Cyclopropanecarboxylic acid, [(3S,4R,4aR,6S,6aS,12R,12aS,12bS)-3-[(cyclopropylcarbonyl)oxy]-1,3,4,4a,5,6,6a,12,12a,12b-decahydro-6,12-dihydroxy-4,6a,12b-trimethyl-11-oxo-9-(3-pyridinyl)-2H,11H-naphtho[2,1-b]pyrano[3,4-e]pyran-4-yl]methyl ester

Endpoint summary

Description of key information

The repeated dose toxicity of the test substance was investigated in several short-term and long-term oral-feeding studies in rats, mice and dogs as well as one 28d dermal study in rats.

SHORT-TERM TOXICITY

The short-term toxicity of Afidopyropen was investigated in 28- and 90-day oral feeding studies in rats, mice and dogs as well as in a 1-year feeding study in dogs. Additionally, a 28-day repeated dose dermal exposure study in rats was conducted.

Administration of Afidopyropen to rats, mice and dogs by the oral route resulted in organ weight increases and histopathological findings. The principal target organ in all studies was the liver, as indicated by organ weight changes and associated histopathological changes at high doses (lipid deposits). In the F344 rat, the uterus (weight decrease) and the heart (vacuolar changes) were additional target organs at high doses. Findings at the LOAEL were typically only mildly adverse (slight changes in clinical chemistry, organ weight changes without severe histopathological findings). At higher doses, particularly with the dog and mice, treatment was not well tolerated, with severe effects on clinical signs and mortality / moribundity. Dermal administration of Afidopyropen to rats for 28-days did not result in any treatment-related changes up to the limit dose of 1000 mg/kg. Based on the findings in short-term studies, a relevant overall short-term NOAEL of 15 mg/kg bw/d is proposed. This value is derived from the NOAEL in the 90-day dog study.

CHRONIC TOXICITY

The chronic toxicity of the test substance was evaluated in long-term studies in rats and mice.

In a chronic toxicity study with rats, Afidopyropen was administered to groups of Fischer rats at dietary concentrations of 0, 75, 150, 300 or 1000 ppm for a period of 1 year. There were no treatment-related effects reported for mortality, detailed and general observations, organ weights, functional examination, ophthalmology, feed efficiency, urinalysis, or body weight ratio in any of the treated groups. At the 1000 ppm dietary level, there were treatment-related decreases in body weights and food consumption of females, and alterations in hematology and biochemistry in both sexes, gross changes in the liver of one female and an increase in the incidence of fatty vacuolar changes in both the liver and the heart of females. Based on the results of this study, the no adverse effect level (NOAEL) was 300 ppm (14.6 mg/kg/day) in males and 150 ppm (8.9 mg/kg/day) in females.

In a second supplemental chronic toxicity study with rats, Afidopyropen was administered to groups of Fischer rats at dietary concentrations of 0, 1000 or 3000 ppm for a period of 1 year. There were no treatment-related clinical signs or neurobehavioral effects. Toxicity was observed in both sexes at both doses resulting in effects on bodyweight, food consumption, hematology, clinical chemistry, urinalysis, organ weights and gross pathology. Histopathological changes were observed in the liver, heart and pituitary of females. The NOAEL for all effects observed in this study was established in a prior chronic rat study.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From August 13, 2009 to August 17, 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: MAFF in Japan, No 12 Nousan No 8147

Version / remarks:

2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.4100 (Chronic Toxicity)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

Version / remarks:

1981

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Lot number: 080722
Expiry: July 25, 2012
Appearance: Pale yellow, green powder

Species:

rat

Strain:

Fischer 344/DuCrj

Details on species / strain selection:

The rat is an animal species used generally for recommended repeated dose oral toxicity studies of agricultural chemicals and abundant biological reference data on this strain have been accumulated in the testing facility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Source: Charles River, Japan, Inc. Kanagawa, Japan
Age at study initiation: 6 weeks at dosing
Weight at dosing: 121-140 g (males); 95-105 g (females)
Housing: 2 of the same sex per cage in stainless steel, wire mesh cages; 21 cm (W) x 35 cm (D) x 20 cm (H)
Diet: Powdered basal diet (Oriental Yeast Co., Ltd., Tokyo, Japan)
Water: Tap water (well water), ad libitum. Water was sterilized with sodium hypochlorite.
Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
Temperature: 20-25 °C
Humidity: 30 - 70%;
Air changes: 10 or more changes per hour
Photoperiod: 12 hours/day (light on at 07:00 and off at 19:00)

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Remarks:

Basal diet without the test material

Details on oral exposure:

DOSE SELECTION
The doses were based on a previous 90-day feeding study rats.

DIET PREPARATION
Appropriate amounts of test substance for the 75 ppm, 150 ppm, 300 ppm and 1000 ppm groups were measured accurately and were mixed with 1997 kg, 1994 kg, 1988 kg and 1960 kg of basal diets, respectively. The test substance was smashed and mixed gradually with small amount of the basal diet, and subsequently a total of 2 kg of the preliminary mixed diet was shaken and mixed in a vinyl bag for approximately 5 minutes. The preliminary mixed diets in each group were mixed with 38 kg of basal diets, to make 40 kg of test diet for each group, using the mixer Mighty 120, for which wing rotation was set at 140 times/minute, for 20 minutes.

The test diet was prepared before the first treatment and 14 times during the study (every 4 weeks).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Three out of 5 samples (approximately 10 g each) collected from the test substance mixed diet in each treated group at the 1st, 8th and 14th (Final) preparations were analyzed for concentration and homogeneity of test substance. One sample from the basal diet in the control group was analyzed in the same manner. A part (approximately 10 g) of the residual mixed diets (the 1st, 8th and 14th prepared diets) in each group was collected after feeding and analyzed for stability of the test substance. The allowable range of the concentration of test substance in the test substance mixed diets was defined to be within ±15 % of selected dose levels.

The mean dose levels of the 75 ppm, 150 ppm, 300 ppm and 1000 ppm groups were 66.5 ppm, 134.4 ppm, 265.4 ppm and 925.6 ppm at the 1st preparation, 73.0 ppm, 146.9 ppm, 289.6 ppm and 979.7 ppm at the 8th preparation and 73.6 ppm, 142.7 ppm, 288.3 ppm and 988.0 ppm at the 14th preparation, respectively. Each analyzed concentration ranged from 87 % to 93 % of the selected dose levels at the 1st preparation, 94% to 99% at the 8th preparation and from 93 % to 99 % at the 14th preparation, being within the allowable range (± 15 %).

Homogeneity of test substance in the test substance mixed diets at the 1st, 8th and 14th preparations was also analyzed by using 3 out of 5 samples in each dose level. The coefficient of variation in all samples from each dose level was within 1.7 % at the 1st preparation, within 2.6 % at the 8th preparation and within 2.5% at the 14th preparation.

Stability of the test substance in the test substance mixed diets was analyzed by using a part (approximately 10 g) of the residual mixed diets (the 1st, 8th and 14th prepared diets) in each dose level. The actual levels ranged from 91% to 94% of the selected dose levels at the 1st preparation, from 95 % to 98 % at the 8th preparation and from 96 % to 99 % at the 14th preparation being within the allowable range (± 15 %).

Duration of treatment / exposure:

About 1 year (52 weeks)

Frequency of treatment:

Daily, 7 days /week.

Dose / conc.:

0 ppm

Remarks:

Plain diet

Dose / conc.:

75 ppm

Remarks:

corresponding to 3.7 mg/kg bw/d in males, 4.4 mg/kg bw/d in females

Dose / conc.:

150 ppm

Remarks:

corresponding to 7.3 mg/kg bw/d in males, 8.9 mg/kg bw/d in females

Dose / conc.:

300 ppm

Remarks:

corresponding to 14.6 mg/kg bw/d in males, 17.7 mg/kg bw/ d in females

Dose / conc.:

1 000 ppm

Remarks:

corresponding to 47.6 mg/kg bw/d in males, 56.1 mg/kg bw/d in females

No. of animals per sex per dose:

24 animals/sex/group

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS
- morbidity and mortality at least twice a day on weekdays and once a day on Saturdays, Sundays, and holidays during the treatment period.
- clinical signs once a day during the treatment period.

DETAILED CLINICAL OBSERVATIONS
Detailed clinical observations were performed in all animals once before the initiation of treatment (test week -1) and weekly during the treatment period. The observations were performed in the afternoon. The following items were observed and findings were recorded using a scoring system.
Home cage: Posture, Respiration, Grooming, Stereotypy behavior, Tremor, Twitch, Convulsion
Open field: Ease of removal from cage, Palpebral closure, Exophthalmos, Fur appearance, Piloerection, Urination (including numbers), Defecations (including numbers), Diarrhea, Alertness, Rears, Abnormal gait, Vocalization, Pinna response, Corneal response, Touch response, Tail pinch
Handling: Positional passivity, Salivation, Lacrimation, Discharge, Dermal and mucosal color, Pupil size, abdominal tone, righting reflex.

FUNCTIONAL EXAMINATIONS
Functional examinations were performed in 10 males and 10 females during test week 49. Sensor-motor response (response to noise of Galton’s whistle, visual placing response and proprioceptive sense), grip strength (forelimb and hind limb) and locomotor activity were examined. Acclimatization of rats to the equipment cages for locomotor activity was not done.

BODY WEIGHT
Body weights of all animals were measured on the day of receipt and on the grouping day. After grouping, body weights were measured in the morning once a week from test week 1 to 13 and once per 4 weeks from test week 16 to the termination of treatment. Final body weights of the animals were measured before euthanasia on each necropsy day.

FOOD CONSUMPTION
Food consumption (3-day or 4-day total amount) in each cage was measured once at test week 1 and twice a week during test week 2 to 13 and twice per 4 weeks from test week 16 to the termination of treatment. The total amount of food consumption was converted to a daily amount of one animal in each group. The weekly mean food consumption (g/rat/day) of males and females in each group was calculated based on the mean food consumption of each cage. The total mean food consumption of males and females in each group during the treatment period was calculated by averaging the weekly mean food consumption.

TEST SUBSTANCE INTAKE
The mean test substance intake (mg/kg/day) of males and females in each dose-group was calculated in each measuring week according to the following formula:
Mean test substance intake = mean food consumption x selected dose level/mean body weight.
In addition, total mean test substance intake during the treatment period in males and females in each group was calculated by averaging the weekly mean test substance intake.

WATER CONSUMPTION
No water consumption data were recorded.

FEED EFFICIENCY
The mean group body weight gain of all dose groups in each treatment week was divided by the mean group food consumption from the initiation of treatment to test week 13 and then the mean group feed efficiency percentage (%) was calculated. In addition, the total mean feed efficiency of males or females in each dose group during the first 13 treatment weeks was calculated by averaging the mean group feed efficiency in each week.

OPHTHALMOLOGY
Ophthalmology was performed for all animals 2 weeks before initiation of treatment (test week -2) and also for the animals in the control and high dose groups at test week 52. In the examination, external, anterior segment, optic media and fundus of both eyes were observed externally and with an indirect ophthalmoscope and a 28-diopter aspheric lens. No animals in other treatment groups were examined because no abnormal findings related to the treatment with the test substance were observed in any animals in the high dose group at test week 52.

HAEMATOLOGY
At test weeks 14 and 27, blood samples (2 mL) were collected from 10 males and 10 females (the same animals as those used for urinalysis) in each group via the cervical vein after an overnight fast. Heparin sodium was used for anticoagulant at the collection. The blood samples collected were examined on the items shown in the following table, except for reticulocyte, prothrombin time (PT) and activated partial thromboplastin time (APTT).

At test week 53, blood samples were collected from 10 males and 10 females in each group, which were used for urinalysis in principle, via the abdominal aorta. Animals were under deep anesthetization with diethyl ether inhalation. The items examined are shown below. EDTA-2K was used as anticoagulant in measurement of the items except for PT and APTT, and 3.2 % sodium citrate solution in measurement of PT and APTT.

Red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV*), mean corpuscular hemoglobin (MCH*), mean corpuscular hemoglobin concentration (MCHC), white blood cell (WBC) and platelet (PLT) were measured by using an automated hematology analyzer. PT and APTT were measured by using a coagulation analyzer. *calculated using values for RBC, HGB and HCT

CLINICAL CHEMISTRY
Blood biochemical examinations were carried out on 10 males and 10 females in each group (the same animals as those used for hematology) at test weeks 14, 27 and 53. The items examined are shown in the following table. Heparinized plasma samples separated from the hematology sample were used. The items were measured by using an auto-analyzer (Accute TBA-40FR, Toshiba Medical Systems, Corp., Tochigi, Japan) except for sodium (Na), potassium (K) and chloride (Cl), which were measured by using an auto-analyzer for electrolyte (EA07, ATWiLL Corp.). Examined parameters: aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), creatine kinase (CK), blood urea nitrogen (UN), creatinine (Creat), total cholesterol (TCHO), triglyceride (TGL), free cholesterol (FCHO), ester ratio (E/T; calculated values), glucose (Glu), total bilirubin (TB), total protein (TP), albumin (Alb), albumin/globulin ratio (A/G ratio; calculated values), calcium (Ca), inorganic phosphorus (IP), sodium (Na), potassium (K), chloride (Cl).

URINALYSIS
Urinalysis was carried out on 10 males and 10 females in each group at test weeks 13, 26 and 51. Fresh urine was collected from the animals which were housed individually in a metabolic cage for urine sampling. Specific gravity, pH, ketones, protein, glucose, occult blood, urobilinogen and bilirubin were measured using Clinitek status with a urinalysis testing paper. Subsequently, urine volume for approximately 24 hours were measured. Examined parameters: turbidity, occult blood, specific gravity, pH, ketone glucose, urobilinogen, bilirubin, sediments (WBC, RBC, casts, epithelium), volume (24 h), protein, color

Sacrifice and pathology:

NECROPSY
The animals were anesthetized by diethyl ether inhalation. The animals killed without blood sampling were euthanized by exsanguination from the neck artery, the sampled animals were exsanguinated form the abdominal artery.

ORGAN WEIGHTS
- absolute organ weights: Heart, Spleen, Liver, Kidneys (bilateral), Testes (bilateral), Epididymides (bilateral), Ovaries (bilateral), Uterus, Brain, Pituitary, Thyroids with parathyroids (bilateral), Adrenal glands (bilateral)
- measured before fixation in 10 males and 10 females killed in each group (same animals as those used for hematology at test week 53).
- relative organ weights were calculated as ratios of organ weights to final body weights.

HISTOPATHOLOGY
The organs and tissues below were removed from all animals at necropsy and fixed in 10 % neutral-buffered formalin except for the eyes and testes, which were fixed in Bouin solution. The lungs were infused with applied volume of 10 % neutral-buffered formalin from the trachea and then fixed in the fixative.
Heart, Aorta, Spleen, Thymus, Bone with bone marrow (sternum, right femur and knee joint), Mandibular lymph node, Mesenteric lymph node, Trachea, Lungs with bronchi, Laryngopharynx, Salivary glands (mandibular gland and sublingual gland), Tongue, Esophagus, Stomach (forestomach and glandular stomach), Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum, Liver, Pancreas, Kidneys (bilateral), Urinary bladder, Testes (bilateral), Epididymides (bilateral), Seminal vesicle and coagulation gland (bilateral), Prostate, Ovaries (bilateral), Uterus, Vagina, Brain (cerebrum, cerebellum, pons and medulla oblongata), Vertebrae with spinal cord (cervical, thoracic and lumbar regions), Ischiadic nerve (right), Eyes (bilateral), Harderian glands (bilateral), Pituitary, Thyroids with parathyroids (bilateral), Adrenal glands (bilateral), Skeletal muscle (right femur), Skin (dorsal region), Mammary gland (the 2nd and 3rd glands), Skull with nasal tissue, oral mucosa and tympanum, All gross lesions

Histopathological examination was conducted on the following designated organs from all animals in the control and high dose (1000 ppm) groups and one male in the 150 ppm group that was found dead. Gross lesions observed in any animals in other groups were also examined. In addition, the heart and liver in females in other treated groups were examined because vacuolar change of the heart and liver were observed in females in the 1000 ppm group.
Heart, Aorta, Spleen, Thymus, Bone with bone marrow (sternum, right femur and knee joint), Mandibular lymph node, Mesenteric lymph node, Nasal cavity, Laryngopharynx, Trachea, Lungs with bronchi, Salivary glands (mandibular gland and sublingual gland), Esophagus, Stomach (forestomach and glandular stomach), Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum, Liver, Pancreas, Kidneys (bilateral), Urinary bladder, Testes (bilateral), Epididymides (bilateral), Seminal vesicle and coagulation gland (bilateral), Prostate, Ovaries (bilateral), Uterus (bilateral horns and cervical portions), Vagina, Brain (cerebrum, cerebellum, pons and medulla oblongata), Vertebrae with spinal cord (cervical, thoracic and lumbar regions), Ischiadic nerve (right), Eyes (retina and optic nerve, bilateral), Harderian glands (bilateral), Pituitary, Thyroids with parathyroids (bilateral), Adrenal glands (bilateral), Skeletal muscle, Skin (dorsal region), Mammary gland (the 2nd and 3rd glands), All gross lesions

Statistics:

The data on detailed clinical observations (numbers of defecations, urination and rears),grip strength, locomotor activity, body weight, food consumption, feed efficiency, urine volume, hematological findings, blood biochemistry findings, organ weights and organ weight to body weight ratio were assessed by Bartlett’s test (significance level: 5 %) at first. As the results, if the variance was homogeneous, the data were assessed by one-way layout analysis of variance (significance level: 5 %). If the result was significant, the data were assessed between the control group and the treated groups by Dunnett's multiple comparison test (two-tailed, significance level: 5% and 1%). Subsequently, if the variance was heterogeneous, the data were assessed by Kruskal-Wallis rank test (significance level: 5 %). If the result was significant, the data were assessed between the control group and the treated groups by Dunnett type joint-ranking test (two-tailed, significance level: 5 % and 1 %).

The data on mortality, general clinical observations, ophthalmological findings, gross pathological findings and histopathological findings were assessed between the control group and the treated groups by Fisher's exact probability test (one-tailed, significance level: 5 % and 1 %).

The data on urinalysis except for urine volume were assessed between the control group and the treated groups by Dunnett type joint-ranking test (two-tailed, significance level: (5 % and 1 %).

SAS and EXSUS statistical evaluation software was used (SAS Institute Japan Ltd., Tokyo, Japan and CAC Corp., Tokyo, Japan)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

In the 1000 ppm and 300 ppm groups, neither males nor females showed any abnormal clinical signs during the treatment period. Effects in the other dose groups were considered not to be treatment-related.

In the 1000 ppm group, the number of rears of males increased significantly at test weeks 2 and 8. In females, the number of rears increased significantly at test weeks 5, 7, 38 and 39. In the 300 ppm group, the number of rears of females increased significantly at test week 8. In the 75 ppm group, the number of rears of males increased significantly at test week 46.The differences in the number of rears were sporadic and unrelated to the dose levels, and not likely to be related to treatment with the test substance.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male in the 150 ppm group died at test week 30. No females died in any treated groups throughout the treatment period. This death was not characterized as being related to treatment.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was a slight reduction in female body weight at 1000 ppm; the mean bodyweight was significantly lower at test weeks 20 and 48. No significant changes were noted in males during the treatment period. No statistically significant differences in the body weight were noted in females of other treated groups or in any treated groups of males.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In the 1000 ppm group there was a consistent decrease in female food consumption throughout the treatment period, the majority of results being statistically significantly lower than the control value.

In the remaining groups there were sporadic statistically significant differences, but these were neither consistent or dose related and were not considered to be related to treatment.

Food efficiency:

no effects observed

Description (incidence and severity):

The total mean feed efficiency (%) during the first 13 treatment weeks in the 0 ppm, 75 ppm, 150 ppm, 300 ppm and 1000 ppm groups were 16.2 %, 16.2 %, 16.3 %, 16.0 % and 16.1 % in males and 9.7 %, 9.7 %, 9.7 %, 9.5 % and 9.6 % in females, respectively.

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

not examined

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In the 1000 ppm group, unilateral retinal atrophy was observed in one male and one female at test week 52, but the incidence was not significantly different compared with that in the control group. These findings were considered to be unrelated to the treatment with the test substance.

In the control group, focal atrophy of the unilateral retina was observed in one male and unilateral lens opacity was observed in one female at test week 52.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Significant decreases of red blood cell (RBC), hemoglobin (HGB) and hematocrit (HCT) at test week 53 in males at 1000 ppm were considered to be related to treatment. In females HGB and HCT at test weeks 27 and 53, mean corpuscular volume (MCV) at test weeks 14, 27 and 53 and mean corpuscular hemoglobin (MCH) at test week 53 in females in the 1000 ppm group were noted in the high dose group and also considered to be test substance related.

The following statistically significant changes were considered in the report not to be related to treatment because they were not dose related, occurred only transiently or changes in associated parameters were not observed.

In the 1000 ppm group, platelets (PLT) at test week 27 and prothrombin time (PT) at test week 53 increased significantly in males. In females, PLT at test weeks 14 and 27 increased significantly. No significant changes were noted in any items in males at test week 14.

In the 300 ppm group, PT at test week 53 increased significantly in males. In females, PLT at test week 14 increased significantly and HGB, HCT and differential monocyte counts (Mono) at test week 27 decreased significantly. No significant changes were noted in any items in males at test weeks 14 and 27 and in females at test week 53.

In the 150 ppm group, HGB and HCT at test week 27 decreased significantly in females. No significant changes were noted in any items in males at test weeks 14, 27 and 53 and in females at test weeks 14 and 53.

In the 75 ppm group, no significant changes were noted in any items in males and females at test weeks 14, 27 and 53.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Significant increases of blood urea nitrogen (UN) in males at test week 53 and in females at test weeks 14 and 27 in the 1000 ppm group and alkaline phosphatase (ALP) in females at test weeks 14 and 53 in the 1000 ppm group and significant decrease of triglyceride (TGL) in males at test weeks 14 and 53 in the 1000 ppm group and total cholesterol (TCHO) in males at test week 53 in the 1000 ppm group were either related to the dose levels or remarkable changes in the high dose level, suggesting to be related to the treatment with the test substance.

Significant decreases of total bilirubin (TB) in males at test week 14 and in females at test week 27 in the 1000 ppm group and alanine aminotransferase (ALT) in males at test weeks 14 and 53 in the 1000 ppm group and in males at test week 14 in the 300 ppm group were decreasing changes, which were considered to be of no toxicological significance. Other significant changes were considered to be unrelated to the treatment with the test substance because those changes were not related to the dose levels or not continuous until test week 53 such as the end of treatment.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related observations in urinalysis for males or females.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

In the 1000 ppm group, locomotor activity in females decreased significantly, but this change was unrelated to the dose levels. Locomotor activity in males in the 1000 ppm group increased but was not significantly different from that in the control group, and was considered to be unrelated to the treatment with the test substance.

No statistically significant differences in any items examined were noted in males or females of other treated groups.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No significant changes were noted in any organ weight and any body weight ratio in males and females in all treated groups.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Effects observed In males were observed in one or a few animals in either control group or each treated group. Thus, they are not considered to be treatment-related.

In females, cloudiness of the liver was observed in one female in the 1000 ppm group. Other effects in females were observed in one or a few females in either control group or each treated group. However, the incidences of those lesions in the treated groups were not significantly different from those in the control group and not related to the dose levels.

In one male in the 150 ppm group that died, swelling and gray foci of the spleen, swelling of the renal lymph node, nodule in the kidney, hydroperitoneum and nodule in the mesentery were observed.

Neuropathological findings:

no effects observed

Description (incidence and severity):

No indications of neurotoxicy from the limited number of examined tissues.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Vacuolar change of hepatocytes in the peripheral portion of the liver (slight degree) was observed in 15 of 24 females in the 1000 ppm group and in two of 24 females in the 300 ppm group.,. Vacuolar change of myocardium of the heart (slight degree) was observed in six of 24 females in the 1000 ppm group, and the incidence of the lesion was significantly higher than that in the control group.

Protein casts of the kidney (slight degree) was observed in 16 of 24 males in the 1000 ppm group and the incidence of the lesion was significantly higher than that in the control group. The lesion was slight in severity and was not related to changes in urinalysis and blood biochemistry. It was considered to be spontaneous and unrelated to the treatment with the test substance.

In one male in the 150 ppm group that died, fibrosis of myocardium (slight degree) in the heart, fibrosis of capsule (slight degree) and extramedullary hematopoiesis (moderate degree) in the spleen, increased hematopoiesis (moderate degree) in the bone marrow, dilatation of sinus (slight degree) in the renal lymph node, brown pigment deposit in the tubular epithelium (slight degree), protein casts (slight degree) and nephroblastoma in the kidney and diffuse hypertrophy of the cortex (slight degree) in the adrenal gland were observed. These findings were not associated with treatment.

Other lesions observed were spontaneously occurring lesions and not related to the test treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: corresponding to 150 ppm

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

gross pathology

histopathology: non-neoplastic

Remarks on result:

other: corresponding to 300 ppm

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

cardiovascular

Organ:

heart

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

8.9 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

GLP and guideline compliant

System:

hepatobiliary

Organ:

liver

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

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From December 5, 2011 to August 06, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Version / remarks:

1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3200 (Repeated Dose Dermal Toxicity -21/28 Days)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 440/2008

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Specific details on test material used for the study:

Lot number: 080722
Expiry: 31 July 2013
Appearance: solid/yellow

Species:

rat

Strain:

other: Crl:WI(Han)

Details on species / strain selection:

The rat is a frequently used Iabaratory animal, and there is comprehensive experience with this animal species. Moreover, the rat has been proposed as a suitable animal species by the OECD and the EPA.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Source: Charles River Laboratories, Research Models and Services GmbH, Germany
Age at treatment start: 63 ± 1 days
Weight at dosing: 260.3-264.0 g (males) and 176.7-180.6 g (females)
Housing: Housed individually in Makrolon type M III cages (Becker &Co., Castrop-Rauxel, Germany, floor area of about 800 cm2).
Diet: ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland. ad libitum
Water: tap water in bottles, ad libitum
Acclimation period: about 6 days

ENVIRONMENTAL CONDITIONS
Temperature (°C): 20 - 24 °C
Humidity (%): 30-70 %;
Air changes (per hr): 15 per hour
Photoperiod (hrs dark/hrs light): 12 hours/day (light on at 06:00 and off at 18:00)

Type of coverage:

semiocclusive

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

Suspension of 1%

Details on exposure:

TEST SUBSTANCE PREPARATION
To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking water containing 1% carboxymethylcellulose was filled up to the desired volume, subsequently released with Ultraturax. During administration of the test substance, preparations were kept homogeneaus by stirring with a magnetic stirrer. The test-substance preparations were produced at least once a week.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in drinking water containing 1 % carboxymethylcellulose at room temperature over a period up to 5 days was demonstrated before the administration period. At the beginning of the administration period samples were taken from the lowest and highest concentration for homogeneity analysis.Additional concentration control analyses of the test substance preparations were performed in samples of all concentrations at the start of the administration period. The test substance was homogeneously distributed ranging form 90-110 % of the nominal concentration.

Duration of treatment / exposure:

4 weeks (males: 21 applications, females: 22 applications); applied uniformly to the clipped dorsal skin (dorsal and dorsolateral parts of the trunk; at least 10 % of the body surface)

Frequency of treatment:

Every week day

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 rats/sex/group

Control animals:

yes, concurrent no treatment

Details on study design:

RANDOMISATION
Prior to the first detailed clinical observation, the rats were distributed according to weight among the individual test groups, separated by sex. The weight variation of the rats used did not exceed 20 percent of the mean weight of each sex. The list of randomization instructions was compiled with a computer.

The test substance was applied uniformly to the clipped dorsal skin (dorsal and dorsolateral parts of the trunk; at least 10 % of the body surface) using 3 mL syringes. The application volume was 5 mL/kg bw, based upon the latest individual body weight determination. The skin was covered for at least 6 hours after administration using a semiocclusive dressing, consisting of 4 layers of porous gauze dressing and a stretch bandage. After removal of the dressing, the treated skin was washed with lukewarm water. Control animals received the vehicle, only.

The first clipping was carried out at least 24 hours before the randomization. The rats were reclipped at least once a week (depending on hair growth).

All rats were sacrificed after a fasting period (withdrawal of food) of at least 16 hours.

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS
- Mortality and moribundity: twice daily on working days, once daily on Saturdays, Sundays and public holidays
- Clinically abnormal signs: daily

DETAILED CLINICAL OBSERVATIONS
- 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

FOOD CONSUMPTION
- weekly and calculated as mean food consumption in grams per animal and per day.

DRINKING WATER CONSUMPTION
- daily visual inspection of the water bottles for any overt changes in volume.

BODY WEIGHT
- before the start of the administration period
- during the administration period 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.

FUNCTIONAL OBSERVATIONAL BATTERY(FOB)
- at the end of the administration period.
- passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests and reflex tests.
- The findings were ranked according to the degree of severity, if applicable.

Home cage:
The animals were observed in their closed home cages for: posture, tremor, convulsions, abnormal movements, impairment of gait, other findings

Open field:
The animals were transferred to a standard arena (50 x 50 cm with sides of 25 cm high) and observed for at least 2 minutes for: 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 assessment
- at the end of the administration period.
- same day as the FOB was performed.
- The examinations were performed using the TSE Labmaster System. The animals were placed in new clean polycarbonate cages for the time of measurement. Eighteen beams were allocated per cage. The numbers of beam interrupts were counted over 12 intervals of 5 minutes. The sequence at which the animals were placed in the polycarbonate cages was selected at random. For each animal, measurement started individually when the 1st beam was interrupted and was finished exactly 1 hour later. The animals did not receive any food or water during the measurements.

OPHTHALMOSCOPY
- prior to the start of the administration period.
- At the end of the administration period, i.e. study day 28, the eyes of animals in test groups 0 (control) and 3 (1000 mg/kg bw/d) were examined for any changes using an ophthalmoscope after administration of a mydriatic.

HAEMATOLOGY
Time schedule for collection of blood: Blood was drawn in the morning
Anaesthetic used for blood collection: Yes ; isoflurane
How many animals: 10 animals/sex/group
Parameters in the table below were examined: Leukocyte count (WBC), Erythrocyte count (RBC), Haemoglobin (HGB), Haematocrit (HCT), Mean corpuscular volume (MCV), Prothrombin time (Hepato Quick’s test; HQT), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes
Blood smears were prepared and stained according to Wright without being evaluated due to non-ambiguous results of the differential blood cell counts

CLINICAL CHEMISTRY
Time schedule for collection of blood: Blood was drawn in the morning
How many animals: 10 animals/sex/group
The following parameters were examined: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT), Sodium (NA), Potassium (K), Chloride (Cl), Inorganic phosphate (INP), Calcium (CA), , Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL)

Sacrifice and pathology:

NECROPSY
The animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.

ORGAN WEIGHTS AND FIXATION
- all animals sacrificed at scheduled dates
- anesthetised animals, adrenal glands, brain, epididymides, heart, kidneys, liver ovaries, spleen, testes, thymus, thyroid glands, uterus with cervix
- fixed in 4 % formaldehyde solution or in modified Davidson’s solution: all gross lesions, adrenal glands, aorta, brain, bone marrow (femur), cecum, cervix, colon, duodenum, epididymides, esophagus, eyes with optic nerve (modified Davidson's solution), heart, ileum, jejunum, kidneys, larynx, liver, lungs, lymph nodes (axillary and mesenteric lymph nodes), mammary gland, nose (nasal cavity), ovaries, pancreas, parathyroid glands, pharynx, pituitary gland, prostate, rectum, salivary glands (mandibular and sublingual glands), sciatic nerve, seminal vesicles, skeletal muscle, skin treated and untreated, spinal cord (cervical, thoracic and lumbar cord), stomach (forestomach and glandular stomach), spleen, target organs, testes (modified Davidson's solution), thymus, thyroid glands, trachea, urinary bladder, uterus

From the livers, one additional slice of the Lobus dexter medialis and the Lobus sinster lateralis was fixed in Carnoy's solution and embedded in paraplast.

HISTOPATHOLOGY
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings.
All organs were stained with hematoxylin and eosin (H&E), lungs as well embedded in paraplast (test group 1 and 2)

Statistics:

CLINICAL EXAMINATIONS
Body weight, body weight change and food consumption: A comparison of each group with the control group was performed using DUNNETT's
test (two-sided) for the hypothesis of equal means
Feces, rearing, grip strength forelimbs, grip strength hindlimbs, foot-splay test, motor activity: Non-parametric one-way analysis using KRUSKAL- WALLIS test (two-sided). lf 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

CLINICAL PATHOLOGY
Blood parameters:
For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. 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 hypothesis of equal medians
For parameters with unidirectional changes: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians

PATHOLOGY
Weight parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). lf 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 the WILCOXON test for the hypothesis of equal medians

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment related clinical signs of toxicity were observed throughout the study.

.

Dermal irritation:

effects observed, non-treatment-related

Description (incidence and severity):

One female animal of test group 2 (300 mg/kg bw/d) and three female animals of test group 3 (1000 mg/kg bw/d) showed lesions caused by repeated shearing. The finding was assessed as being not related to the test substance.

Mortality:

no mortality observed

Description (incidence):

No mortality was observed in this study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight and body weight changes were not affected by treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No clear treatment-related effects on food consumption were observed.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

No test-substance influence on water consumption was observed.

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects observed.

All apparent findings were assessed as being incidental in nature since they occurred in individual animals only and did not show a dose-response relationship.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes in the hematological parameters were found.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes in the clinical chemistry parameters were found.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

FOB
There were no treatment related effects observed.

Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, were without a doseresponse relationship or occurred in single rats only, these observations were considered to have been incidental.

MOTOR ACTIVITY MEASUREMENT
The single interval No. 5 was significantly decreased in male animals of test groups 1, 2 and 3 ( 100, 300 and 1000 mg/kg bw/d). The overall motor activity was significantly increased in female animals of test group 1 (100 mg/kg bw/d).

However, there were no significant deviations in the overall motor activity (summation of all intervals) in male animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) and no significant deviations in the single intervals in female animals of test group 1 (100 mg/kg bw/d) in comparison to the concurrent control group. Thus, these findings were assessed as being not relevant.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related changes in absolute or relative organ weight were observed.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All gross lesions observed in test animals occurred singularly and were considered to be spontaneaus in origin and are not related to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In the treated skin, minimal multifocal hyperkeratosis of parakeratotic type (parakeratosis) was observed through all test groups including also control animals. The following incidence was found:
1000 mg/kg bw/d
Male: 4/10; female 5/10
300 mg/kg bw/d
Male 2/10; female: 4/10
100 mg/kg bw/d
Male: 2/10; female: 2/10
Control
Male: 1/10; female 1/10

The increased incidence observed in females of the 300 mg/kg bw/d dose group and males and females the 1000 mg/kg bw/d dose groups rather regarded as incidental since the severity of the parakeratosis remained minimal in both treated as well as control animals.

All other histopathological findings occurred either individually or were biologically equally distributed over the control group and the treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Remarks on result:

other: There were no adverse local and systemic effects at any dose level, including the limit dose.

Key result

Critical effects observed:

no

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP and guideline compliant

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From December 5, 2011 to August 06, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Version / remarks:

1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3200 (Repeated Dose Dermal Toxicity -21/28 Days)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 440/2008

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Specific details on test material used for the study:

Lot number: 080722
Expiry: 31 July 2013
Appearance: solid/yellow

Species:

rat

Strain:

other: Crl:WI(Han)

Details on species / strain selection:

The rat is a frequently used Iabaratory animal, and there is comprehensive experience with this animal species. Moreover, the rat has been proposed as a suitable animal species by the OECD and the EPA.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Source: Charles River Laboratories, Research Models and Services GmbH, Germany
Age at treatment start: 63 ± 1 days
Weight at dosing: 260.3-264.0 g (males) and 176.7-180.6 g (females)
Housing: Housed individually in Makrolon type M III cages (Becker &Co., Castrop-Rauxel, Germany, floor area of about 800 cm2).
Diet: ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland. ad libitum
Water: tap water in bottles, ad libitum
Acclimation period: about 6 days

ENVIRONMENTAL CONDITIONS
Temperature (°C): 20 - 24 °C
Humidity (%): 30-70 %;
Air changes (per hr): 15 per hour
Photoperiod (hrs dark/hrs light): 12 hours/day (light on at 06:00 and off at 18:00)

Type of coverage:

semiocclusive

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

Suspension of 1%

Details on exposure:

TEST SUBSTANCE PREPARATION
To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking water containing 1% carboxymethylcellulose was filled up to the desired volume, subsequently released with Ultraturax. During administration of the test substance, preparations were kept homogeneaus by stirring with a magnetic stirrer. The test-substance preparations were produced at least once a week.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in drinking water containing 1 % carboxymethylcellulose at room temperature over a period up to 5 days was demonstrated before the administration period. At the beginning of the administration period samples were taken from the lowest and highest concentration for homogeneity analysis.Additional concentration control analyses of the test substance preparations were performed in samples of all concentrations at the start of the administration period. The test substance was homogeneously distributed ranging form 90-110 % of the nominal concentration.

Duration of treatment / exposure:

4 weeks (males: 21 applications, females: 22 applications); applied uniformly to the clipped dorsal skin (dorsal and dorsolateral parts of the trunk; at least 10 % of the body surface)

Frequency of treatment:

Every week day

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 rats/sex/group

Control animals:

yes, concurrent no treatment

Details on study design:

RANDOMISATION
Prior to the first detailed clinical observation, the rats were distributed according to weight among the individual test groups, separated by sex. The weight variation of the rats used did not exceed 20 percent of the mean weight of each sex. The list of randomization instructions was compiled with a computer.

The test substance was applied uniformly to the clipped dorsal skin (dorsal and dorsolateral parts of the trunk; at least 10 % of the body surface) using 3 mL syringes. The application volume was 5 mL/kg bw, based upon the latest individual body weight determination. The skin was covered for at least 6 hours after administration using a semiocclusive dressing, consisting of 4 layers of porous gauze dressing and a stretch bandage. After removal of the dressing, the treated skin was washed with lukewarm water. Control animals received the vehicle, only.

The first clipping was carried out at least 24 hours before the randomization. The rats were reclipped at least once a week (depending on hair growth).

All rats were sacrificed after a fasting period (withdrawal of food) of at least 16 hours.

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS
- Mortality and moribundity: twice daily on working days, once daily on Saturdays, Sundays and public holidays
- Clinically abnormal signs: daily

DETAILED CLINICAL OBSERVATIONS
- 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

FOOD CONSUMPTION
- weekly and calculated as mean food consumption in grams per animal and per day.

DRINKING WATER CONSUMPTION
- daily visual inspection of the water bottles for any overt changes in volume.

BODY WEIGHT
- before the start of the administration period
- during the administration period 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.

FUNCTIONAL OBSERVATIONAL BATTERY(FOB)
- at the end of the administration period.
- passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests and reflex tests.
- The findings were ranked according to the degree of severity, if applicable.

Home cage:
The animals were observed in their closed home cages for: posture, tremor, convulsions, abnormal movements, impairment of gait, other findings

Open field:
The animals were transferred to a standard arena (50 x 50 cm with sides of 25 cm high) and observed for at least 2 minutes for: 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 assessment
- at the end of the administration period.
- same day as the FOB was performed.
- The examinations were performed using the TSE Labmaster System. The animals were placed in new clean polycarbonate cages for the time of measurement. Eighteen beams were allocated per cage. The numbers of beam interrupts were counted over 12 intervals of 5 minutes. The sequence at which the animals were placed in the polycarbonate cages was selected at random. For each animal, measurement started individually when the 1st beam was interrupted and was finished exactly 1 hour later. The animals did not receive any food or water during the measurements.

OPHTHALMOSCOPY
- prior to the start of the administration period.
- At the end of the administration period, i.e. study day 28, the eyes of animals in test groups 0 (control) and 3 (1000 mg/kg bw/d) were examined for any changes using an ophthalmoscope after administration of a mydriatic.

HAEMATOLOGY
Time schedule for collection of blood: Blood was drawn in the morning
Anaesthetic used for blood collection: Yes ; isoflurane
How many animals: 10 animals/sex/group
Parameters in the table below were examined: Leukocyte count (WBC), Erythrocyte count (RBC), Haemoglobin (HGB), Haematocrit (HCT), Mean corpuscular volume (MCV), Prothrombin time (Hepato Quick’s test; HQT), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes
Blood smears were prepared and stained according to Wright without being evaluated due to non-ambiguous results of the differential blood cell counts

CLINICAL CHEMISTRY
Time schedule for collection of blood: Blood was drawn in the morning
How many animals: 10 animals/sex/group
The following parameters were examined: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT), Sodium (NA), Potassium (K), Chloride (Cl), Inorganic phosphate (INP), Calcium (CA), , Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL)

Sacrifice and pathology:

NECROPSY
The animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.

ORGAN WEIGHTS AND FIXATION
- all animals sacrificed at scheduled dates
- anesthetised animals, adrenal glands, brain, epididymides, heart, kidneys, liver ovaries, spleen, testes, thymus, thyroid glands, uterus with cervix
- fixed in 4 % formaldehyde solution or in modified Davidson’s solution: all gross lesions, adrenal glands, aorta, brain, bone marrow (femur), cecum, cervix, colon, duodenum, epididymides, esophagus, eyes with optic nerve (modified Davidson's solution), heart, ileum, jejunum, kidneys, larynx, liver, lungs, lymph nodes (axillary and mesenteric lymph nodes), mammary gland, nose (nasal cavity), ovaries, pancreas, parathyroid glands, pharynx, pituitary gland, prostate, rectum, salivary glands (mandibular and sublingual glands), sciatic nerve, seminal vesicles, skeletal muscle, skin treated and untreated, spinal cord (cervical, thoracic and lumbar cord), stomach (forestomach and glandular stomach), spleen, target organs, testes (modified Davidson's solution), thymus, thyroid glands, trachea, urinary bladder, uterus

From the livers, one additional slice of the Lobus dexter medialis and the Lobus sinster lateralis was fixed in Carnoy's solution and embedded in paraplast.

HISTOPATHOLOGY
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings.
All organs were stained with hematoxylin and eosin (H&E), lungs as well embedded in paraplast (test group 1 and 2)

Statistics:

CLINICAL EXAMINATIONS
Body weight, body weight change and food consumption: A comparison of each group with the control group was performed using DUNNETT's
test (two-sided) for the hypothesis of equal means
Feces, rearing, grip strength forelimbs, grip strength hindlimbs, foot-splay test, motor activity: Non-parametric one-way analysis using KRUSKAL- WALLIS test (two-sided). lf 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

CLINICAL PATHOLOGY
Blood parameters:
For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. 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 hypothesis of equal medians
For parameters with unidirectional changes: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians

PATHOLOGY
Weight parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). lf 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 the WILCOXON test for the hypothesis of equal medians

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment related clinical signs of toxicity were observed throughout the study.

.

Dermal irritation:

effects observed, non-treatment-related

Description (incidence and severity):

One female animal of test group 2 (300 mg/kg bw/d) and three female animals of test group 3 (1000 mg/kg bw/d) showed lesions caused by repeated shearing. The finding was assessed as being not related to the test substance.

Mortality:

no mortality observed

Description (incidence):

No mortality was observed in this study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight and body weight changes were not affected by treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No clear treatment-related effects on food consumption were observed.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

No test-substance influence on water consumption was observed.

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects observed.

All apparent findings were assessed as being incidental in nature since they occurred in individual animals only and did not show a dose-response relationship.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes in the hematological parameters were found.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes in the clinical chemistry parameters were found.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

FOB
There were no treatment related effects observed.

Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, were without a doseresponse relationship or occurred in single rats only, these observations were considered to have been incidental.

MOTOR ACTIVITY MEASUREMENT
The single interval No. 5 was significantly decreased in male animals of test groups 1, 2 and 3 ( 100, 300 and 1000 mg/kg bw/d). The overall motor activity was significantly increased in female animals of test group 1 (100 mg/kg bw/d).

However, there were no significant deviations in the overall motor activity (summation of all intervals) in male animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) and no significant deviations in the single intervals in female animals of test group 1 (100 mg/kg bw/d) in comparison to the concurrent control group. Thus, these findings were assessed as being not relevant.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related changes in absolute or relative organ weight were observed.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All gross lesions observed in test animals occurred singularly and were considered to be spontaneaus in origin and are not related to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In the treated skin, minimal multifocal hyperkeratosis of parakeratotic type (parakeratosis) was observed through all test groups including also control animals. The following incidence was found:
1000 mg/kg bw/d
Male: 4/10; female 5/10
300 mg/kg bw/d
Male 2/10; female: 4/10
100 mg/kg bw/d
Male: 2/10; female: 2/10
Control
Male: 1/10; female 1/10

The increased incidence observed in females of the 300 mg/kg bw/d dose group and males and females the 1000 mg/kg bw/d dose groups rather regarded as incidental since the severity of the parakeratosis remained minimal in both treated as well as control animals.

All other histopathological findings occurred either individually or were biologically equally distributed over the control group and the treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Remarks on result:

other: There were no adverse local and systemic effects at any dose level, including the limit dose.

Key result

Critical effects observed:

no

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP and guideline compliant.

Justification for classification or non-classification

The Afidopyropen PK studies revealed that at high doses there was high oral absorption coupled with saturated saturation of elimination and metabolism. This saturation leads to a disproportionate increase in plasma concentration of Afidopyropen as doses increase. Because the disproportionate increases in plasma concentrations occur at dose levels used in the Afidopyropen toxicology studies, the PK results are relevant to assess high-dose-specific toxicological effects observed with Afidopyropen.

The dose at which the inflection point for onset of saturated pharmacokinetics is observed is well separated from projected human doses resulting from Afidopyropen use. It is unlikely humans will be exposed to a dose level where superlinear kinetics would occur. Toxicological effects observed with Afidopyropen that occur only at dose levels above a kinetically derived maximum tolerated dose (KMD) are of questionable human relevance.

Therefore, classification according to Regulation (EU) 1272/2008, is not warranted.