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Description of key information

In a 28-Day oral toxicity study in rats, a NOAEL was established at 300 mg/kg/day. In a 90-day oral (in-feed) toxicity study in rats with a read-across substance, a NOEL was not achieved since body weight effects were noted in all treated groups. However, no other effects were detected. The same substance showed effects on body weight gain decrease and liver hypertrophy when tested in dogs, with a NOAEL of 69 mg/kg bw. The use of data obtained with the structural analogue to cover subchronic toxicity of the target substance was agreed upon in ECHA decision CCH-D-2114384240-56-01/F from January 8th, 2018.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 Sep 2009 - 10 Jun 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
GLP compliance:
yes (incl. certificate)
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany
- Age at study initiation: 41-43 days (males/females)
- Weight at study initiation: males: approx. 170g; females: approx. 135 g
- Housing: 5 animals per cage (H-Temp polysulfonate) with dust-free bedding
- Diet: ad libitum, ground Kliba maintenance diet
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was administered as a solution. For preparation, the appropriate amount of test substance was weighed out depending on the desired concentration and filled up with the vehicle (corn oil). The preparations were homogenized using a magnetic stirrer. The testsubstance preparations were prepared weekly.

VEHICLE
- Amount of vehicle (if gavage): 5 ml/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analyses of the test-substance preparations were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany. The stability of the test substance in corn oil at room temperature for a period of 7 days was proven before the start of the administration period (Study No.: 01Y0356/098022). omogeneity analyses of the test-substance preparations were performed in samples of all concentrations at the start of the administration period. These samples also served for concentration control analyses.
The formulations were shown to be homogeneous and the concentrations under the conditions of administrations were found within the range of 87.8 and 105.8% of the nominal concentrations.
Duration of treatment / exposure:
28 consecutive days
Frequency of treatment:
once daily
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
males & females
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
males & females
Dose / conc.:
750 mg/kg bw/day (actual dose received)
Remarks:
males
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
females
No. of animals per sex per dose:
5/sex/group plus additional 5/sex in control and high-dose groups for a 2-week recovery period
Control animals:
yes, concurrent vehicle
Details on study design:
- Post-exposure recovery period in satellite groups: 2 weeks for selected male and female animals of the control and high-dose groups
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied. All animals were checked daily for any abnormal clinically signs before, up to 1 hour and about 3-4 hours after the administration. Abnormalities and changes were documented for each animal.

DETAILED CLINICAL OBSERVATIONS: Yes
Detailed clinical observations (DCO) were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 × 37.5 cm with sides of 25 cm high). The following parameters were examined: abnormal behavior during “handling”, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmus,, feces (appearance/consistency), urine, pupil size.

BODY WEIGHT: Yes
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration and the recovery periods the body weight was determined on day 0 (start of the administration period) and thereafter at weekly intervals. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as body weight change.

FOOD CONSUMPTION: Yes
Individual food consumption was determined weekly over a period of 1 day and calculated as mean food consumption grams per animal and day.

WATER CONSUMPTION: Yes
Water consumption was observed daily by visual inspection of the water bottles for any overt changes in volume.

CLINICAL PATHOLOGY: Yes
Blood was taken from the retroorbital venous plexus in the morning from fasted animals. The animals were anaesthetized using isoflurane (Isoba®, Essex GmbH Munich, Germany). The blood sampling procedure and the subsequent analysis of the blood and serum samples were carried out in a randomized sequence. For urinalysis (main groups only) the individual animals were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight. The urine samples were evaluated in a randomized sequence The assays of blood and serum parameters were performed under internal laboratory quality control conditions with commercial reference controls to assure reliable test results. The results of the clinical pathology examinations are expressed in units of the International System (SI). The following examinations were carried out in all animals per test group and sex at the end of the administration period and in the remaining animals at the end of the recovery period (except for enzymes and urinalysis).

HEMATOLOGY:
The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes, Prothrombin time
(Hepato Quick’s test; HQT). Furthermore, blood smears were prepared and stained according to Wright but were not evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument.

CLINICAL CHEMISTRY:
Parameters analyzed: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT), Sodium (NA), Potassium (K), Chloride (CL), Inorganic phosphate (INP), Calcium (CA), Urea, Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL), Magnesium (MG), Bile acids (BILE)

URINALYSIS:
Parameters analyzed: pH, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Specific Gravity, Sediment, Color, turbidity, Volume.

NEUROBEHAVIOURAL EXAMINATION: Yes
A functional observational battery (FOB) was performed in all animals at the end of the administration (main groups) and the recovery period starting at about 10.00 h. At least one hour before the start of the FOB the animals were transferred singly to polycarbonate cages. Drinking water was provided ad libitum whereas no food was offered during the measurements. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.
Home cage observations: The animals were observed in their closed home cages; any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to: posture, tremor, convulsions, abnormal movements, impairment of gait, other findings.
Open field observations: The animals were transferred to a standard arena (50 × 50 cm with sides of 25 cm high) and observed for at least 2 minutes. Following parameters were examined: behavior when removed from cage, fur, skin, salivation, nose discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements, impairment of gait,, activity/arousal level, feces (number of fecal pellets/appearance/consistency) within two minutes, urine (appearance/quantity) within two minutes, number of rearings within two minutes.
Sensorimotor Tests/Reflexes: The animals were removed from the open field and subjected to following sensorimotor or reflex tests: approach response, touch response, vision ("visual placing response"), pupillary reflex, pinna reflex, audition ("startle response"), coordination of movements ("righting response"), behavior during "handling", vocalization, pain perception ("tail pinch"), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test, other findings.
Motor activity measurements: The motor activity (MA) measurement was carried out on all individual animals at the end of the administration (main groups) and the recovery period (in a randomized sequence on each examination day). The examinations were performed using the Multi-Varimex system supplied by Columbus Instruments Int. Corp., Ohio, U.S.A. For this purpose, the animals were placed in cages for the time of measurement. Four beams were allocated per cage. The number of beam interrupts was counted over 12 intervals for 5 minutes in each case. The sequence at which the animals were placed in the cages was selected at random. The first measurement of the main groups was started at about 12:45 h and the second measurement of the main groups was started at about 14:00 h. The measurement of the recovery groups was started at about 14:00 h. On account of the measuring variant "staggered", the starting time was varied by the time needed to place the animals in the cages. For each animal, measurement was started individually when the 1st beam was interrupted and ended exactly 1 hour later. The animals received no food or water during the measurements. After the transfer of the last animal in each case, the room where the measurements were carried out was darkened.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
The animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology. Weight assessment was carried out on all animals sacrificed at scheduled dates. The following weights were determined: Anesthetized animals, Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Prostate, Seminal vesicles with coagulation glands, Spleen, Testes, Thymus, Thyroid glands, Uterus with cervix.

HISTOPATHOLOGY: Yes
The immunorelevant organs and tissues were evaluated according to the following parameters:
Thymus:
• Increased/decreased grade of cortico-medullar ratio (related only to area)
• Increase of starry sky cells
• Changes of cellular density in the cortex
• Changes of cellular density in the medulla
Spleen:
• Changes of the cellularity of PALS, lymphoid follicles, marginal zone, red pulp
• Altered cellular composition of follicles
• Altered number of germinal centers
Lymph nodes (mesenteric and axillary lymph nodes):
• Changes in the cellularity of follicles, interfollicular area, paracortical area, medulla
• Altered cellular composition of paracortex
• Altered number of germinal centers
• Hyperplasia of high endothelial venules
Peyer's patches (of the jejunum):
• Changes of the cellularity of follicles (including mantle zone and germinal centers)
• Changes of the cellularity of interfollicular area
Bone marrow:
• Changes of the cellularity
• Changes of the myeloid/erythroid ratio
Whenever the histopathologic evaluation of the immunorelevant organs and tissues did not reveal a morphologic alteration of these items and/or whenever no other pathologic finding was noted, these organs were diagnosed as "no abnormalities detected”.
Special attention was given for the synchrony of the morphology of the estrous cycle in ovaries, uterus, cervix, and vagina.
Statistics:
Body weight: A comparison of each group with the control group was performed using DUNNETT's test (two-sided) for the hypothesis of equal means.
Feces, FOB parameters, Clinical pathology parameters, urine volume, urine specific gravity, Clinical weight parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians.
Urinalysis, except color, turbidity, volume and specific gravity: Pairwise comparison of each dose group with the control group using FISHER's exact test for the hypothesis of equal proportions.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Salivation after treatment was seen in all animals of test group 3 (750/1000 mg/kg bw/d), test group 2 (300 mg/kg bw/d) and in all males and 3 females of test group 1 (100 mg/kg bw/d). This finding was observed on day 8 for the first time and afterwards on several days of the study. No salivation was seen in the recovery period.
Mortality:
no mortality observed
Description (incidence):
No animal died prematurely within the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight and body weight change were significantly lower in males of test group 3 (750 mg/kg bw/d) on day 7 (body weight: -7.2%; body weight change: -33.0%).
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption was increased in both sexes of test groups 2 (300 mg/kg bw/d) and 3 (750/1000 mg/kg bw/d) during the administration period. No changes in food consumption were seen in the recovery period.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No test substance-related, overt changes in water consumption were observed.
Haematological findings:
no effects observed
Description (incidence and severity):
No treatment-related changes among hematological parameters were measured. All altered hematology parameter values were regarded as being incidental and not treatment-related.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No treatment-related changes among clinical chemistry parameters were measured. All observed alterations among clinical chemistry parameters were regarded as being incidental and not treatment-related.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No treatment-related changes among urinalyses parameters were measured.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Home cage observations: No test substance-related findings were observed. Deviations from "zero values" were obtained in several animals. However, as most findings were equally distributed between treated groups and controls, were without a dose-response relationship or occurred in single animals only, most observations were considered to have been incidental.
Open field observations: Slight closure eyelid(s) (palpebral closure) was observed in 4 males and 7 females of test group 3 (750/1000 mg/kg bw/d) on study day 23/24. This finding was assessed as being related to treatment. However, no test substance-related findings were observed after recovery.
Sensorimotor tests/reflexes: On study day 23/24, retarded adaptation of the pupil to light (pupillary reflex) was seen in 1 male and 3 females of test group 3 (750/1000 mg/kg bw/d). In addition, no adaptation of the pupil to light (pupils permanently dilated) was observed for 7 females of test group 3 (1000 mg/kg bw/d). Two female animals of test group 3 (1000 mg/kg bw/d) showed very frequent vocalizations when touched. Both types of findings were assessed as being related to dosing. However, no test substance-related findings were observed after recovery.
Quantitative parameters: On study day 23/24 animals of the test group 3 (750/1000 mg/kg bw/d) showed reduced rearing by -37% (males) and significantly reduced rearing -45.1% (females). A relation to dosing was assumed. On study day 41 the grip strength of forelimbs was increased in females in the test group 3 (1000 mg/kg bw/d) up to 17.8%. This finding was assessed as being incidental as grip strength of hindlimbs and foot splay test did not reveal any differnces.
Motor activity measurement: The single interval number 1 was significantly increased in female animals of test group 2 (300 mg/kg bw/d) and decreased in female animals of test group 3 (1000 mg/kg bw/d) on study day 24. The single interval number 6 was significantly decreased in male animals of test group 3 (750 mg/kg bw/d) on study day 23. Furthermore the single interval number 8 was significantly decreased in female animals of test groups 1 (100 mg/kg bw/d) and 3 (1000 mg/kg bw/d) on study day 24. On study day 41, the single interval number 12 was significantly increased in female animals of test group 3 (1000 mg/kg bw/d). These findings were assessed as being incidental and not related to treatment.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Absolute weights: A statistically significant increase of the mean absolute liver weight in females of test group 3 (1000 mg/kg bw/d) was regarded as treatment-related. A significant decrease of the ovaries weight was observed in females of test group 2 (300 mg/kg bw/d). This finding occurred without dose-relationship and was therefore considered to be incidental. All other mean absolute weight parameters did not show relevant differences compared to the control group and were regarded to be within the normal range of test animals of that age. In the recovery group, mean absolute organ weights in test groups 1-3 did not show relevant changes compared to the control group.
Relative weights: A statistically significant increase of the spleen weight was noted in males of test group 3 (750 mg/kg bw/d). Since no histopathological findings were observed in the spleen of males of test group 3 (750 mg/kg bw/d), this increase in the relative weight of the spleen was not considered to be treatment-related.The significant increase in the relative weight of the liver in females of test group 3 (1000 mg/kg bw/d) was regarded as treatment-related. The significant decrease of the ovaries weight in females of test group 2 (300 mg/kg bw/d) occurred without dose-relationship and was therefore considered to be incidental. All other mean relative weight parameters of the treated animals did not show relevant differences compared to the control groups. In the recovery group, the brain weight in males of test group 3 (750 mg/kg bw/d) revealed a significant increase (117%, p ≤ 0.01) compared to controls (set to 100%). This increase was considered to be in relation with a non-significant decrease of the terminal body weight. All other mean relative weight parameters did not show relevant differences compared to the control group.
See also tables below.
Gross pathological findings:
no effects observed
Description (incidence and severity):
All gross findings observed in test animals occurred singularly. They were considered to be spontaneous in origin and were not related to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment-related histopathological findings were observed in test animals. All findings noted were either single observations or were biologically equally distributed between control and treated rats. All of them were considered to be incidental and/or spontaneous in origin.
Histopathological findings: neoplastic:
not examined
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: eye findings (mydriasis) at dose levels of 750 mg/kg bw/d (males) and 1000 mg/kg bw/d (females).
Critical effects observed:
not specified

ORGAN WEIGHTS

Compared to controls (set to 100%) following values (expressed in %) were significantly increased:

Male animals Female Animal
Test group 1 2 3 1 2 3
Liver 98% 101% 117%*
Ovaries 94% 82%* 92%

* : p ≤ 0.05; **: p ≤ 0.01

Compared to controls (set to 100%) following values (expressed in %) were significantly increased:

Male animals Female Animal
Test group 1 2 3 1 2 3
Spleen 100% 111% 128%*
Liver 99% 100% 108%
Ovaries 94% 81%* 84%

* : p ≤ 0.05; **: p ≤ 0.01

Conclusions:
Under the conditions of the present study, the no observed adverse effect level (NOAEL) of the test substance was 300 mg/kg bw/d for male and female Wistar rats taking into account the eye findings (mydriasis) at dose levels of 750 mg/kg bw/d (males) and 1000 mg/kg bw/d (females). Increased liver weights in females were assessed as adaptive and non-adverse as no histopathological findings were observed. Salivation was considered to be related to either the bad taste of the test substance or local affection of the upper digestive tract. This finding was not considered to be an adverse and toxicologically relevant effect.
Executive summary:

The test substance was administered daily by gavage to male and female Wistar rats at dose levels of 0 (test group 0; corn oil served as vehicle), 100 (test group 1), 300 (test group 2) and 750 (test group 3, males)/1000 (test group 3, females) mg/kg body weight/day (mg/kg bw/d) over a period of 28 days. Control and test group 3 (750 mg/kg bw/d [males]/1000 mg/kg bw/d [females]) consisted of each 10 animals per sex, whereas test groups 1 (100 mg/kg bw/d) and 2 (300 mg/kg bw/d) consisted of each 5 animals per sex. After 4 weeks of treatment 5 animals per sex of all test groups were sacrificed (main groups). The remaining 5 animals per sex of control and test group 3 (750 mg/kg bw/d [males]/1000 mg/kg bw/d [females]) were maintained for another 14 days without administration of the test substance (recovery groups).

Food consumption and body weight were determined weekly. The animals were examined for signs of toxicity and mortality at least once a day. Detailed clinical examinations in an open field were conducted prior to the start of the administration period and weekly thereafter. Additionally, a functional observational battery (FOB) as well as measurement of motor activity (MA) was carried out at the end of the administration as well as the end of the recovery period. Clinicochemical and hematological examinations were performed towards the end of the administration and the end of the recovery period. Urinalyses were only done for the main groups. All animals were assessed by gross pathology, followed by histopathological examinations.

The following test substance-related, adverse findings were noted in test group 3 during clinical examinations: no adaptation of the pupil to light in 7 female animals, retarded adaptation of the pupil to light in 1 male and 3 female animals, slight closure of eyelid(s) in 4 males and 7 females. During clinical pathology, no test substance-related, adverse findings were observed. No test substance-related, adverse findings were observed during pathology. Increased liver weights in females were assessed as adaptive and non-adverse as no histopathological findings were observed. Adverse findings were confined to the high-dose group, only and displayed predominantly as effect on pupil adaptation to light. Thus, under the conditions of the present study, the no observed adverse effect level (NOAEL) of th test article was 300 mg/kg bw/d for male and female Wistar rats taking into account the eye findings (e.g. mydriasis) at dose levels of 750 mg/kg bw/d (males) and 1000 mg/kg bw/d (females).

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1974
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity in Non-Rodents)
Deviations:
yes
Remarks:
limited parameters, stability, homogeneity and accuracy of the test substance formulation was not determined
GLP compliance:
no
Limit test:
no
Species:
dog
Strain:
Beagle
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: internal breeding facility
- Age at study initiation: 7-13.5 months
- Weight at study initiation: males 11-15.1 kg, females 8.7-13 kg
- Housing: individually housed in metabolism cages
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
not specified
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
The diet was prepared from powdered Purina Dog meal mixed with 15% dry malt (Diamalt OCD) and 10% water.
A 2 kg pre-mix of the dog meal with compound at the appropriate levels was prepared and mixed for 1 hour. This was then added to the bulk diet and the whole mixed for a further 0.5 hour (water being added during this time). The mixture was then pelleted and dried for 12 hours at a temperature not exceeding 45°C. Control diet was prepared in the same manner without addition of test substance.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
continuously
Dose / conc.:
800 ppm
Remarks:
corresponding to actual intake males 27 mg/kg bw and females 27 mg/kg bw
Dose / conc.:
2 600 ppm
Remarks:
corresponding to actual intake males 69 mg/kg bw and females 78 mg/kg bw
Dose / conc.:
5 000 ppm
Remarks:
corresponding to actual intake males 150 mg/kg bw and females 155 mg/kg bw
The initial high dose of 8000 ppm was unpalatable and was therefore reduced to 2600 ppm on day 43 and increased to 5000 ppm on day 50 until the end of th estudy period.
No. of animals per sex per dose:
4/sex/dose for low and intermediate dose
5/sex/dose for control and high dose (extra animals for recovery period)
Control animals:
yes
Details on study design:
Post-exposure period: 4 weeks
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE
- Time schedule for examinations: daily

FOOD EFFICIENCY:
Calculated for 2 weeks pre-test and then for each month of the trial using the formula (Body weiqht gain x 100)/ (Total food consumed)

OPHTHALMOSCOPIC EXAMINATION: Yes
An ophthalmic and hearing examination was carried out on all animals pre-test and during weeks 6, 9 and 13. The animals on the recouery experiment were also examined during week 17.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: pre-test and during weeks 5, 9 and 13. Samples were also taken from the dogs on the recovery experiment (two animals from each of groups 1 and 4) during week 17.
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: all animals
- Parameters checked: Haemoglobin, Erythrocytes, Haematocrit, Reticulocytes, Inclusion Bodies, Thrombocytes, Leucocytes (total count and differential count), Prothrombin time, ESR

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: pre-test and during weeks 5, 9 and 13. Samples were also taken from the dogs on the recovery experiment (two animals from each of groups 1 and 4) during week 17.
- Animals fasted: No data
- How many animals: all animals
- Parameters checked: Sodium, Potassium, Glucose, Urea, SGOT, SGPT, SAP, Serum proteins (total and electrophoresis), Cholesterol

URINALYSIS: Yes
- Time schedule for collection of urine: pre-test and during weeks 5, 9 and 13. Samples were also taken from the dogs on the recovery experiment (two animals from each of groups 1 and 4) during week 17.
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked: pH, specific gravity, protein, glucose, bilirubin, ketones, blood, urine sediment

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
- Organ weights: adrenals, brain, heart, kidneys, liver, ovaries, pituitary, spleen, testes, thymus, thyroid, prostate and uterus

- Macroscopic: on all animals: adrenals, aorta, brain, colon, eye, heart, kidneys, liver, lungs, lymph nodes, mammary gland, ovaries, pancreas,
peripheral nerve (sciatic), pituitary, prostate (or uterus), small intestine, skeletal muscle, spleen, spinal cord, stomach, testes, thymus, thyroid and
urinary bladder

- Microscopic: on all animals: adrenals, aorta, bone marrow, brain, colon, eye, heart, kidneys, liver, lungs, lymph nodes, mammary gland, ovaries,
pancreas, peripheral nerve (sciatic), pituitary, prostate (or uterus), small intestine, skeletal muscle, spleen, spinal cord, stomach, testes, thymus,
thyroid and urinary bladder
Statistics:
Laboratory parameters and organ weight data were analysed by the Mann Whitney U test, in those cases where it was considered that meaningful results might be obtained.
Clinical signs:
no effects observed
Description (incidence and severity):
No mortalities and no treatment related abnormalities occurred.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 8000 p.p.m. the diet proved so unpalatable that the dogs refused to eat it, consequently food intake and body weight fell and became so extreme as to threaten survival. On day 43 the dietary concentration of the test compound was reduced to 2600 p.p.m. At this level food intake and body weights improved dramatically. On day 50 the dietary concentration was increased to 5000 p.p.m. At this level food intake fell slightly but improved as the trial progressed. From this time body weight gains were satisfactory. At 2600 p.p.m. and 800 p.p.m. there was no significant difference in food consumption and body weight gains compared to controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
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):
Mild conjunctivitis in all groups (including controls), not related to treatment.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In the high dose group at week 5 a slight decline in red cell parameters (haemoglobin, erythrocyte and haematocrit) was recorded in females only (P < 0.05). The ESR was increased in a few individuals. By week 13 these differences were no longer significant and were considered to be due to the poor food intake during the first few weeks of the trial. At week 5 there was a marked increase in ESR in one male animal of the 2600 ppm group only.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In female animals of the high dose the serum protein levels were slightly below controls throughout the test. This may also be attributed to poor food intake. At 2600 ppm, serum proteins in females were marginally lower than controls, all values were within normal limits.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No treatment related effects recorded.
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Differences observed in some organ weights (adrenals, ovaries, uterus, heart and spleen) could not be attributed to treatment due to high inter-individually variations and small group size with large standard deviations.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At autopsy no changes caused by the administration of the test compound were seen. Lungs granuloma in 1 female at 8000 ppm, 1 female at 2600 ppm and 2 males and 1 female at 8000 ppm.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
On histopathological examination the only change due to the administration of the test compound was a minimal hypertrophy of periportal hepatocytes in high dose animals which is considered to be an adaptive phenomenon.
Dose descriptor:
NOAEL
Effect level:
69 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
histopathology: non-neoplastic
Dose descriptor:
LOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
histopathology: non-neoplastic
Critical effects observed:
not specified
Conclusions:
NOAEL 2600 ppm (69-78 mg/kg bw) based on effects on body weight gain decrease and liver hypertrophy.
Executive summary:

In a repeated dose toxicity study similar to OECD TG 409, male and female dogs (4/sex/dose) were exposed to the test substance at 0, 800, 2600 and 8000 (the latter reduced to 2600 ppm in week 7 and then increased up to 5000 ppm from week 8 onwards) ppm via their diet during 90 days. This equals daily dosages of 0, 27, 69 and 150 mg/kg bw for males and 0, 27, 78 and 155 mg/kg bw for females. In addition, two animals (one male and one female) in the control and high-dose groups were included for a 4-week recovery period. No mortality occurred. No treatment-related clinical signs were observed. At 8000 ppm the diet proved so unpalatable that the dogs refused to eat it, consequently food intake and body weight decreased and threatened survival. In consequence, the dietary concentration of the test compound was adjusted as described above. However, the average daily test substance intake in the high dose group remained relatively constant. After 13 weeks, body weight gain was decreased in males and females in the high dose group (11 and 16%, respectively). A slight and transient (5th and 9th week) decrease in red cell parameters (haemoglobin, packed cell volume and red blood cells) was noted in females in the high dose group (week 5 and 9). Differences observed in some organ weights could not be attributed to treatment due to high inter-individual variations and small group size with large standard deviations. Histopathological examination revealed lung granulomas (males 2/4 in the high-dose group; females 1/4 at each treatment level) which is considered as a background finding in that species. In addition, minimal hepatic periportal hypertrophy in the high dose group was noted. No treatment related changes were seen in the recovery group after 4 weeks without exposure. The NOAEL was established to be 2600 ppm (69-78 mg/kg bw/day) based on decreased body weight and liver hypertrophy.

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
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
limited number of parameters investigated compared to current guidelines, stability and homogeneity of the test substance formulation was not determined, Inflammation observed in all animals, including control group
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: not indicated
- Weight at study initiation: males 156-210 g, females 148-187 g
- Housing: five to a cage (unless the number was reduced by mortality) In suspended cages with wire-mesh floors.
- Diet: powdered laboratory rat food, Spratt's Laboratory Diet 2, ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 50 ± 5
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
A pre-mix containing 20000 ppm was prepared each week and from this various dietary concentrations were obtained by direct dilution with further quantities of diet, homogeneity being achieved by mixing for 10 minutes in a rotary double-cone blender. The diets were then stored until use in heat-sealed, opaque polythene bags.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the diets fed to the rats containing test material were added to Methyl-ethylketon and boiled for 30 minutes at reflux. After cooling the mixture was made up with Methyl-ethylketon to the starting-weight and the content of test substance was determined by gaschromatography. The estimated relative reproducibility of this method is ± 10% or better. Within the limits of error of the sampling technique and the analytical method there was good correspondence between the concentrations found in the diets and the nominal values.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
continuously
Dose / conc.:
400 ppm
Remarks:
corresponding to 26 mg/kg bow in males and 29 mg/kg body weight in females
Dose / conc.:
1 300 ppm
Remarks:
corresponding to 80 mg/kg bow in males and 90 mg/kg body weight in females
Dose / conc.:
4 000 ppm
Remarks:
corresponding to 261 mg/kg bow in males and 277 mg/kg body weight in females
No. of animals per sex per dose:
- 20/sex/dose
- 5/sex additionally for control and high dose groups for recovery
Control animals:
yes, plain diet
Details on study design:
- Post-exposure recovery period in satellite groups: 4 weeks on 5 males and 5 females from control and high dosage level groups.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
All signs of ill-health or reaction to treatment together with any behavioural changes, were recorded. Any rat showing signs of severe debility or intoxication was isolated. Animals in extremis were sacrificed to prevent cannibalism. All mortalities were recorded as date and nature of death. All rats found dead, or killed for humane reasons, were subjected to detailed macroscopic examination in an attempt to define the cause of death. A full spectrum
of tissue samples was preserved and macroscopically abnormal tissues were subjected to histological examination.

BODY WEIGHT: Yes
The weight of each rat was recorded initially and at weekly intervals thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
The quantity of food consumed by each cage of rats was recorded and the mean weekly intake calculated.

FOOD EFFICIENCY:
The efficiency with which food was utilized was assessed by calculation of mean food conversion ratios (FCR values), as weights of food consumed per unit gains in bodyweight.

WATER CONSUMPTION: Yes
Water consumption was measured during weeks 5 and 12 for each cage of Groups 1 (Control) and 4 (4000 ppm). and the mean daily intake calculated.

OPHTHALMOSCOPIC EXAMINATION: Yes
Before treatment commenced and during weeks 4, 8 and 12 the eyes of all male and female rats from Groups 1 (Control) and 4 (4000 ppm) were examined using a Keeler indirect ophthalmoscope.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During weeks 4, 8 and 12
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: 10 males and 10 females from Groups 1 (Control) and 4 (4000 ppm)
- Parameters examined: Packed cell volume, Haemoglobin, Red cell count, Mean corpuscular haemoglobin concentration, mean cell volume, total white cell count, differential count, platelet count, thrombotest.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: During weeks 4, 8 and 12
- Animals fasted: Yes
- How many animals: 10 males and 10 females from Groups 1 (Control) and 4 (4000 ppm)
- Parameters examined: Plasma Urea, Plasma Glucose, Total serum proteins, Serum protein electrophoresis and AG ratio, Serum alkaline phosphatase, Serum glutamic - pyruvic transaminase, Sodium, Potassium.

URINALYSIS: Yes
- Time schedule for collection of urine: During weeks 4, 8 and 12
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters examined: pH, Specific gravity, Protein, Reducing substances, Glucose, Ketones, Bile pigments, Urobilin, Blood Pigments. Microscopy of spun deposit - after centrifugation at 1000 rev/minute for 10 minutes, the deposit was examined for: epithelial cells, polymorphonuclear leucocytes, mononuclear leucocytes, erythrocytes, organisms, casts, abnormal constituents, sperms.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
After treatment for 13 weeks, 20 males and 20 females from each group were killed. The remaining 5 female and 5 male rats from the control and high dosage levels were killed after a further 4 week observation period.
- Organ weights: adrenals, brain, heart, kidneys, liver, ovaries, pituitary, spleen, testes, thymus, thyroid.
- Macroscopic: on all animals adrenals, aorta, brain, caecum, colon, duodenum, eye, femur, heart, ileum, jejunum, heart, kidneys, liver, lungs, lymph nodes, mammary gland, oesophagus, ovaries, pancreas, peripheral nerve (sciatic), pituitary, prostate (or uterus), salivary glands, seminal vesicles, skeletal muscle, skin, spleen, stomach, testes, thymus, thyroid, tongue, trachea and urinary bladder.

HISTOPATHOLOGY: Yes
Samples of the following tissues (together with any other macroscopically abnormal entity) were preserved in buffered 10% formalin (except eyes, which were preserved in Davidson's fixative): adrenals, aorta, bone marrow, brain, caecum, duodenum, eye, heart, ileum, heart, kidneys, liver, lungs, lymph nodes, oesophagus, ovaries, pancreas, pituitary, prostate (or uterus), spleen, stomach, testes, thymus, thyroid, tongue and urinary bladder. In the first instance, microscopic examination was confined to: (i) all rats that died, in an attempt to determine cause of death, (ii) all rats of Groups 1 (Control) and 4 (4000 ppm) killed after treatment for 13 weeks, (iii) all rats of Groups 1 (Control) and 4 (4000 ppm) killed at the end of the 4 week recovery period.
Statistics:
Student's t test was employed to assess the significance of intergroup differences where the data suggested evidence of a response to treatment.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no visible signs of reaction to treatment.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One control male rat died during the removal of a blood sample during week 8. One female receiving 400 ppm was found dead in week 5 and one female receiving 1300 ppm was sacrificed during week 11 because of a suspected broken jaw. Autopsy of these rats revealed no macroscopic change that could be related to treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Male rats receiving the test article at 4000 ppm and all three treated female sub-groups had a lower rate of bodyweight gain than that of the controls (statistically significant decreased (17%) in males at 4000 ppm and 13%, 23% and 24% in females at 400, 1300 and 4000 ppm respectively). Males receiving 400 or 1300 ppm gained weight at a rate comparable with that of the controls. During the four week withdrawal period, both males and females from the high dosage level group had a rate of bodyweight gain which was superior to that of the controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A marginally lower food intake was recorded during the treatment period for both sexes receiving the test material at 1300 or 4000 ppm, although the difference from the control intake only attained a level of statistical significance in females receiving 4000 ppm (reduction of 8%). During the withdrawal period, food consumption among the high dosage level males remained at a similar level, whereas females which had received 4000 ppm had a superior food intake to that of the controls.
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
During the treatment period, male rats receiving 4000 ppm and all females receiving the test item had an inferior efficiency of food utilization when compared with that of the controls. During the withdrawal period males and females which had received 4000 ppm had a superior efficiency of food utilization when compared with that of the controls.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Water intake recorded during weeks 5 and 12 was comparable in controls and rats receiving test material at 4000 ppm.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No abnormalities of the eyes were detected that were considered to be related to treatment.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
When compared with control values, minimal increases in RBC count, PCV and Hb levels were observed at weeks 4, 8 and 12 in females receiving 4000 ppm. Although attaining a level of statistical significance, all values were within 'normal' limits for the strain and age of rat employed. The differences were, therefore, considered to be fortuitous and without biological significance. All other parameters measured were within 'normal' limits.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Some small differences between values for controls and rats receiving the test compound at 4000 ppm attained a level of statistical significance. As these differences were not seen consistently at the 4, 8 and 12 weeks investigations and all values were within 'normal' limits, these intergroup differences were not considered to be of toxicological significance.
Urinalysis findings:
no effects observed
Description (incidence and severity):
All parameters studied were comparable in controls and rats receiving test material at 4000 ppm.
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no organ weight changes recorded in rats sacrificed at 13 weeks which could clearly be attributed to treatment. The differences between control and treated rats in both absolute and relative organ weight values, which attained a level of statistical significance were considered to reflect the lower bodyweight of the treated rats rather than a direct effect of treatment (liver decreased in males (13%) at 4000 ppm and in females (10%) at 4000 ppm; kidney statistically significant decreased in males (15%) at 4000 ppm and in females (9-12%) at 1300 and 4000 ppm; adrenals statistically significant decreased in females (15-16%) at 1300 and 4000 ppm). When expressed relative to body weight, the brain was statistically significant increased in males (7%) at 4000 ppm and in females (8-13%) at 1300 and 4000 ppm. The gonads were statistically significant increased in males (9%) at 4000 ppm and in females (14%) at 1300 ppm. When expressed relative to brainweight, the only values which differed significantly from control values were organs in which a decreased weight was recorded from control values in the absolute data. Thus indicating that organ weight differences were primarily the result of disparities in the group bodyweights. At the end of the recovery period, there were no treatment-related differences in organ weights between control rats and rats that had received the test substance at 4000 ppm. The increased thyroid weight recorded in the treated male group was considered to be fortuitous, since at the 13 week kill, the weight of this organ in treated rats was comparable with that of the controls.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related macroscopic findings at autopsy after 13 weeks of treatment and after the recovery period.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
After both 13 weeks and 17 weeks, no histopathological change or variation from normal was seen in the tissues examined that was considered to be related to the administration of test material. In both treated and control animals foci of inflammatory cell infiltration, peribronchiolar lymphoid hyperplasia and perivascular accumulation of lymphocytes in the lungs, vacuolated and distended hepatocytes (males) and incidentally minimal chronic inflammatory cell infiltration in the interstitial tissue of the kidneys were observed. These changes were considered to be those that occur commonly in the rat and were of no significance.

Details on histopathology, week 13
The following comments are made in summary:
Lungs: Foci of chronic inflammatory cell infiltration, peribronchiolar lymphoid hyperplasia and perivascular accumulations of lymphocytes were seen in the lungs of the majority of animals and were associated in some instances with patchy consolidation, groups of distended macrophages, bronchiectasis, collapse and bronchiectatic abscess formation. In one male control rat pleurisy was seen; there was evidence of haemorrhage in a male high dose rat and sparse cellular exudate in the bronchioles in a female high dose rat. Foci of dystrophic mineralisation were seen in the media of the pulmonary artery of several animals from all groups. These changes were recognised as those that occur commonly in the lungs of the rat. They were related to chronic respiratory infection and were of no toxicological significance.
Liver: Vacuolated and distended hepatocytes were seen in males from both control and treated groups and foci of chronic inflammatory cell Infiltration were also noted in occasional animals. An Infarct was seen in a female control rat and a trace of fat was seen in another female control rat. These changes were considered to be those that occur commonly in the liver of the rat and were of no significance.
Kidneys: Minimal chronic Inflammatory cell infiltration was seen in the Interstitial tissue of the kidneys of occasional animals from both control and treated groups and was associated with occasional distended tubules containing eosinophilic material and tubules characterised by the basophilic staining of the cells of the epithelium. Foci of dystrophic mineralisation were seen at the corticomedullary junction in several females from all groups. Hydronephrosis was noted in female control rat. These changes were considered to be those that occur commonly in the kidneys of the rat and were of no significance.
Other morphological entities observed but not considered to be significant included:
chronic inflammatory cell Infiltration in the pancreas in one male control and one make high dose rat; macrophages containing brown pigment and foci of extra-medullary haemopoiesis in the spleens of occasional animals from both control and treated groups; foci of chronic inflammatory cell infiltration In the myocardium and the pericardium In occasional rats from both control and treated groups; hyperplastic lymph nodes from occasional animals in both control and
treated groups; congestion in thymus of a frmale control and a male high dose rat; epithelial whorl In the thyroid of another male control rat.

Details on histopathology, week 17
The following comments are made in summary:
No histopathological change or variation from normal was seen in the tissues examined that was considered to be related to the administration of the compound under test.
Lungs: Peribronchiolar lymphoid hyperplasia and perivascular and subpleural accumulations of lymphocytes were seen in the lungs of the majority of animals examined. Bronchopneumonic consolidation was seen in two high dose males. Foci of dystrophic mineralisation were seen In the media of the pulmonary artery in a female control. These changes were recognised as those that occur commonly in the lungs of laboratory rats, they showed no evidence of relationship to treatment and hence were disregarded from a point of view of the experiment.
Dose descriptor:
NOEL
Effect level:
< 29 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
body weight and weight gain
Dose descriptor:
LOEL
Effect level:
29 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
body weight and weight gain
Critical effects observed:
not specified

Organ weights (absolute) - group mean values (g) in rats sacrificed after 13 weeks

Males

Body

Weight

Brain Pituitary (mg) Heart Liver Spleen Thymus Kidney Thyroid (mg) Adrenals (mg) Gonads
Group 1 527 2 16 1.5 21.6 0.9 0.5 3.9 26 63 5
Group 2 506 2 17 1.5 20.1 0.8 0.5 3.7 26 62 5
Group 3 521 2.1 15 1.5 21.1 0.9 0.5 3.6 24 58 5.2
Group 4 469** 2 15 1.4 18.7** 0.8 0.4 3.3** 24 61 4.9
Females

Body

Weight

Brain Pituitary (mg) Heart Liver Spleen Thymus Kidney Thyroid (mg) Adrenals (mg) Gonads
Group 1 306 1.9 17 1 11 0.6 0.3 2.4 20 82 87
Group 2 287* 1.9 17 0.9 10.4 0.6 0.3 2.3 22 75 88
Group 3 274*** 1.8 16 0.9 10.3 0.5 0.3 2.2* 22 69*** 89
Group 4 266*** 1.9 16 0.9 9.9** 0.6 0.3 2.1** 19 70** 84

Organ weights (relative to body weight, expressed as percentage of body weight x 100) - group mean values in rats sacrificed at 13 weeks

Males Body Weight Brain Pituitary Heart Liver Spleen Thymus Kidney Thyroid Adrenals Gonads
Group 1 527 39 0.3 28 411 17 9 74 0.5 1.2 96
Group 2 506 41 0.3 30 639 16 10 73 0.5 1.2 100
Group 3 521 40 0.3 29 404 17 9 70 0.05 1.1 99
Group 4 469** 42* 0.3 29 399 18 9 71 0.5 1.3 105**
Females Body Weight Brain Pituitary Heart Liver Spleen Thymus Kidney Thyroid Adrenals Gonads
Group 1 306 62 0.5 33 360 19 11 78 0.7 2.7 2.9
Group 2 287* 65 0.6 33 362 19 12 80 0.8* 2.6 3.1
Group 3 274*** 67* 0.6 34 375 20 11 80 0.8* 2.5 3.3*
Group 4 266*** 70*** 0.6 36* 372 22* 11 80 0.7 2.6 3.2

Organ weights (relative to brain weight, expressed as percentage of brain weight x 100) - group mean values in rats sacrificed at 13 weeks

Males Body Weight Pituitary Heart Liver Spleen Thymus Kidney Thyroid Adrenals Gonads
Group 1 527 0.8 73 1068 45 23 193 1.3 3.1 248
Group 2 506 0.8 73 983 41* 24 180 1.3 3 245
Group 3 521 0.7 71 1014 43 23 176 1.2 2.8 249
Group 4 469** 0.8 70 955* 43 22 171 1.2 3.1 250
Females Body Weight Pituitary Heart Liver Spleen Thymus Kidney Thyroid Adrenals Gonads
Group 1 306 0.9 53 589 32 18 128 1.1 4.3 4.7
Group 2 287* 0.9 51 562 30 18 123 1.2 4.1 4.8
Group 3 274*** 0.9 51 564 30 14 120 1.2 3.8** 4.9
Group 4 266*** 0.8 51 535* 31 16 115* 1 2.8** 4.5

* p<0.05

** p< 0.01

*** p<0.001

Conclusions:
A LOEL of 29 mg/kg was derived based on body weight changes.
Executive summary:

In a 90-day oral similar to OECD 408, study groups of 20 male and 20 female Sprague-Dawley rats were exposed daily to the test article at 0, 400, 1300 and 4000 ppm (males 0, 26, 80 and 261 mg/kg body weight; females 0, 29, 90 and 277 mg/kg body weight) via their diet during 90 days. Additional 5 male and female animals in the control and high-dose group (4000 ppm) were allowed a 4-week recovery period. No treatment-related effect was seen on mortality or clinical observations. A statistically significant decrease in body weight gain compared to the control group was observed in males exposed to 4000 ppm (17%) and all treated females (13%, 23% and 24% at 400, 1300 and 4000 ppm respectively). Food consumption was statistically significantly reduced in females (8%) during week 1-13 at 4000 ppm. No treatment-related effect was observed for hematological parameters, urinalysis and clinical chemistry (conducted only in control and highest dose group). Liver, kidney, adrenals, showed decreased absolute weights compared to the control group at 4000 ppm in males and 1300 and 4000 ppm in females, however this is expected to be due to the body weight gain decrease rather than to be due to the test substance and is also reflected in an relative increase compared to control of brain and gonads. No treatment-related effects were noted after the recovery period. At macroscopic or histopathological examination no treatment-related abnormalities were noted. The LOEL was determined at 29 mg/kg bw based on the decreased body weight gain in females. The study had some limitations due to a background inflammation in all animals including control (inflammatory cell infiltration in lungs and kidney). However these findings did not seem to impair the results of the study and were considered of no significance.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
29 mg/kg bw/day
Study duration:
subchronic
Species:
rat
System:
other: general toxicity: body weight gain

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

In a repeated-dose 28-day oral toxicity study with a 14-day recovery the test substance was administered daily by gavage to male and female Wistar rats at dose levels of 0, 100, 300 and 750/1000 mg/kg bw/d. Regarding clinical examinations, no signs of general systemic toxicity were observed at dose levels up to 750/1000 mg/kg bw/d. Salivation was seen after dosing all rats of test groups 2 and 3 (300 and 750/1000 mg/kg bw/d) and sporadically in 5 male animals and three females animals of test group 1 (100 mg/kg bw/d). From the temporary, short appearance immediately after dosing it is likely, that this finding was induced by the taste of the test substance or local affection of the upper digestive tract. This finding was not considered to be an adverse and toxicologically relevant effect. Dilation of pupils or delayed closure observed in several animals of test group 3 (750/1000 mg/kg bw/d). In addition, ptosis of eyelids was observed and was considered as secondary to the mydriatic effect. It was suggested that the test substance and/or its metabolite(s) interact with the neurotransmitter system provided sufficiently high plasma concentrations could be obtained. For chemically-related substances, binding to acetylcholine receptors was shown (PAPKE et al., 1994; GRAHAM et al., 2005). This receptor activity might lead to inhibitory effects (e.g. mydriasis) modulated by the parasympathic nervous system. How the neurological changes induced ultimately causes pupil dilation is unknown. This effect was obviously concentration-dependent and was not observed during the recovery period. Post mortem examinations did not indicate a damage of the involved organ(s). Therefore, this effect represents a transient functional alteration only induced at peak concentrations not leading to tissue damage. Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose of the compound of 750 mg/kg bw/d in male rats and 1000 mg/kg bw/d in female rats. With regard to pathology, no clear histopathological correlate was found in the liver of female rats of test group 3 (1000 mg/kg bw/d) that could explain the slight increase of the absolute and relative organ weight. Nevertheless, the weight increase was regarded as a treatment related adaptive effect due to an enzyme induction as a detoxifying mechanism in the hepatocytes. No histopathological findings were observed in the spleen of males of test group 3 (750 mg/kg bw/d). Therefore, the increase in the relative weight of the spleen was not considered to be treatment-related. In conclusion, a 4 week oral administration of test substance up to 750 mg/kg/day in males and 1000 mg/kg/day in females led to no pathological findings in the rat. Thus, under the conditions of the present study, the no observed adverse effect level (NOAEL) was 300 mg/kg bw/d for male and female Wistar rats taking into account the eye findings (mydriasis) at dose levels of 750 mg/kg bw/d (males) and 1000 mg/kg bw/d (females). Increased liver weights in females were assessed as adaptive and non-adverse as no histopathological findings were observed. Salivation was considered to be related to either the bad taste of the test substance or local affection of the upper digestive tract. This finding was not considered to be an adverse and toxicologically relevant effect.

REFERENCES

RL Papke, AG Craig and SF Heinemann: Inhibition of nicotinic acetylcholine receptors by bis (2,2,6,6- tetramethyl- 4-piperidinyl) sebacate (Tinuvin 770), an additive to medical plastics, Volume 268, Issue 2, pp. 718-726,1994

John H. Graham, Roger L. Papke and Jerry J. Buccafusco: Functional Central Nicotinic Acetylcholine Receptor Antagonism by Systemic Administration of Tinuvin 770 (BTMPS) Current Alzheimer Research Volume 2, Number 2, 2005

In addition, several repeated dose studies are available with a structurally related compound (read-across justification attached). The use of data obtained with the structural analogue to cover subchronic toxicity of the target substance was agreed upon in ECHA decision CCH-D-2114384240-56-01/F from January 8th, 2018.

In a 90-day oral study groups of 20 male and 20 female Sprague-Dawley rats were exposed daily to the test article at 0, 400, 1300 and 4000 ppm (males 0, 26, 80 and 261 mg/kg body weight; females 0, 29, 90 and 277 mg/kg body weight) via their diet during 90 days. Additional 5 male and female animals in the control and high-dose group (4000 ppm) were allowed a 4-week recovery period. A statistically significant decrease in body weight gain compared to the control group was observed in males exposed to 4000 ppm (17%) and all treated females (13%, 23% and 24% at 400, 1300 and 4000 ppm respectively). Food consumption was statistically significantly reduced in females (8%) during week 1-13 at 4000 ppm. No treatment-related effect was observed for hematological parameters, urinalysis and clinical chemistry (conducted only in control and highest dose group). Liver, kidney, adrenals, showed decreased absolute weights compared to the control group at 4000 ppm in males and 1300 and 4000 ppm in females, however this is expected to be due to the body weight gain decrease rather than to be due to the test substance and is also reflected in an relative increase compared to control of brain and gonads. No treatment-related effects were noted after the recovery period. At macroscopic or histopathological examination no treatment-related abnormalities were noted. The LOEL was determined at 29 mg/kg bw based on the decreased body weight gain in females. The study had some limitations due to a background inflammation in all animals including control (inflammatory cell infiltration in lungs and kidney). However, these findings did not seem to impair the results of the study and were considered of no significance. No treatment-related effect was seen on mortality or clinical observations.

 

The read-across substance was further tested in a 90 day feeding study in dogs at concentrations of 0, 800, 2600 and 8000/5000 ppm (corresponding to actual intake of 0, 27, 69 and 150 mg/kg in males and 0, 27, 78 and 155 mg/kg in females). At a concentration of 8000 ppm the test article was so unpalatable that dogs refused to eat. On day 43 the dietary concentration of the test compound was reduced to 2600 ppm and on day 50 it was increased again to 5000 ppm, whereupon a reasonable food intake was maintained. A slight reduction in red cell parameters until the 12th week and a reduction of serum protein throughout the test were recorded in females only. These changes in the laboratory parameters were probably caused by the poor food intake during the first part of the test. A minimal hepatocyte hypertrophy was observed. This is considered to be a phenomenon of adaptive nature. At 2600 ppm and at 800 ppm no changes were seen which could be considered as related to the administration of test material. In a recovery experiment of four weeks (8000 -> 5000 ppm) neither changes of laboratory parameters nor histopathological changes were seen. The NOAEL was set at 2600 ppm.

 

In a 28d repeated dose toxicity study, male and female CFY rats (10/sex and group) were orally exposed to test material (suspended in 0.5% CMC) via gavage at 0, 50, 200 and 600 mg/kg body weight. One male in the control group (day 17) and two females in the 600 mg/kg group (day 8) were found dead. Reduced grooming, urine-stained fur, salivation after dosing and respiratory distress during week 3 and 4 were observed at 600 mg/kg. Statistically significant reduction of body weight gain was observed in males and females at 600 mg/kg (25 and 45%, respectively) and in females exposed at 200 mg/kg (21%). Body weight was decreased in males at 200 mg/kg/day, but did not reach statistical significance. No treatment-related food consumption effects were observed. No treatment-related effects were observed for hematological parameters, clinical chemistry or urinalysis. Absolute and relative adrenal weights were statistically significantly increased at 600 mg/kg bodyweight (16 and 29%, respectively) in males only without histopathological changes. Necropsy examinations showed distension of small intestine in one and two males at 200 and 600 mg/kg, respectively, and in one female at 200 mg/kg. Histopathology showed no relevant effects. The NOAEL was established to be 50 mg/kg bodyweight based on the reduced body weight gain and gross pathology.

 

In a second 28 day repeated dose toxicity study performed in rats the test compound was administered orally by gavage at daily doses of 600, 1000 and 2000 mg/kg (5 animals per sex and group). All animals of the 2000 mg/kg group died between day 2 and 17. Two male rats of the 1000 mg/kg group died on day 5 or 28. No mortalities occurred at the 600 mg/kg dose level. The main symptoms observed in all dosed groups were ptosis of eye lids, muscular hypotonia and rough coat. Sedation, brownish eye discharge and kyphotic carriage were seen in the 1000 and 2000 mg/kg group. The latter showed also soiled snout and dyspnea. Some individual rats of all dosed groups showed salivation, stiff movements, slow or irregular respiration, ventrior laterocumbency, slight cyanosis, tremor, meteorism, dyspnea, sedation, diarrhea and soiled snout. Loss of bodyweight was observed in the male rats of the 2000 mg/kg group. The females of this group showed stagnation of growth during the first days of the study followed by reduced weight gain up to day 12 and practically normal weight until day 16 after which all animals of this group had died. Male and female rats of the 1000 and 600 mg/kg group showed a physiological pattern of growth. Food consumption was low in the males of the 1000 mg/kg group during the first two weeks of the experiment. In all other dosed groups this parameter was found to be within physiological limits. Laboratory investigations revealed an increase of neutrophilic granulocytes and a relative decrease of lymphocytes in the differential blood count. All other parameters investigated were found to be within physiological limits valid for the strain of rats used. At termination of the study the animals were referred to pathology and autopsied. Special attention was paid to the sympathetic nervous system and ganglia. In the latter, transmitter function was checked in some rats by histochemical techniques. Histologically increased amount of eosinophilic and neutrophilicleucocytes in the spleen and in the blood vessels and perivascular tissue in the lungs was observed in all treated animals. The quantitative neurohistochemical examination showed that the average noradrenaline content of the principal perikarya of the superior cervical ganglion of treated rats was distinctly lower than in the controls. The measured values recorded in the striatum and the vas deferens did not deviate from the control values. No NOAEL was established.

The following table gives an overview on the relevant toxicological effects observed for the two compounds and shows that the effects are very comparable:

  Target Substance Source Substance
28-day oral toxicity, rat, gavage; 0, 100, 300 and 750/1000 mg/kg - Dilation of pupils and delayed closure, mydriasis and ptosis of eyelids secondary to the mydriatic effect at 750/1000 mg/kg.
- Significantly lower body weight change on day 7 in males at 750 mg/kg.
 
90-day oral toxicity, rat, feed; 0, 400, 1300 and 4000 ppm   -Decrease in body weight gain in males exposed to 4000 ppm and all treated females
90-day oral toxicity, dog, feed; 0, 800, 2600 and 5000 ppm   - body weight gain decrease
- liver hypertrophy
28-day oral toxicity, rat, gavage; 0, 50, 200 and 600 mg/kg   - Reduction of body weight gain was observed in males and females at 600 mg/kg and females at 200 mg/kg
28-day oral toxicity, rat, gavage; 600, 1000 and 2000 mg/kg   - Ptosis of eye lids in all dose groups (> 600 mg/kg)
- At 2000 mg/kg, loss of bodyweight and stagnation of growth in males and females, respectively

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008. Based on the present data, classification for repeated dose toxicity is not warranted under Regulation (EC) No.1272/2008.