance (estimated to be 15.9 mg/kg/day for males and 16.8 mg/kg/day for females) represents a "no observable effect level" (NOEL) in rats when administered in their feed over a period of three months

Executive summary:

In a subchronic toxicity study test substance was administered to groups of 20 male and 20 female Tif:RAIf rats in diet at dose levels of 0, 240, 1200 and 6000 ppm for up to three months.  Mortality, clinical observations, body weights and food consumption were measured throughout the study. Blood samples were collected from all animals at 4, 8 and 13 weeks for haematology and clinical chemistry analysis; urinalysis was done in the weeks of blood collection. Brain, heart, liver, kidneys adrenals and gonads were weighed. A comprehensive range of organs and tissues from all animals was examined histopathologically.

No animals died. No clinical symptoms and no signs of local and/or systemic toxicity were observed. The body weight gain of all male and female rats of the highest concentration (6000 ppm) and that of the females of the intermediate group (1200 ppm) were significantly decreased when compared to the controls. The mean food consumption of all treated male and female rats was comparable to the controls. Due to the reduced body weight gain the mean food conversion of the males and females of group 4 (6000 ppm) was slightly increased when compared with the controls. The results of the haematological investigation, blood chemistry data and the urinalysis were generally unremarkable for both treated rats and controls. The terminal body weights of all male and female rats from the highest concentration and that of the females of the intermediate concentration were significantly decreased when compared with those of the control animals. The organ weights, organ to body weight and organ to brain weight ratios were changed accordingly. In the spleen of all female rats of the highest concentration slightly increased haemosiderosis was seen. No other gross or microscopical changes which could be related to treatment were found.

It can be inferred from the observations made during the above study that 240 ppm of the test substance (estimated to be 15.9 mg/kg/day for males and 16.8 mg/kg/day for females) represents a "no observable effect level" (NOEL) in rats when administered in their feed over a period of three months.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

15.9 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

In line with current guideline standards

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (90-Day (Subchronic) Inhalation Toxicity Study

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

other: RAIf SPF

Sex:

male/female

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

head only

Vehicle:

other: Acetone

Duration of treatment / exposure:

6 hours/day

Frequency of treatment:

5 days per week for 13 weeks

Dose / conc.:

20 mg/m³ air (nominal)

Remarks:

21 mg/m3 air (actual concentration)

Dose / conc.:

80 mg/m³ air (nominal)

Remarks:

85 mg/m3 air (actual concentration)

Dose / conc.:

200 mg/m³ air (nominal)

Remarks:

191 mg/m3 air (actual concentration)

Control animals:

yes, sham-exposed

Dose descriptor:

NOEC

Effect level:

20 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

Remarks on result:

other: Equivalent to 21 mg/m3 air (acutal dose received)

Critical effects observed:

no

Table 1. Summary of study test atmosphere characteristics

Parameter
Group	Negative control	Vehicle control	Exposure level
Gravimetric concentration	0 (air)	10 mg/m3 acetone	21±2 mg/m3	85±7 mg/m3	191±10 mg/m3
Particle size distribution	>80% smaller than 7 µm
Air flow (m/sec)	0.4	0.41	0.4	0.4	0.41
Temperature (°C)	24.8	24.9	24.8	24.9	24.9
Humidity (%)	63.9	66.3	66.3	65.9	64.5
Oxygen content (%)	20.3	20.4	20.4	20.3	20.2

Mortality: There were no treatment related mortalities.
Clinical observations: No clinical symptoms and no signs of local and/or systemic toxicity were observed.
Body weight and gain: Slightly lower body weights in the males at 85±7 mg/m3 and females at 21±2 and 191±10 mg/m3 were seen on some occasions during the treatment period achieving statistical significance (p≤0.01) when compared with the controls.

Table 2. Intergroup comparison of mean body weight (g)

Study week		Males				Females
	Negative control	Vehicle control	Exposure level			Negative control	Vehicle control	Exposure level
	0 (air)	10 mg/m3 acetone	21±2 mg/m3	85±7 mg/m3	191±10 mg/m3	0 (air)	10 mg/m3 acetone	21±2 mg/m3	85±7 mg/m3	191±10 mg/m3
1	227	232	222	222	216	186	192	184	191	184
2	259	262	251	249	245	199	205	189	196	193
3	292	293	284	278	279	211	214	200	203	201
4	319	318	309	229*	302	222	229	209*	212	209*
5	319	33	327	319	321	228	236	212*	219	215*
6	332	335	331	321	328	236	239	219*	225	217*
7	354	356	349	336*	349	240	247	225*	229	226*
8	371	367	361	347*	358	246	249	228*	236	227*
9	387	390	380	366*	379	250	257	232*	243	234*
10	401	413	396	385	399	254	268*	242	251	240
11	417	429	417	401	413	260	273*	248	253	246*
12	429	434	430	414	427	263	274	249*	258	250
13	433	448	446	425	439	265	273	250*	258	249*

* Statistically significant difference from control group mean (p≤0.01).

Food consumption and utilisation: No effect on food consumption or food utilisation was noted.
Food consumption and compound intake: The mean food consumption of all treated male and female rats was comparable to the controls.
Ophthalmoscopic examination: No treatment related effects noted.
Haematology: There were no differences in haematological parameters which were considered to be related to treatment.
Blood clinical chemistry: There were no differences in blood clinical chemistry parameters which were considered to be related to treatment.

Sacrifice and pathology:
Organ weights: Some organ weight, organ to body weight and organ to brain weight ratios showed some intra-group variation, but none were considered to be toxicologically significant.
Macroscopic findings: There were no treatment related macroscopic abnormalities.
Microscopic findings: There were no treatment related microscopic abnormalities.

Conclusions:

A NOEC of the test substance for male rats was established at 20 mg/m3 (21 mg/m3 acutal dose received) but not for females because of the body weight differences noted at this exposure level.

Executive summary:

In this inhalation toxicity study, test substance was administered by aerosol exposure (to the head only) to groups of 20 male and 20 female RAIf SPF rats at dose levels of 0 (air), 10 (acetone, vehicle control), 21±2, 85±7 or 191±10 mg/m3 for 6 hours per day, 5 days per week for a period of 90 days.Test atmospheres were analysed daily for particulate concentration and test substance. Mortality, clinical observations, body weights and food consumption were measured throughout the study. Blood samples were collected from 10 fasted rats per sex per group at 6 and 13 weeks for haematology and clinical chemistry analysis. Brain, heart, liver, lungs and kidneys were weighed. A comprehensive range of organs and tissues from all animals was examined histopathologically.
There were no treatment related mortalities. No clinical symptoms and no signs of local and/or systemic toxicity were observed. No effects on food consumption or food utilisation were noted. Slightly lower body weights in the males at 85±7 mg/m3and in females at 21±2 mg/m3 and 191±10 mg/m3 were seen on some occasions during the treatment period when compared with the controls. The results of the haematological investigation and blood chemistry data were generally unremarkable for both treated rats and controls. There were no treatment related macroscopic or microscopic changes. Some organ weight, organ to body weight and organ to brain weight ratios showed some intra-group variation, but none were considered to be toxicologically significant.

A NOEC of test substance technical for male rats was established at 21 mg/m3 but not for females because of the body weight differences noted at this exposure level.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (90-Day (Subchronic) Inhalation Toxicity Study

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

other: RAIf SPF

Sex:

male/female

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

head only

Vehicle:

other: Acetone

Duration of treatment / exposure:

6 hours/day

Frequency of treatment:

5 days per week for 13 weeks

Dose / conc.:

20 mg/m³ air (nominal)

Remarks:

21 mg/m3 air (actual concentration)

Dose / conc.:

80 mg/m³ air (nominal)

Remarks:

85 mg/m3 air (actual concentration)

Dose / conc.:

200 mg/m³ air (nominal)

Remarks:

191 mg/m3 air (actual concentration)

Control animals:

yes, sham-exposed

Dose descriptor:

NOEC

Effect level:

20 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

Remarks on result:

other: Equivalent to 21 mg/m3 air (acutal dose received)

Critical effects observed:

no

Table 1. Summary of study test atmosphere characteristics

Parameter
Group	Negative control	Vehicle control	Exposure level
Gravimetric concentration	0 (air)	10 mg/m3 acetone	21±2 mg/m3	85±7 mg/m3	191±10 mg/m3
Particle size distribution	>80% smaller than 7 µm
Air flow (m/sec)	0.4	0.41	0.4	0.4	0.41
Temperature (°C)	24.8	24.9	24.8	24.9	24.9
Humidity (%)	63.9	66.3	66.3	65.9	64.5
Oxygen content (%)	20.3	20.4	20.4	20.3	20.2

Mortality: There were no treatment related mortalities.
Clinical observations: No clinical symptoms and no signs of local and/or systemic toxicity were observed.
Body weight and gain: Slightly lower body weights in the males at 85±7 mg/m3 and females at 21±2 and 191±10 mg/m3 were seen on some occasions during the treatment period achieving statistical significance (p≤0.01) when compared with the controls.

Table 2. Intergroup comparison of mean body weight (g)

Study week		Males				Females
	Negative control	Vehicle control	Exposure level			Negative control	Vehicle control	Exposure level
	0 (air)	10 mg/m3 acetone	21±2 mg/m3	85±7 mg/m3	191±10 mg/m3	0 (air)	10 mg/m3 acetone	21±2 mg/m3	85±7 mg/m3	191±10 mg/m3
1	227	232	222	222	216	186	192	184	191	184
2	259	262	251	249	245	199	205	189	196	193
3	292	293	284	278	279	211	214	200	203	201
4	319	318	309	229*	302	222	229	209*	212	209*
5	319	33	327	319	321	228	236	212*	219	215*
6	332	335	331	321	328	236	239	219*	225	217*
7	354	356	349	336*	349	240	247	225*	229	226*
8	371	367	361	347*	358	246	249	228*	236	227*
9	387	390	380	366*	379	250	257	232*	243	234*
10	401	413	396	385	399	254	268*	242	251	240
11	417	429	417	401	413	260	273*	248	253	246*
12	429	434	430	414	427	263	274	249*	258	250
13	433	448	446	425	439	265	273	250*	258	249*

* Statistically significant difference from control group mean (p≤0.01).

Food consumption and utilisation: No effect on food consumption or food utilisation was noted.
Food consumption and compound intake: The mean food consumption of all treated male and female rats was comparable to the controls.
Ophthalmoscopic examination: No treatment related effects noted.
Haematology: There were no differences in haematological parameters which were considered to be related to treatment.
Blood clinical chemistry: There were no differences in blood clinical chemistry parameters which were considered to be related to treatment.

Sacrifice and pathology:
Organ weights: Some organ weight, organ to body weight and organ to brain weight ratios showed some intra-group variation, but none were considered to be toxicologically significant.
Macroscopic findings: There were no treatment related macroscopic abnormalities.
Microscopic findings: There were no treatment related microscopic abnormalities.

Conclusions:

A NOEC of the test substance for male rats was established at 20 mg/m3 (21 mg/m3 acutal dose received) but not for females because of the body weight differences noted at this exposure level.

Executive summary:

In this inhalation toxicity study, test substance was administered by aerosol exposure (to the head only) to groups of 20 male and 20 female RAIf SPF rats at dose levels of 0 (air), 10 (acetone, vehicle control), 21±2, 85±7 or 191±10 mg/m3 for 6 hours per day, 5 days per week for a period of 90 days.Test atmospheres were analysed daily for particulate concentration and test substance. Mortality, clinical observations, body weights and food consumption were measured throughout the study. Blood samples were collected from 10 fasted rats per sex per group at 6 and 13 weeks for haematology and clinical chemistry analysis. Brain, heart, liver, lungs and kidneys were weighed. A comprehensive range of organs and tissues from all animals was examined histopathologically.
There were no treatment related mortalities. No clinical symptoms and no signs of local and/or systemic toxicity were observed. No effects on food consumption or food utilisation were noted. Slightly lower body weights in the males at 85±7 mg/m3and in females at 21±2 mg/m3 and 191±10 mg/m3 were seen on some occasions during the treatment period when compared with the controls. The results of the haematological investigation and blood chemistry data were generally unremarkable for both treated rats and controls. There were no treatment related macroscopic or microscopic changes. Some organ weight, organ to body weight and organ to brain weight ratios showed some intra-group variation, but none were considered to be toxicologically significant.

A NOEC of test substance technical for male rats was established at 21 mg/m3 but not for females because of the body weight differences noted at this exposure level.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Additional information

All available data was assessed and the studies representing the worst-case effects were included as key or weight-of-evidence studies. Other studies are included as supporting information. The key studies are considered to be worst-case and were selected for the CSA.

 

90 - day subchronic oral toxicity study in rats, Sachsse 1979

In a subchronic toxicity study test substance was administered to groups of 20 male and 20 female Tif:RAIf rats in diet at dose levels of 0, 240, 1200 and 6000 ppm for up to three months. Mortality, clinical observations, body weights and food consumption were measured throughout the study. Blood samples were collected from all animals at 4, 8 and 13 weeks for haematology and clinical chemistry analysis; urinalysis was done in the weeks of blood collection. Brain, heart, liver, kidneys adrenals and gonads were weighed. A comprehensive range of organs and tissues from all animals was examined histopathologically.

No animals died. No clinical symptoms and no signs of local and/or systemic toxicity were observed. The body weight gain of all male and female rats of the highest concentration (6000 ppm) and that of the females of the intermediate group (1200 ppm) were significantly decreased when compared to the controls. The mean food consumption of all treated male and female rats was comparable to the controls. Due to the reduced body weight gain the mean food conversion of the males and females of group 4 (6000 ppm) was slightly increased when compared with the controls. The results of the haematological investigation, blood chemistry data and the urinalysis were generally unremarkable for both treated rats and controls. The terminal body weights of all male and female rats from the highest concentration and that of the females of the intermediate concentration were significantly decreased when compared with those of the control animals. The organ weights, organ to body weight and organ to brain weight ratios were changed accordingly. In the spleen of all female rats of the highest concentration slightly increased haemosiderosis was seen. No other gross or microscopical changes which could be related to treatment were found.

It can be inferred from the observations made during the above study that 240 ppm of the test substance (estimated to be 15.9 mg/kg/day for males and 16.8 mg/kg/day for females) represents a "no observable adverse effect level" (NOAEL) in rats when administered in their feed over a period of three months.

 

1 year subchronic oral toxicity study in dogs, Johnson 1985
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.

13 weeks subchronic oral toxicity study in rats, Herberth 2013a
The test substance was administered on a continuous basis in the diet for approximately 13 weeks to Crl:CD(SD) rats in Groups 2 - 4 at concentrations of 200, 600, and 3500 ppm for males and 200, 600, and 1500 ppm for females. This study was conducted according to OECD TG 424 and followed GLP criteria. A concurrent control group (Group 1) was offered the basal diet on a comparable regimen. Animals were approximately 6 weeks old at the initiation of test diet administration. Each group consisted of 12 rats/sex. All animals were observed twice daily for mortality and moribundity. Detailed physical examinations, body weights, and food consumption were recorded weekly. Functional observational battery (FOB) and locomotor activity (LMA) data were recorded for all animals during the pre-test period and the second, fourth, eighth, and thirteenth weeks of test diet administration (study weeks 1, 3, 7, and 12, respectively). Ophthalmic examinations were performed prior to the start of test diet administration (study week -2) and study week 12 (near the end of test diet administration). Five rats/sex/group were deeply anesthetised and perfused in situ during study week 13; brain weights and brain dimensions (excluding olfactory bulbs) were recorded. Of the rats perfused in situ, those in the control and high dose groups (3500 ppm males; 1500 ppm females) were subjected to a neuropathological evaluation of selected tissues from the central and peripheral nervous systems. Animals not selected for in situ perfusion were euthanised and discarded without macroscopic examination.
Mean test substance consumption for the 200, 600, and 3500 ppm males was 13, 38, and 222 mg/kg bw/day, respectively, over the entire study (study days 0-91). Mean test substance consumption for the 200, 600, and 1500 ppm females was 15, 45, and 111 mg/kg bw/day, respectively, over the entire study. All animals survived to the scheduled necropsy. No test substance-related clinical findings were noted during the weekly observations. Findings attributed to treatment with test substance were limited to the 3500 ppm males, and included a statistically significant decrease in mean body weight gain, with a corresponding reduction in mean food consumption, during study days 0-7. Overall (study days 0-91) mean body weight gain for the 3500 ppm males was 10.9% lower than the control group. At the end of treatment (study day 91), the mean absolute body weight for the 3500 ppm males was 6.9% lower than the control group. Mean absolute body weights, body weight gains, and food consumption for the 200 and 600 ppm males and the 200, 600, and 1500 ppm females were unaffected by test substance exposure. FOB parameters, including home cage, handling, open field, sensory, neuromuscular, and physiological parameters, were unaffected by test substance exposure. There were no test substance-related effects on mean total and ambulatory LMA counts for males and females at any dose level. No remarkable shifts in the pattern of habituation occurred in any of the test substance-treated groups during the evaluation periods of study weeks 1, 3, 7, and 12. There were no test substance-related ophthalmologic changes or macroscopic/microscopic findings. There were no effects on brain weights or brain measurements at any test substance concentration.
There was no evidence of neurotoxicity associated with exposure to test substance based on FOB evaluations, LMA assessments, mean brain weights and measurements, and macroscopic and microscopic findings. A lower mean body weight gain, with a corresponding reduction in food consumption, was noted for the 3500 ppm males during study days 0-7. As a result, mean absolute body weight for the 3500 ppm males at the end of treatment (study day 91) was 6.9% lower than the control group. No effects on body weights or food consumption were noted for the 200 and 600 ppm males or the 200, 600, and 1500 ppm females. Based on these data, the NOAEL values for systemic toxicity in males and females were 600 ppm (38 mg/kg bw/day) and 1500 ppm (111 mg/kg bw/day), respectively.

Justification for classification or non-classification

Based on the available information classification upon repeated exposure is not warranted according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.