Registration Dossier

Administrative data

Description of key information

Repeated dose toxicity was assessed with data obtained for a structural analogue:

In a 90-day feeding study with dogs (Beagle) a NOAEL of 84.1 (males) and 90.2 mg/kg bw/day (females) was derived.

No treatment-related effects have been observed. in a 90-day feeding study with Sprague-Dawley rats, a NOEL of 122 (males) and 136 mg/kg bw/day (females) was derived. Slight impairment of growth during the first 6 weeks of treatment, lower efficiency of food utilisation and elevated SAP levels among males were reported.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
No purity of test substance available
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
12 May 1981
Deviations:
no
GLP compliance:
no
Remarks:
study predates GLP regulations but QUA statement included
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at 20°C in the dark
Species:
rat
Strain:
other: CD strain of Sprague-Dawley origin
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, Mass., USA
- Age at study initiation: 28 days
- Weight at study initiation: 150-155 g (females), 202-205 g (males)
- Housing: 5 animals per cage
- Diet: Spratt´s Laboratory Diet 2 (low fat) + 20 ppm Hetrazeen (Vitamin K supplement)
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23+/-3
- Humidity (%): 50+/-5
- Photoperiod: 12 hrs dark / 12 hrs light
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was weighed out and ground in a pestle and mortar, then a similar quantity of Spratt's Laboratory Diet 2 (low fat) was added and mixed well. A further quantity of diet was added and mixed well. The mixture was transferred to a bowl and further quantities of diet added until the required quantity was reached. The mixture was stirred thoroughly after each addition. This premix was then mixed in an air-filled polythene bag for 3-4 minutes. The dietary concentrations required were obtained from this premix by direct dilution with further quantities of diet, homogeneity being achieved by mixing for 10 minutes in the rotary double-cone blender.

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): Spratt´s Laboratory Diet 2 (low fat) supplemented with Hetrazeen
- Storage temperature of food: 4 °C
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before treatment commenced a specimen sample of each test diet was sent to the sponsor for stability analysis and verification of dietary levels. Dietary analysis of all test diets for test item content was performed by the sponsor on diets fed during weeks 1 and 13. For each treatment level a 200 g sample was obtained at the time of preparation of the diets. On each sampling occasion, a sample (5 g) of the test compound was sent to the sponsor. These samples were taken at the time of preparation of the test diets.
Duration of treatment / exposure:
7 days/week; 13 weeks
Frequency of treatment:
Continuous dosing with feed.
Dose / conc.:
40 mg/kg bw/day (actual dose received)
Remarks:
mean achieved dosage for males recieving 700 ppm in the diet
Dose / conc.:
48 mg/kg bw/day (actual dose received)
Remarks:
mean achieved dosage for females recieving 700 ppm in the diet
Dose / conc.:
122 mg/kg bw/day (actual dose received)
Remarks:
mean achieved dosage for males recieving 2000 ppm in the diet
Dose / conc.:
136 mg/kg bw/day (actual dose received)
Remarks:
mean achieved dosage for females recieving 2000 ppm in the diet
Dose / conc.:
376 mg/kg bw/day (actual dose received)
Remarks:
mean achieved dosage for males recieving 6000 ppm in the diet
Dose / conc.:
420 mg/kg bw/day (actual dose received)
Remarks:
mean achieved dosage for females recieving 6000 ppm in the diet
No. of animals per sex per dose:
Main study: 20 animals per sex and dose
Withdrawal group: 5 animals per sex in the control and high dose group
Control animals:
yes, plain diet
Details on study design:
- Post-exposure recovery period in satellite groups: 4 weeks
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS and DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule for examinations: Daily check of animals for dead or moribund animals. Principal observations of individual animals during the first four weeks of a study were carried out daily at approximately the same time each day. After four weeks of treatment there was no reason to continue daily observations and the frequency was reduced to once a week for the remainder of the study, except for a period during weeks 8 and 9 when, due to on outbreak of sialodacryoadenitis, the frequency of observation was increased for the duration of the outbreak.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each rat was recorded one week before the commencement of treatment and at weekly intervals 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
- 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

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
Daily monitoring by visual examination of the water bottles was maintained throughout the study. Consumption for each cage in Groups 1 (Control) and 4 (6000ppm) was measured daily for a 5-day period during weeks 5 and 11. The measurement was repeated in week 7 among rats in Groups 1 (Control) and 4 (6000ppm) and the investigation was extended in week 11 to rats of all groups. In the light of findings during the treatment period, consumption was measured in week 16 (3rd week of withdrawal) among all surviving rats of Groups 1 (Control) and 4 (previously receiving 6000ppm).

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before treatment commenced and during weeks 4 , 8 and 13
- Dose groups that were examined: the eyes of all animals in Groups 1 (Control) and 4 (6000 ppm) were examined by indirect ophthalmoscopy

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During weeks 4 and 12 samples of blood were withdrawn from the orbital sinus of all surviving rats in all groups while under light ether anaesthesia. In the light of findings during the treatment period, samples of blood were withdrawn in week 17 (4th week of withdrawal) from all surviving rats in Groups 1 (Control) and 4 (previously receiving 6000 ppm) for investigation of white blood cell parameters only.
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- Parameters checked: packed cell volume, haemoglobin, red cell count, mean corpuscular haemoglobin concentration, mean cell volume, mean corpuscular haemoglobin, differential count (neutrophils, lymphocytes, eosinophils, basophils, monocytes), platelet count, thrombotest.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: During weeks 4 and 12 samples of blood were withdrawn from the orbital sinus of all surviving rats of each group under light ether anaesthesia. In the light of findings in the treatment period, samples of blood were withdrawn in week 17 (4th week of withdrawal) from all surviving males in groups 1 (Control) and 4 (previously receiving 6000 ppm) for estimation of total serum proteins, albumin, and serume alkaline phosphatase, and from all surviving females in groups 1 (Control) and 4 (previously receving 6000 ppm) for estimation of plasma glucose, total serum proteins and albumin.
- Animals fasted: Yes
- Parameters checked: blood urea nitrogen, plasma glucose, serum total protein, serum albumin, serum alkaline phosphatase, serum glutamic-pyruvic transaminase, serum creatine, serum cholesterol, electrolytes (sodium, potassium, chloride, calcium, inorganic phosphorus).

URINALYSIS: Yes
- Time schedule for collection of urine: During weeks 4 and 12 individual overnight urine samples were collected from all surviving males and females from each group. In the light of findings in the treatment period, samples of urine were obtained in week 17 (4th week of withdrawal) from all surviving females of Groups 1 (Control) and 4 (previously receiving 6000 ppm) for estimation of volume, pH, specific gravity and protein only. The volume of each sample was measured.
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes
- Parameters checked: pH, specific gravity, protein, reducing substances, glucose, ketones, bile pigments, urobilinogen, haemogolbin, microscopy of spun deposit.

other:
HEARING TEST
Before treatment commenced and during weeks 4 , 8 and 13, hearing function was assessed using a Galton whistle, set at 10 KHz and placed at 1 metre from the head. Each rat of Groups 1 (Control) and 4 (6000 ppm) was isolated and examined for response to the stimulus. In those rats In which a clearly positive response was not observed, the test was normally repeated.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All rats were killed in random order by carbon dioxide asphyxiation. The appearance of the tissues was noted and the weights of the following organs were recorded: adrenals, brain, heart, kidneys, liver, ovaries, testes, thymus.
All gross lesions were recorded in narrative, descriptive terms including location, size (mm), number, shape, colour and texture. Distended urinary bladders were opened and examined under a dissecting microscope before fixation. Empty, contracted urinary bladders were partly distended with formalin and, after fixation, opened and examined under a dissecting microscope.

ORGAN WEIGHTS
adrenals, brain, heart, kidneys, liver, ovaries, testes, thymus

HISTOPATHOLOGY: Yes
Prior to microscopic examination, tissues were embedded in paraffin wax and sections cut at 5 µm and stained with haematoxylin and eosin. An additional section of kidney was stained with periodic acid-Schiff reagent (PAS) for basement membrane. Frozen cryostat sections of liver and kidney, previously fixed in buffered 10% formalin were sectioned at 12 µm and stained for fat with Oil Red O and fresh frozen liver sections were also stained using PAS for glycogen. Microscopic examination was performed on the following tissue from all animals in each group:
adrenals, aorta, bone, brain (medullary, cerebellar, thalamic nuclei, cortical sections), caecum, duodenum, eyes (retina, optic nerve), heart, Ileum, jejunum, kidneys, thymus, thyroid, liver, lungs (with main stem bronchi), lymph nodes, (cervical and mesenteric), mammary gland, mid-colon, nasal cavity, oesophagus, ovaries, pancreas, parathyroid, trachea, urinary bladder, pituitary, prostate, salivary gland, sciatic nerve, skeletal muscle (thigh), skin
spinal cord (thoracic and lumbar sections), spleen, sternum (bone marrow), stomach (glandular and non-glandular), testes (with epididymides), uterus.
Statistics:
- Analysis of variance followed by Student's t- test was used to assess the significance of intergroup differences in the food consumption, water consumption, bodyweight and clinical laboratory data.
- Analysis of the food consumption data was performed using the means of all the cage total food intakes in each group over specified time period. The cage totals were a sum of the weekly cage mean food intakes. The weekly cage means were calculated using the amounts of food given to and left by each cage in each week and the number of animals surviving in the cage for the majority of days in the week. The cage mean food consumption figures were rounded to the nearest whole number, but the cage totals were calculated using the exact mean weekly figures.
- Analysis of the bodyweight data was performed using the individual weight gains of rats surviving throughout the specified time period.
- Analysis of organ weights was performed using analysis of covariance. Organ weights were adjusted for final bodyweight as covariate where this was the more efficient method of analysis. Group means were compared using both Student's t-test and Williams' test.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Evidence of reduced grooming activity among the majority of males and females in the high dose group (6000 ppm in diet) during the second half of the treatment period, and to some extent during the withdrawal period. Reduced grooming was no longer recorded for females after 15 weeks.
Symptoms of sialodacryoadenitis among a number of males and females of all groups during weeks 8 and 9. There was no clinical evidence of the disease during week 10 and all rots showed total recovery with no residual effects
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Growth of males and females receiving 6000 ppm was Inferior to that of the controls during the first six weeks of treatment. For the remainder of the treatment period, the gains recorded
for rats of this group were similar to those of the controls.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
The appetite of the five males and five females previously receiving 6000 ppm of the test substance was marginally lower than that of the controls. However, in the absence of an effect on food consumption
during the treatment period, the finding was considered to be of no toxicological significance.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Minimally lower efficiency of food utilisation was recorded during the initial six weeks of treatment among males and females receiving 6000 ppm. This was partly associated with the
lower bodyweight gain recorded for these rats over the same period. For the remainder of the treatment period, food utilisation efficiency of rats receiving 6000 ppm was similar to that of
the controls. The performance of males and females receiving 700 or 2000 ppm was not affected by
treatment. During the withdrawal period, food utilisation efficiency of rats previously treated with the test substance showed no evidence of a treatment-related effect.
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
Increased water consumption was recorded for rats receiving 6000 ppm during weeks 5 and 11, this finding was not confirmed during week 7 nor during the withdrawal period, and In the absence of any corroborative evidence from the food consumption or urinalysis data the toxicological significance of these results remains obscure
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the week 4 Investigation, a white blood cell count revealed an apparently increased number of neutrophils in males and females receiving 6000 ppm. Males and females of other treated groups were not thus affected. However as this finding
was not confirmed by investigations made during the 12th week of treatment, nor by Investigations made during the 4th week of the withdrawal period. It was considered to be of doubtful toxicological significance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
No consistent or marked effect on blood urea nitrogen, serum cholesterol, plasma glucose or protein levels was observed. It was considered that the
only finding which could be clearly related to treatment was elevation of SAP levels among males receiving 6000 ppm of the test substance at weeks 4 and 12. The toxicological significance of other blood chemistry findings was considered to be obscure.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Marginally higher urine volumes and lower specific gravity readings than are normally encountered In rats of this age and strain, were recorded for males of all
groups. Including the controls. At week 12, urine of slightly lower volume and higher specific gravity was voided by
female controls, resulting In on apparent treatment-related effect on urine from treated females. However, as there was no clear evidence of on effect on the concentrating ability of the kidneys of treated males or females at weeks 4 or 12, this finding was considered to have arisen fortuitously.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Changes observed, but considered to be of no toxicological significance were as follows:
Peribronchiolar lymphoid hyperplasia was seen in the lungs of the majority of animals examined from all groups and was associated with perivascular and subpleural accumulations of lymphocytes in most instances.
Vacuolated hepatocytes were seen in centrilobular areas most frequently in male animals from all groups, but were also present in a few female animals. Minimal chronic inflammatory cell infiltration was seen in small foci in the parenchyma and in portal tracts in a majority of rats from all groups; a focus of necrosis was seen in one male rat of the 2000 ppm group and distended sinusoids were noted in one male rat of the 700 ppm group.
Minimal chronic inflammatory cell infiltration was seen in the interstitial tissue of the cortex of the kidneys in a few animals from both Control and treatment groups and occasional tubules characterised by the basophilic staining of the cells of the epithelium were also noted.
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Description (incidence and severity):
Hearing test: Hearing function, as assessed by the Galton whistle test was not affected by treatment
Dose descriptor:
NOEL
Effect level:
122 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
food efficiency
clinical biochemistry
Dose descriptor:
NOEL
Effect level:
136 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
food efficiency
Dose descriptor:
NOAEL
Effect level:
376 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: highest tested dose
Dose descriptor:
NOAEL
Effect level:
420 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: highest tested dose
Critical effects observed:
no

Based on the data obtained it may be concluded that a dietary level of 2000 ppm test substance, equivalent to a calculated intake of 122 mg/kg/day for males and 136 mg/kg/day for females, was without effect under the conditions of this study and can be taken as the NOEL. The elevation of serume alkaline phosphatase (SAP) levels may be due to an adaptive response to general liver changes, which have been observed in all treatment groups, but most frequently in males. Vacuolated hepatocytes were seen in centrilobular areas most frequently in male animals from all groups, but were also present in a few female animals. Minimal chronic inflammatory cell infiltration was seen in small foci in the parenchyma and in portal tracts in a majority of rats from all groups. Also other liver enzymes as the glutamate-pyruvate-transaminase (GPT) were slightly increased, thus rather indicating an overall instead of a specific effect on the animals. Thus, a NOAEL of 376-420 mg/kg bw (6000 ppm, nominal) can be deduced, which was the highest dose tested.

Tab. 1 Number of rats showing clinical signs/ Number of rats examined

 male control  male 700 ppm  male 2000 ppm  male 6000 ppm  female control  female 700 ppm  female 2000 ppm  female 6000 ppm
10 / 25   14 / 20 6 /20   12 / 25 11 / 25  9 / 20  7 / 20  7 / 25 
Conclusions:
Based on the data obtained it may be concluded that a dietary level of 2000ppm, equivalent to a calculated intake of 122 mg/kg/day for males and 136 mg/kg/day for females, was without effect under the conditions of this study.
Executive summary:

In a 90 day subchronic feeding study the test article was administered at levels of 700, 2000 or 6000 ppm in the diet to groups of 20 male and 20 female CD rats. A similar group received the diet without test article and acted as a control. Additionally, five male and five female animals were assigned to the control and high dose groups and kept untreated for 4 weeks after termination of the study for recovery observations. The observed parameters were the following: Clinical signs, mortality, ophthalmologic and hearing examinations, body weight, food uptake, food efficiency, hematology, clinical chemistry, urine analysis, detailed gross pathology, organ weights and detailed histopathology. No deaths occurred during the study. At 6000 ppm there was evidence of reduced grooming activity among the majority of males and females, slight impairment of growth during the first 6 weeks of treatment, lower efficiency of food utilisation, increases in the number of neutrophilic cells at week 4 (but not at week 12 or 17), elevated SAP levels among males at weeks 4 and 12 (but not during the recovery period). At 700 and 2000 ppm no treatment related effects were noted. Based on the results of this study, the NOEL is 2000 ppm corresponding to 122 mg/kg bw for male and 136 mg/kg bw for female rats.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 Oct 1979 - 12 Feb 1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
No purity of test substance available
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity in Non-Rodents)
Version / remarks:
12 May 1981
Deviations:
no
GLP compliance:
no
Remarks:
study predates GLP regulations but QUA statement included
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Room temperature
- Stability under test conditions:
A structural similar material is stable in dietary mixtures for at least 4 weeks at room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Moistened with 20 % w/w of tap water

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

ENVIRONMENTAL CONDITIONS
- Temperature (°C): mean daily maximum = 19 ± 2 °C, mean daily minimum = 13 ± 4 °C
- Humidity (%): 78 ± 8
- Photoperiod: 12 hrs dark / 12 hrs light
- Air changes: 15 per hour
Dogs were held in a limited access facility. Personnel entering were required to wear protective clothing. Each room was kept at positive pressure with respect to the outside and had its own supply of filtered fresh air which was passed to the atmosphere and not re-circulated.
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
On the first four occasions of formulation, the initial dispersal of the test substance into dry powdered diet was made by mixing the required quantity of the test substance with 100 g of diet in a Kenwood planetary mixer. Thereafter this was achieved by shaking the constituents of the admixture in a closed container and homogenising this product by passage through a grinder. This modification was adopted to minimise the generation of dust and hence possible loss of compound. The resulting pre-mix was further diluted with dry powdered diet in a Kenwood Chef planetary mixer for ten minutes followed by further dilution with diet in a Hobart A200 planetary mixer (15 minutes) to give a final pre-mix of 8800 ppm. The final admixtures for Group 3 (600 ppm) and Group 4 (2000 ppm) were achieved by diluting the required amount of pre-mix with diet in a Gardner 50 L trough mixer with interrupted spiral agitator (15 minutes). The final admixture for Group 2 (200 ppm) was achieved by prediluting the required amount of pre-mix with diet in a Hobart A200 planetary mixer (ten minutes) with a final dilution with diet in the Gardner trough mixer (15 minutes). Each product, and untreated control diet, was wetted with tap water (200 mL/kg): and mixed in the Gardner trough mixer (15 minutes). At the request of the Sponsor, the mixing times were extended from 17 January 1980 (Week 12) by five minutes for each mixing procedure (excluding the wetting stage) involving the Kenwood planetary, Hobart A200 planetary and Gardner trough mixers. The final products were stored at 4°C for no more than four days and issued daily to the animal house.

DIET PREPARATION
- Rate of preparation of diet (frequency): At least every 4 days
- Storage temperature of food: 4 °C
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the unwetted diet (200 g of the single common pre-mix and of each test group final mix and 500 g of control diets) prepared before the commencement of treatment and for use in weeks 1, 5, 8/9, 13 and the last week of treatment were supplied to the sponsor for analysis. In addition, a further 50 g sample of each test diet was retained for at least three months in sealed polythene sachets and stored at room temperature in these laboratories pending possible future analysis as required by Section 58.113 of the Good Laboratory Practice regulations. These samples were not analysed and were eventually discarded.
Duration of treatment / exposure:
7 days/week; 13 weeks (The treatment was intended to continue for 13 weeks. In fact the animals were held for at least one additional day before the terminal sacrifice commenced. Necropsies took up to 13 days to complete for all animals. Throughout this additional period, daily treatment continued)
Frequency of treatment:
Continuous dosing with feed
Dose / conc.:
8.5 mg/kg bw/day (actual dose received)
Remarks:
mean achieved dosage for males recieving 200 ppm in the diet
Dose / conc.:
9.2 mg/kg bw/day (actual dose received)
Remarks:
mean achieved dosage for females recieving 200 ppm in the diet
Dose / conc.:
24.7 mg/kg bw/day (actual dose received)
Remarks:
mean achieved dosage for males recieving 600 ppm in the diet
Dose / conc.:
28.3 mg/kg bw/day (actual dose received)
Remarks:
mean achieved dosage for females recieving 600 ppm in the diet
Dose / conc.:
84.1 mg/kg bw/day (actual dose received)
Remarks:
mean achieved dosage for males recieving 2000 ppm in the diet
Dose / conc.:
90.2 mg/kg bw/day (actual dose received)
Remarks:
mean achieved dosage for females recieving 2000 ppm in the diet
No. of animals per sex per dose:
6 animals per sex and dose
Control animals:
yes, plain diet
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
All dogs were inspected at least twice daily for any evidence of reaction to treatment or ill-health. A detailed record was maintained of any deviations from normal in respect of nature and severity, date and time of onset, duration and progress of the observed response.

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

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food residues, if any, were weighed and recorded daily for each pen. The total weekly intake per dog was calculated and, from these individual data, mean values for each group were derived.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
- The achieved intake of test compound was calculated from the nominal dietary concentrations and the group mean values for bodyweight and food consumption

BODY WEIGHT: Yes
The weight of each dog was recorded weekly for two weeks before commencement of treatment and throughout the study.

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: No

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before commencement and after five, ten, and 12 weeks of treatment
- Dose groups that were examined: All animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Before commencement and after six and 12 weeks of treatment
- Anaesthetic used for blood collection: Yes (identity) / No / Not specified
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked: Packed cell volume (PCV), hemoglobine concentration (Hb), Erythrocyte count (RBC), Mean cell haemoglobin concentration (MCHC), Mean cell volume (MCV), leucocyte count (WBC; total and differential), Platelet count, prothrombin time (PT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Before commencement and after six and 12 weeks of treatment
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked: Alkaline phosphatase (AP), Alanine amino-transferase activity (ALT:GPT), Aspartate amino-transferase activity (AST:GOT), Urea concentration, Glucose concentration, Direct bilirubin concentration, Total cholesterol concentration, Total protein concentration, Electrophoretic protein fractions, sodium (Na), potassium (K), chloride (Cl), calcium (Ca), lactate dehydrogenase (LDH). The majority of samples taken without anticoagulant before commencement of treatment were haemolysed. Repeat samples were obtained five days later and results for these repeat samples are reported only.

URINANALYSIS: Yes
- Time schedule for collection of urine: Before commencement and after six and 12 weeks of treatment
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: Appearance, pH, specific gravity, protein, glucose, ketone, bile pigments, urobilin, blood pigments. The sediment from centrifugation at 3,400 rpm. for ten minutes was examined for epithelial cells (E), polymorph (P) and mononuclear (M) leucocytes, red :blood cells (R), casts (C), or other abnormalities (A).

NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No

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

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

HISTOPATHOLOGY: Yes
Samples from each dog, together with any abnormality identified at necropsy, were preserved in buffered 4% formaldehyde except where otherwise specified. For processing, the tissues were dehydrated, embedded in paraffin wax, sectioned (at 5 µm thickness) and stained with haematoxylin and eosin. Four sections of the brain (to include cerebellum, cerebral cortex, thalamic nuclei, mid-brain and medulla) were prepared and examined. The spinal cord, in transverse and longitudinal sections, was prepared and examined. In addition a costal bone marrow smear was prepared, airdried, fixed in methanol and stained by the May-Gruenwald-Giemsa procedure.
The following tissues were examined microsopically: Adrenals, Aorta (thoracic), Brain, Colon, Duodenum, Epididymides, Eyes and optic nerves, Gall bladder, Heart (auricle, ventricle, ventricular septum, Ileum, Jejunum, Kidneys, Liver, Lungs (including mainstem bronchi), Lymph nodes (axillary, cervical, mesenteric), Mammary gland (caudal, cranial), Oesophagus, Ovaries, Pancreas, Parathyroids, Pituitary, Prostate, Salivary glands (left), Sciatic nerve (left), Skeletal muscle (thigh), Skin (dorsum), Spinal cord (cervical region, lumbar region), Spleen, Sternum, Stomach (cardia, fundus, pyloru), Testes, Thymus, Thyroids, Tongue, Trachea, Urinary bladder, Uterine cervix, Uterus
Statistics:
The significance of inter-group differences in food consumption, bodyweight, blood composition and organ weights was assessed by Students 't' test using a pooled variance.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The superior food consumption of treated females was also apparent before the treatment period and reflected the low
appetite of two control animals. The food consumption of treated and control males was similar throughout the study.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Almost all animals suffered conjunctivitis of varying severity and isolated incidences of mild keratitis were observed before and
during treatment. It is considered that these conditions resulted from the feeding of powdered diet and were not related to the administration of the test substance
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Lower packed cell volume, haemoglobin concentration and erythrocyte count were found after six and 12 weeks of treatment in the treated males; these values were
similar to those obtained before the commencement of the study and the deviations from control values are therefore attributed to chance.
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Unilateral cryptorchidism was present in two animals treated with 2000 ppm in diet. In one of these dogs the epididymis was also small.
A small number of other changes was present'and all are-considered incidental and those commonly found in dogs.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There was a range of banal degenerative and inflammatory lesions present, similar in type and incidence to those considered usual in beagle dogs of this age. These
included hypercellularity of Peyer's patches in the ileum; craniopharyngeal cysts in the anterior pituitary gland, and mammary
hyperplasia and secretion which can be correlated with the middle and late metoestral phase of the oestrous cycle. Two male dogs in the highest dosage group had a unilateral juvenile epididymis and testis due to cryptorchidism.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Dose descriptor:
NOAEL
Effect level:
84.1 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Highest dose tested.
Dose descriptor:
NOAEL
Effect level:
90.2 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Highest dose tested.
Critical effects observed:
no

The calculated values for achieved dosage were consistent within each group throughout the treatment period; mean dosages were, for males and females respectively, 8.5 or 9.2, 24.7 or 28.3 and 84.1 or 90.2 mg/kg/day for those receiving 200, 600 or 2000 ppm. The slightly higher values for female groups compared with the corresponding male groups reflected the similar appetites of the males and females combined with the lower group mean bodyweights of the females.

Conclusions:
Based on the results of the present study a NOAEL of 84.1 and 90.2 mg/kg bw/ day for male and female dogs, respectively, can be concluded after daily oral exposure to the test substance.
Executive summary:

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

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
84 mg/kg bw/day
Study duration:
subchronic
Species:
dog

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

No data are available for the test article. Repeated dose toxicity is therefore assessed with data obtained with a structural analogue. Please refer to the Annex of the CSR for the read across justification. Three studies are available for the read across substance:

In the key study (LSR, 1990) six Beagle dogs per sex were dosed daily with 200, 600, and 2000 ppm test substance (nominal concentration in food) for 13 weeks. Actual doses were 8.5, 24.7, 84.1 mg/kg bw/day for males and 9.2, 28.3, 90.2 mg/kg bw/day for females. No mortalities occurred and treated and control dogs remained similar in all aspects of behavior and appearance. No treatment-related findings were reported for body-weights, food consumption or blood and urine parameters. After pathology unilateral cryptorchidism was present in two animals in the high dose group. This finding confirmed similar observations recorded during physical observation before the commencement of treatment. Due to the observed cryptorchidism, both dogs had a unilateral juvenile epididymis and testis. In one of these dogs, the epididymis was also found to be small. In addition, a small number of other changes was present, and all are considered incidental and those commonly found in dogs. Comparison of organ weights, expressed either in absolute or bodyweight-relative terms, gave no indication of any differences attributable to test substance administration. After histopathology no changes were detected that could be related to treatment with the test substance. There was a range of banal degenerative and inflammatory lesions present, similar in type and incidence to those considered usual in beagle dogs of this age. In conclusion, no treatment-related adverse effects have been observed, thus a NOAEL of 84.1 (males) and 90.2 mg/kg bw/day (females), respectively, was derived, which was the highest concentration tested.

In the second key study a total of 180 Sprague-Dawley rats of the CD strain (20 males and 20 females per dose group with a 4-weeks recovery group of 5 males and 5 females in the control and high dose group) were fed with a diet for 13 weeks containing 0, 700, 2'000 and 6'000 ppm of the test substance in the feed (Huntingdon, 1979). No mortality occurred throughout the whole study period. Evidence of reduced grooming activity among the majority of males and females in the high dose group during the second half of the treatment period, and to some extent during the withdrawal period was observed. The growth of males and females of the high dose group was impaired during the first six weeks but was similar to that of the controls thereafter. Associated in part with the lower growth rate was a lower efficiency of food utilization, recorded among males and females of the high dose group during the first six weeks. An increased number of neutrophils was recorded in males and females of the high dose group at week 4, but not at weeks 12 or 17 (4th week of withdrawal). Elevated serum alkaline phosphatase levels were recorded among males of the high dose group at weeks 4 and 12, but not during recovery period. No organ weight changes considered to be related to treatment were seen at the 13 or 17 week examinations. No macroscopic lesions attributable to treatment with the test substance were recorded at the end of the treatment or withdrawal period, and no treatment-related changes were seen in the sections examined histopathologically. Based on the data recorded an NOEL was derived at the dietary level of 2000 ppm in feed, equivalent to a calculated average intake of 122 mg/kg/day for males and 136 mg/kg/day for females.

Additionally, a subacute oral 28-day study with Sprague-Dawley rats was performed. The animals were dosed daily by gavage with 100, 300, and 1000 mg/kg bw (Huntingdon, 1977). No mortality was observed at any dose level. Higher liver weights were recorded for males and females of the high dose group. Histological examination revealed a minimal increase in cytoplasmic basophilia of periportal hepatocytes in 3 males. Minimal enlargement of centrilobular and mid-zonal hepatocytes was seen in 2 males. Macroscopic examination after 4 weeks of treatment revealed thickening of the nonglandular region of the stomach in 5 males and 1 female of the high dose group. Histological examination revealed minimal hyperplasia of the squamous epithelium of the non-glandular region in 6 males and 1 female. Caecal enlargement not associated with any morphological change was seen in 4 males and 3 females. All of these findings were reversible. The effects observed in stomach and caecum were also reported in the mid dose group (300 mg/kg), however in less animals. Based on the results of this study, a NOEL of 100 mg/kg body weight was derived for the test substance when administered to rats for 28 consecutive days.

Justification for classification or non-classification

Classification,Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is considered to be not classified for repeated dose toxicity under Regulation (EC) No. 1272/2008.