Registration Dossier
Registration Dossier
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EC number: 620-056-5 | CAS number: 874195-61-6
Endpoint summary
Administrative data
Description of key information
NOAEL (oral, dog, 1 year): 2.8 and 3.0 mg/kg bw/day for males and females, respectively
NOAEL (oral, rat, 90 days): 16.4 and 20 mg/kg bw/day for males and females, respectively
NOAEL (oral, mouse, 90 days): 1001 and 1170 mg/kg bw/day for males and females, respectively
LOAEL (oral, dog, 90 days): 27.2 and 30.3 mg/kg bw/day for males and females, respectively
NOAEL (dermal, rat, 28 days): 500 mg/kg bw/day for males and females
Key value for chemical safety assessment
Repeated dose toxicity: via oral route - systemic effects
Link to relevant study records
- Endpoint:
- chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 10 Mar 2011 - 30 Mar 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 452 (Chronic Toxicity Studies)
- Version / remarks:
- adopted in September 2009
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.30 (Chronic Toxicity Studies)
- Version / remarks:
- adopted 1992
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.4100 (Chronic Toxicity)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: M.A.F.F N°8147 (Japan, notification 12 Nousan N°8147), adopted in November 2000
- Deviations:
- no
- GLP compliance:
- yes
- Species:
- dog
- Strain:
- Beagle
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Green Hill 2001 Srl, Montichiari, Italy
- Age at study initiation: 8-9 months
- Weight at study initiation: 8.8-11.7 kg (males), 6.9-10.2 kg (females)
- Housing: individually in stainless steel kennels with a floor surface area of 2 m², pair housed overnight when possible (2 dogs from the same sex and dose group)
- Diet: certified canine meal 125C3-P1 (Scientific Animal Food and Engineering, Augy, France),
- Water: tap water, ad libitum
- Acclimation period: at least 19 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-21
- Humidity (%): 40-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 2011-03-10 To: 2012-03-30 - Route of administration:
- oral: feed
- Vehicle:
- unchanged (no vehicle)
- Details on oral exposure:
- DIET PREPARATION
- Mixing appropriate amounts with (Type of food): ground diet
- Storage temperature of food: at room temperature
The test item was ground to a fine powder before being incorporated into the diet by dry mixing. Nine diet formulations (F1 to F9) were prepared at each dose level to provide the treated diet required for the study. Test diet formulations at 800 ppm were stored at room temperature and those at 300 and 100 ppm were stored at approximately -18 °C. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The stability of the test item in the diet at 600 and 15000 ppm was determined in a previous study under storage and usage conditions similar to those of the current study. Stability of the test substance in the diet at 100 ppm was determined during the chronic study for a period that covers the storage conditions used. Homogeneity was verified for all concentrations on the first preparation of the first formulation and for the lowest and highest concentrations on the first preparation of the fifth formulation to demonstrate adequate formulation procedures. In addition, the homogeneity of the second load of the first formulation (F1) at 100 ppm was checked in error, due to a mistake of labeling or of order of formulation.
Results:
AE 1887196 was stable at concentrations of 600 and 15000 ppm in the dry diet stored for at least 59 days at room temperature. In addition, the stability of the test substance at concentrations of 600 and 15000 ppm in the moistened diet (as distributed to the dog) was demonstrated for a period of 4 hours which corresponded approximately to the time of food preparation and distribution. The test substance was found to be stable at 100 ppm in the dry diet stored frozen for up to 50 days followed by 15 days of storage at room temperature. In addition, the stability of AE 1887196 at concentrations of 100 ppm in the moistened diet as distributed to the dog was demonstrated for a period of 4 hours which corresponded approximately to the time of food preparation and distribution.
Homogeneity reached 88-101% of the nominal concentration and measured concentrations of the formulations ranged between 86 and 99% of nominal concentrations and were therefore within the in-house target ranges (85-115%). According to the available results, homogeneity and concentration of AE 1887196 in diet mixtures were acceptable. - Duration of treatment / exposure:
- 1 year
- Frequency of treatment:
- daily (7 days/week)
- Remarks:
- Doses / Concentrations:
100, 300 and 800 ppm
Basis:
nominal in diet - Remarks:
- Doses / Concentrations:
2.8, 8.8 and 21.9 mg/kg bw/day (males)
Basis:
other: mean dose value as calculated from food intake - Remarks:
- Doses / Concentrations:
3.0, 9.7 and 24.3 mg/kg bw/day (females)
Basis:
other: mean dose value as calculated from food intake - No. of animals per sex per dose:
- 4
- Control animals:
- yes, plain diet
- Details on study design:
- - Dose selection rationale: the dose levels were selected based on the results from a previous subacute toxicity study in dogs
- Rationale for animal assignment (if not random): similar body weight distribution among groups for each sex, whilst ensuring full siblings were not placed in the same treatment group (computerized randomization procedure (Pristima System, version 6.1.0 Build 19)) - Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: clinical signs: at least once daily; detailed physical examinations: weekly in an open area; behavioral changes, ill-health, moribundity and mortality: twice daily or once daily on weekends and public holidays
- Elevated parameters: general behavior and appearance, skin and fur, teeth and gum, eyes, ears, mucous membranes, gait, posture and response to handling
PHYSICAL EXAMINATION: Yes
- Time schedule: monthly except on Study months 4 and 12 (in error)
- Elevated parameters: fur and skin, eyes, ears, teeth, gum, mucous membranes, rectal temperature, gait, stance, general behavior, chest including heart and respiratory rate, abdomen including palpitation, external genitalia and mammary glands, mental state, posture, gait and motor function, muscle tone, postural reactions, spinal nerve reflexes, sensation and cranial nerve reflexes
BODY WEIGHT: Yes
- Time schedule for examinations: at least weekly during the acclimatisation phase and treatment period, and before final necropsy (terminal body weight)
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during the acclimatisation period and at the end of treatment
- Dose groups that were examined: all animals from all dose groups
HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to dosing and on Study days 87-88, 178-179 and 360-361
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: all animals
- Parameters checked: red blood cell count, white blood cell count, hemoglobin concentration, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, reticulocyte count and % reticulocytes, leukocyte differential count, platelet count, activated partial thromboplastin time, prothrombin time
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to dosing and on Study days 87-88, 178-179, 248-249 and 360-361
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: all animals
- Parameters checked: total bilirubin, glucose, urea, creatinine phosphokinase, total protein, total cholesterol, aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase activities, cholinesterase, creatinine, lactic acid dehydrogenase, Gamma glutamyltransferase, sorbitol dehydrogenase, glutamate dehydrogenase, albumin, globulins, trigylcerides and serum protein electrophoresis, calcium, chloride, inorganic phosphorous, potassium and sodium
URINALYSIS: Yes
- Time schedule for collection of urine: prior to dosing and on Study days 85, 176-177, 354, 358-359 in the morning
- Animals fasted: access to water was restricted
- Parameters checked: appearance, volume, specific gravity, osmolality, refractive index, pH, sediment, protein, glucose, ketones, bilirubin, blood/red blood cells, nitrate, urobilinogen
OTHER:
Bioanalytical examination:
- Time schedule: test animals: at the end of the study just prior to food distribution and 1, 2 and 4 hours after food distribution (2 dogs/sex/group); . control animals: at the end of the study just prior to food distribution (2 dogs)
-Elevated parameter: plasma was prepared for determination of the test item and its major metabolites - Sacrifice and pathology:
- SACRIFICE: Study days 365-368 - 103 (animals were diet fasted prior to sacrifice)
GROSS PATHOLOGY: Yes
- Parameters checked: tongue, submandibular (salivary) gland, esophagus, stomach, duodenum, jejunum, ileum, ceacum, colon, rectum, liver, pancreas, gallbladder, trachea, lung, pharynx, larynx, aorta, heart, bone marrow, sternum, lymph node (retropharyngeal and mesenteric), spleen, thymus, kidney, urinary bladder, testis, epididymis, prostate gland, ovary, uterus (with cervix), mammary gland, vagina, oviduct, brain, sciatic nerve, spinal cord (cervical, thoracic and lumbar), eyes, optic nerves, pituitary gland, adrenal gland, parathyroid gland, thyroid gland (weighed with parathyroid), bone, sceletal muscle, skin, all gross lesions, articular surface (femur head)
HISTOPATHOLOGY: Yes
- Parameters checked: tongue, submandibular (salivary) gland, esophagus, stomach, duodenum, jejunum, ileum, caecum, colon, rectum, liver, pancreas, gallbladder, trachea, lung, aorta, heart, bone marrow, sternum, lymph node (retropharyngeal and mesenteric), spleen, thymus, kidney, urinary bladder, testis, epididymis, prostate gland, ovary, uterus (with cervix), mammary gland, vagina, oviduct, brain, sciatic nerve, spinal cord (cervical, thoracic and lumbar), eyes, optic nerves, pituitary gland, adrenal gland, parathyroid gland, thyroid gland (weighed with parathyroid), bone, sceletal muscle, skin, all gross lesions, articular surface (femur head) - Statistics:
- Mean and standard deviations were calculated for each group. All statistical analyses were carried out separately for males and females using Path/Tox System V4.2.2. (Module Enhanced Statistics). For determination of statistically significant differences, the data were analysed with Bartlett Test followed by ANOVA Test and DUNNETT Test (if not significant in BARTLETT Test) or KRUSKAL-WALLIS Test and DUNN Test (if significant in BARTLETT Test). Quantitative urinalysis parameter (pH) were analysed with KRUSKAL-WALLIS TEST followed by DUNN TEST in case of a significant result. If one or more group variance(s) equaled 0, means were compared using non-parametric procedures. Comparison between the high-dose group and the control group was performed with F test combined with Modified t-test (in case of a significant result) or T-test (in case of a non-significant result).
- Clinical signs:
- no effects observed
- Mortality:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- In the high dose females, there was a minor decrease of 11% in mean food consumption throughout the study in comparison to the controls.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- no effects observed
- Haematological findings:
- no effects observed
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Lower mean total cholesterol concentrations in males at 800 ppm; lower mean albumin concentrations, lower total protein concentrations and albumin/globulin ratio at 800 and 300 ppm in both sexes; data not always statistically significant
- Urinalysis findings:
- no effects observed
- Behaviour (functional findings):
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Higher mean absolute and relative liver weights were considered to be toxicologically relevant only in males at 800 ppm and in females at 800 and 300 ppm, since these changes were associated with histopathological findings.
- Gross pathological findings:
- no effects observed
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Treatment-related histopathological findings were observed in the liver and thyroid gland.
- Histopathological findings: neoplastic:
- not examined
- Details on results:
- CLINICAL SIGNS AND MORTALITY
There were no mortalities during the study.
At 800, 300 and 100 ppm, no treatment-related clinical signs were noted in either sex. The clinical signs recorded in both sexes were those commonly recorded in untreated dogs of this strain and age or distributed between different groups with no evidence of a dose-related effect and/or with sporadic occurrence and were thus considered not to be related to AE 1887196 administration. Some females from different groups, including the control, were recorded with genital discharge, which correlated with animals being in estrus at the time of clinical examination.
No changes at physical examination were noted in either sex. Rectal temperature recordings in all animals revealed no treatment-related changes.
BODY WEIGHT AND WEIGHT GAIN
At 800, 300 and 100 ppm, mean body weight parameters were unaffected by the treatment in both sexes throughout the study. The few changes observed had no dose-relationship and were thus considered to be incidental.
FOOD CONSUMPTION AND COMPOUND INTAKE
At 800 ppm in females, mean food consumption was marginally reduced during the first week of treatment (-20%, not statistically significant) and at several other occasions during the study (maximum of -26%, not statistically significant), when compared to the controls. Overall in the high dose females, there was a minor decrease of 11% in mean food consumption throughout the study in comparison to the controls.
At 800 ppm in males and at 300 and 100 ppm in both sexes, mean food consumption was unaffected by treatment throughout the study. The few increases in weekly mean food consumption noted in the high and mid dose male groups compared to the controls were considered to be incidental and within the range of expected variations for Beagle dogs of this age kept under laboratory conditions.
OPHTHALMOSCOPIC EXAMINATION
No changes at the ophthalmological examination were noted at any dietary level in either sex.
HAEMATOLOGY
No treatment-related changes were noted at any dietary level in either sex throughout the study.
The few differences noted, even if statistically significant, were not considered to be treatment-related in view of their incidental or sporadic occurrence and/or their low magnitude.
CLINICAL CHEMISTRY
Higher mean alkaline phosphatase activities were observed at all dietary levels in both sexes throughout the study compared to the controls, though no clear dose-effect relationship was seen between 800 and 300 ppm in females. The differences were lower in males than in females.
At 100 ppm in both sexes, the increase in mean alkaline phosphatase activities was not considered to be an adverse effect, because it was the only clinical pathology alteration potentially indicative of liver toxicity and had no associated histopathological changes.
Slightly lower mean total cholesterol concentrations were noted at 800 ppm in males only: -29%, -40%, -34% and -30% at Month 3, 6, 9 and 12, respectively, compared to the controls (p ≤ 0.05).
Lower mean albumin concentrations were observed at 800 and 300 ppm in males (between -9 and -13%) and in females (between -10 and -21%), compared to the controls. As a consequence, lower total protein concentrations (between -5 and -10% in males and -3 and -16% in females) and lower albumin/globulin ratios (between -5 and -13% in males and -10 and -18% in females) were noted in these groups. As the data were non-homogeneous, the differences were not always statistically significant. Lower mean total bilirubin concentrations were noted at 800 and 300 ppm in males and at any dietary level in females, compared to the controls. However, lower total bilirubin concentrations are not considered to be adverse effects of the test item as they do not represent any functional impairment to the test organism. Other changes, even if statistically significant, were not considered to be treatment-related in view of their sporadic occurrence.
URINALYSIS
No relevant change was noted in the parameters assayed. The few differences noted were not considered to be treatment-related in view of their incidental or sporadic occurrence and/or their low magnitude.
TERMINAL BODY WEIGHT AND ORGAN WEIGHTS
There were no treatment-related changes in mean terminal body weight. The lower mean terminal body weight observed in high dose females (-6% compared to the controls, not statistically significant), was in line with the lower mean body weight seen at start of treatment for this group (-8% compared to the controls, not statistically significant). At 800 and 300 ppm in males and at all doses in females, a tendency towards higher mean absolute and relative liver weights was observed, when compared to the controls (not statistically significant).
These changes were considered to be toxicologically relevant only in males at 800 ppm and in females at 800 and 300 ppm, since they were associated with histopathological findings. The few other organ weight changes were considered to be incidental and not treatment-related.
GROSS PATHOLOGY
There were no treatment-related macroscopic changes in treated animals, when compared to the controls.
HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment-related histopathological findings were observed in the liver and thyroid gland.
In the liver, hepatocellular hypertrophy (with eosinophilic cytoplasmic change) was noted at 800 ppm in both sexes and at 300 ppm in females. Concomitantly, a slight decreased incidence and/or severity of hepatocellular glycogen accumulation was noted at 800 and 300 ppm in both sexes.
In the thyroid gland, minimal follicular cell hypertrophy was noted at 800 ppm in one female only.
BIOANALYTICAL EXAMINATION
AE 1887196 individual plasma concentrations on Month 12 prior to food distribution, and 1, 2 and 4 h after food distribution were below the method validation limit of 0.1 mg/L in all dose groups.
For the metabolite BCS-AA10030, individual plasma concentrations at the same time points were below the validation limit of 0.5 mg/L in all dose groups. For the metabolite BCS-BQ87904, individual plasma concentrations were below the validation limit of 0.5 mg/L prior to food distribution in all dose groups and after food distribution in the low and mid dose groups. In the high dose groups, concentrations after food distribution ranged from the validation limit of 0.5 mg/L up to a maximum of 1.08 mg/L and were similar at the 3 times points (1, 2 and 4 h) .
For the metabolite BCS-BP19252, individual plasma concentrations were similar between the two sexes and at the 4 times points (prior to food distribution and 1, 2 and 4 h after food distribution) and ranged from the validation limit of 0.5 mg/L up to 1.36 mg/L in the low dose groups, from 1.07 mg/L up to 4.95 mg/L in the mid dose groups and from 1.69 mg/L up to 11.0 mg/L in the high dose groups. - Dose descriptor:
- NOAEL
- Effect level:
- 100 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: based on liver effects (reduced absolute and relative liver weights; hepatocellular hypertrophy)
- Dose descriptor:
- NOAEL
- Effect level:
- 2.8 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: based on liver effects (reduced absolute and relative liver weights; hepatocellular hypertrophy); corresponds to a nominal dose of 100 ppm as calculated for males in regard to group mean food consumption and group mean body weight
- Dose descriptor:
- NOAEL
- Effect level:
- 3.2 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- other: based on liver effects (reduced absolute and relative liver weights; hepatocellular hypertrophy); corresponds to a nominal dose of 100 ppm as calculated for females in regard to group mean food consumption and group mean body weight
- Critical effects observed:
- not specified
Reference
Table 1: Body weight and weight gain (kg)
|
Dose level (ppm) |
||||
Control |
100 |
300 |
800 |
||
Males |
Initial BW (Day 1) (%C) |
10.1 ± 0.4 |
0.2 ± 0.6 (101%) |
10.5 ± 0.4 (104%) |
10.4 ± 1.3 (103%) |
BWG Week 1 (Days 1-8) |
0.1 ± 0.2 |
-0.1 ± 0.1 |
-0.2 ± 0.3 |
-0.2 ± 0.4 |
|
BWG Weeks 1-13 (Days 1-92) |
1.4 ± 0.4 |
0.7 ± 0.5 |
0.4 ± 1.0 |
1.1 ± 0.5 |
|
BWG Weeks 13-26 (Days 92-183) |
0.6 ± 0.5 |
0.3 ± 0.5 |
0.3 ± 0.5 |
0.7 ± 0.4 |
|
BWG Weeks 26-52 (Days 183-364) |
0.6 ± 1.3 |
-0.3 ± 0.2 |
0.3 ± 0.7 |
0.9 ± 0.6 |
|
BWG Weeks 1-52 (Days 1-364) |
2.6 ± 2.0 |
0.7 ± 1.0 |
1.0 ± 1.9 |
2.7 ± 1.1 |
|
Final BW (Day 364) (%C) |
12.7 ± 2.2 |
10.9 ± 1.3 (86%) |
11.5 ± 2.1 (91%) |
13.1 ± 1.7 (103%) |
|
Females |
Initial BW (Day 1) (%C) |
8.3 ± 1.4 |
8.3 ± 1.3 (99%) |
7.9 ± 1.0 (95%) |
7.7 ± 0.8 (92%) |
BWG Week 1 (Days 1-8) |
0.0 ± 0.1 |
-0.2 ± 0.2 |
0.1 ± 0.1 |
-0.1 ± 0.1 |
|
BWG Weeks 1-13 (Days 1-92) |
0.6 ± 0.6 |
0.8 ± 0.3 |
1.2 ± 0.4 |
0.9 ± 0.4 |
|
BWG Weeks 13-26 (Days 92-183) |
0.2 ± 0.2 |
0.7 ± 0.5 |
0.6 ± 0.6 |
0.3 ± 0.4 |
|
BWG Weeks 26-52 (Days 183-364) |
1.0 ± 0.4 |
0.3 ± 0.1 |
0.6 ± 1.0 |
0.6 ± 0.4 |
|
BWG Weeks 1-52 (Days 1-364) |
1.8 ± 1.1 |
1.8 ± 0.9 |
2.4 ± 1.7 |
1.8 ± 0.4 |
|
Final BW (Day 364) (%C) |
10.1 ± 2.3 |
10.1 ± 0.9 (100%) |
10.3 ± 2.5 (102%) |
9.5 ± 1.0 (94%) |
|
BW: body weight; BWG: body weight gain; %C: % versus control, calculated on raw data
Table 2: Mean food consumption (g/day)
|
Dose level (ppm) |
||||
Control |
100 |
300 |
800 |
||
Males |
Week 1 (days 1-8) (%C) |
767 |
739 (96%) |
779 (102%) |
750 (98%) |
Week 1-52 (days 1-364) (%C) |
767 |
744 (97%) |
793 (103%) |
791 (103%) |
|
Females |
Week 1 (days 1-8) (%C) |
673 |
636 (94%) |
614 (91%) |
539 (80%) |
Week 1-52 (days 1-364) (%C) |
719 |
680 (95%) |
737 (102%) |
643 (89%) |
|
%C: % versus control
Table 3: Compound intake: The mean dosage intake of AE1887196 per group for each sex was as follows:
|
Mean achieved dietary intake of AE 1887196 [mg/kg bw/d] |
|||
Dosage level of AE1887196 |
100 |
300 |
800 |
|
Males |
Week 1 to 13 |
3.0 |
9.3 |
24.4 |
Week 1-52 |
2.8 |
8.8 |
21.9 |
|
Females |
Week 1-13 |
3.2 |
10.4 |
25.3 |
Week 1-52 |
3.0 |
9.7 |
24.3 |
|
Table 4: Organ weights
INCIDENCE AND SEVERITY OF MICROSCOPIC CHANGES IN THE LIVER |
||||||||
Sex |
Males |
Females |
||||||
Dosage level [ppm] |
Control |
100 |
300 |
800 |
Control |
100 |
300 |
800 |
Absolute liver weight (g) |
383.78 ± 77.213 |
336.02 ±45.458 (-12%) |
411.02 ±53.465 (+7%)
|
429.85 ±74.562 (+12%) |
304.92 ±62.100
|
329.29 ±53.712 (+8%)
|
330.30 ±68.990 (+8%) |
363.19 ±44.998 (+19%)
|
Liver to body weight ratio (%) |
3.19 ±1.171 |
3.18 ±0.872 (0%) |
3.73 ±0.706 (+17%)
|
3.30 ±0.474 (+3%) |
3.09 ±0.385
|
3.27 ±0.401 (+6%) |
3.29 ±0.312 (+6%)
|
3.92 ±0.598 (+27%) |
Liver to brain weight ratio (%) |
473.58 ±91.697 |
443.84 ±72.354 (-6%) |
534.07 ±48.239 (+13%) |
561.60 ±96.386 (+19%) |
427.87 ±97.954 |
455.67 ±67.570 (+6%)
|
423.53 ±85.174 (-1%) |
505.23 ±44.866 (+18%) |
Table 6: Microscopic changes in the liver
INCIDENCE AND SEVERITY OF MICROSCOPIC CHANGES IN THE LIVER |
||||||||
Sex |
Males |
Females |
||||||
Dosage level [ppm] |
Control |
100 |
300 |
800 |
Control |
100 |
300 |
800 |
Number of examined animals |
4 |
4 |
4 |
4 |
4 |
4 |
4 |
4 |
Hepatocellular hypertrophy: centrilobular to panlobular |
||||||||
Minimal |
0 |
0 |
0 |
3 |
0 |
0 |
3 |
1 |
Slight |
0 |
0 |
0 |
1 |
0 |
0 |
0 |
3 |
Total |
0 |
0 |
0 |
4 |
0 |
0 |
3 |
4 |
Hepatocellular glycogen accumulation: Mainly centrilobular |
||||||||
Minimal |
0 |
3 |
1 |
2 |
0 |
0 |
2 |
2 |
Slight |
1 |
0 |
0 |
0 |
2 |
4 |
2 |
0 |
Moderate |
2 |
1 |
0 |
0 |
1 |
0 |
0 |
0 |
Marked |
0 |
0 |
0 |
0 |
1 |
0 |
0 |
0 |
Total |
3 |
4 |
1 |
2 |
4 |
4 |
4 |
2 |
Table 7: Microscopic changes in the thyroid galnd
INCIDENCE AND SEVERITY OF MICROSCOPIC CHANGES IN THE THYROID |
||||||||
Sex |
Males |
Females |
||||||
Dosage level [ppm] |
Control |
100 |
300 |
800 |
Control |
100 |
300 |
800 |
Number of examined animals |
4 |
4 |
4 |
4 |
4 |
4 |
4 |
4 |
Follicular cell hypertrophy |
||||||||
Minimal |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
Total |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEL
- 2.8 mg/kg bw/day
- Study duration:
- chronic
- Species:
- dog
- Quality of whole database:
- The available information comprises adequate, reliable (Klimisch score 1-2) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.
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
- Endpoint:
- short-term repeated dose toxicity: dermal
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 30 May - 29 Jun 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.3200 (Repeated Dose Dermal Toxicity -21/28 Days)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- ORSZÄGOS GYÖGYSZERESZETI INTEZET, National Institue of Phyrmacy, Budapest, Hungary
- Species:
- rat
- Strain:
- other: Crl:Wistar rats
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany
- Age at study initiation: approx. 9 weeks
- Weight at study initiation: 302-341 g (males), 195-236 g (females)
- Housing: 5 animals of the same sex per cage in type II and/or III polycarbonate cages; individual caging was employed after the onset of treatment to avoid ingestion of the jackets/bandage/test item between the animals housed in the same cage; deep wood sawdust bedding was used (Lignocel® Hygienic Animal Bedding, J. Rettenmaier & Söhne GmbH+Co.KG, Germany).
- Diet: ssniff® SM R/M “Autoclavable Complete diet for Rats and Mice – Breeding and Maintenance” (Ssniff Spezialdiäten GmbH, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 16 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6–24.3
- Humidity (%): 30-65
- Air changes (per hr): 15-20
- Photoperiod (hrs dark/hrs light): 12/12
IN-LIFE DATES: From: 2012-05-31 To: 2012-06-29 - Type of coverage:
- semiocclusive
- Vehicle:
- other: unchanged (the test substance was moistened sufficiently with water (no more than 300 µL))
- Details on exposure:
- TEST SITE
- Area of exposure: clipped flank (area starting approximately at the scapulae (shoulders) to the wing of the ileum (hipbone) and half way down the flank on each side of the animal
- % coverage: 10% of body surface area
- Type of wrap if used: test substance was held in contact with the skin with a porous gauze dressing (less than or equal to 8 plies); the test site was further covered with non-irritating tape to retain the gauze dressing
- Time intervals for shavings or clipplings: approximately 24 hours before the test (repeated as required and as practical depending upon hair growth of individual animals and/or the effects observed)
REMOVAL OF TEST SUBSTANCE
- Washing (if done): test substance was gently wiped from the skin with lukewarm water
- Time after start of exposure: 6 hours
USE OF RESTRAINERS FOR PREVENTING INGESTION: yes (Lomir jackets with inserts and collars as required), but no complete immobilization was used - Duration of treatment / exposure:
- 28 days
- Frequency of treatment:
- daily (7 days/week)
- Remarks:
- Doses / Concentrations:
250, 500 and 1000 mg/kg bw/day
Basis: - No. of animals per sex per dose:
- 10
- Control animals:
- other: control animals were similarly treated with gauze patches and restrainers (Lomir jackets with inserts and collars).
- Details on study design:
- - Route selection rationale: The dermal route was selected to provide information for hazard assessment as skin contact is one of the possible routes of human exposure.
- Dose selection rationale: Based the absence of signs of toxicity following a single dermal application of 2000 mg/kg bw for 24 hours to the shaved flank of male and female Wistar rats, the applied concentrations were selected.
- Animal assignment: according to body weight - Observations and examinations performed and frequency:
- DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: inspection for morbidity and mortality: twice daily, general clinical observation: at least daily, detailed examinations performed outside the home cage: prior to first exposure and and once weekly thereafter
DERMAL IRRITATION: Yes
- Time schedule for examinations: daily, following patch removal
BODY WEIGHT: Yes
- Time schedule for examinations: weekly
FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to treatment and during the last week of exposure (Day 26)
- Dose groups that were examined: prior to treatment: all animals, Day 26: control and high-dose groups (as no effects were observed in the high-dose group, the low- and mid-dose groups were not further examined)
HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to necropsy on Day 28
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: red blood cell count (RBC), haemoglobin concentration (Hgb), haematocrit (Het), mean corpuscular (erythrocyte)
volume (MCV), mean corpuscular (erythrocyte) haemoglobin (MCH), red cell (erythrocyte) volume (RDW), platelet (thrombocyte) count (Plt), mean platelet (thrombocyte) volume (MPV), reticulocyte count (RETIC), white blood cell (leukocyte) count (WBC); coagulation: activated partial thromboplastin time (APTT), prothrombin time (PT)
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to necropsy on Day 28
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: glucose, total bilirubin concentration (T-BIL), urea, cholesterol (Chol), triglycerides (TRIG), creatinine concentration (CREAT), phosphorus concentration (Phos), sodium, potassium, calcium and chloride concentrations, total protein concentration (Tot Prot), albumin (Alb), Alb/glob ratio (A/G), aspartate aminotransferase activity (U/L), alanine aminotransferase activity (U/L), alkaline phosphatase activity (ALKP), gamma glutamyltransferase activity (GGT), bile acids
URINALYSIS: Yes
- Time schedule for collection of urine: prior to necropsy on Day 28
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: leukocyte (LEU), nitrite (NIT), pH, protein (Pro), glucose (GLU), urobilinogen (UBG), bilirubin (BIL), ketones (KET), blood/erythrocytes (BLD/ERY), specific gravity (SG), sediment (SED), volume (VOL)
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during the last week of exposure (on Day 26)
- Dose groups that were examined: all dose groups
- Battery of functions tested: functional observation battery / grip strength / motor activity / other: measurements of landing foot splay, sensory reactivity
OTHER:
Examination of vaginal smears prior to necropsy - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
Cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed macroscopically.
Organs checked: brain, epididymides, heart, kidney, liver, spleen, prostate incl. seminal vesicles with coagulating glands, testes, thymus, uterus incl. cervix, adrenals, ovaries, pituitary and thyroids with parathyroids
HISTOPATHOLOGY: Yes (full histopathology was performed in the control and high-dose group and in all organs with macroscopic abnormalities.
Organs checked: gross findings, larynx, skeletal muscle (quadriceps), treated skin area, liver (all dose groups), skin and subcutis (inguinal), adrenals, lungs with bronchi, small intestine, lymph nodes, spinal cord, aorta, mammary gland, spleen, brain, nose/nasal cavity, sternum with marrow, epididymides, ovaries with oviduct, stomach, eyes with the optic nerves, pharynx, testes, external lachrymal glands, pancreas, thymus, oesophagus, pituitary, thyroid with parathyroids, femur with marrow, prostate, tongue, harderian glands, salivary glands, trachea, heart, sciatic nerve, urinary bladder, kidneys, seminal vesicles with coagulating glands, uterus, large intestine, vagina - Statistics:
- Mean and standard deviations were calculated and statistical analysis was performed using SPSS PC+4.0. The heterogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan’s Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. If the data was not normal distributed, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using Mann-Whitney U-test.
- Clinical signs:
- no effects observed
- Dermal irritation:
- effects observed, treatment-related
- Description (incidence and severity):
- all dose groups: slight skin lesions (scab and scar) were observed due to friction with the restraint (non-adverse)
- Mortality:
- no mortality observed
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- 500 mg/kg bw/day: transient statistically increased body weight in females during Week 3 (non-adverse)
- Food consumption and compound intake (if feeding study):
- no effects observed
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- no effects observed
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- males: all dose groups: lower PTT values; 500 mg/kg bw/day: lower WBC counts and higher platelet counts, 1000 mg/kg bw/day: higher platelet counts (non-adverse); females: 250 mg/kg bw/day: lymphocyte count statistically different (non-adverse)
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- 1000 mg/kg bw/day: increased cholesterol in males and females: all dose groups: increased creatinine (non-adverse)
- Urinalysis findings:
- effects observed, treatment-related
- Description (incidence and severity):
- males, all dose group: slight differences in urine specific gravity (non-adverse)
- Behaviour (functional findings):
- effects observed, treatment-related
- Description (incidence and severity):
- grip strength test: increased values for males from all dose groups and high-dose females (non-adverse)
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- 1000 mg/kg bw/day: increased relative liver weight in males and females, increased weight of thyroid glands in females (non-adverse)
- Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- all dose groups: incidental gross abnormalities (non-adverse)
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- 1000 mg/kg bw/day: minimal centrilobular hepatocellular hypertrophy and minimal/mild diffuse hepatocellular vacuolation in males and females
- Histopathological findings: neoplastic:
- no effects observed
- Details on results:
- CLINICAL SIGNS AND MORTALITY
No mortality or clinical signs of toxicity were observed during the study period. No signs of skin irritation were noted. Occasionally slight skin lesions (scab and scar) were observed with similar low incidence across the groups in males (2/10, 1/10, 1/10 and 0/10) and females (3/10, 0/10, 1/10 and 0/10) in the control, low, mid and high dose groups, respectively. These observations were ascribed to minor effects of friction with the restraint and not related to the test item.
BODY WEIGHT AND WEIGHT GAIN
No treatment-related effect on body weight and weight gain was noted. Due to the transient occurence, the statistically significant higher body weight gain in mid-dose females only during Week 3 (p < 0.05) is not considered as toxicologically significant or related to treatment (Table 1).
FOOD CONSUMPTION
Food consumption was comparable among the groups.
OPHTHALMOSCOPIC EXAMINATION
Opthalmology did not reveal differences between control and test groups.
HAEMATOLOGY
Variations, attaining statistical significance were noted in males in lower WBC counts in the mid-dose group (p < 0.05) and higher platelet counts in the mid- and high-dose groups (p < 0.05 and p < 0.01, respectively). Lower PTT values were noted in all treated males, due to relatively high variations in the control individual values. Furthermore, statistical difference (p < 0.05) was noted for relative lymphocyte count in low-dose females (Table 2). As all parameters fall within the normal physiological and historical control ranges for the age, strain and sex of rats, these effects are not considered as adverse.
CLINICAL CHEMISTRY
High-dose males and females revealed a slight but statistically significant increase in cholesterol concentration around 23-24% , respectively. In mid-dose females, 2/10 animals reached cholesterol values above the control range. As both values still remained within the physiological range, the effect in the mid-dose group is not considered as adverse. Creatinine levels were slightly increased in all dose groups. In the high-dose group, no more than 17-19% difference was noted without any effect on urea concentration in males. As the observed changes in creatinine were of small magnitude, the mean and individual values remained within normal ranges and no consequent effect on urea concentration was observed, the effect on creatinine is not considered as adverse (Table 3). Other minor variations noted in chloride concentration, total protein or urea achieved statistical significance occasionally (Table 2). Due to the absence of consistent patterns or other correlates, these changes were regarded as incidental or individual findings, and are therefore not considered as treatment-related or of toxicological significance.
URINALYSIS
Slight differences were observed in urine specific gravity in males from all dose groups, which attained statistical significance in low- and high-dose males (p < 0.05 and p < 0.01, respectively). As no dose-dependency was present, and due to the lack of any evident renal pathology, these observations were regarded as incidental findings.
No abnormalities were observed in the remaining evaluated parameters.
NEUROBEHAVIOUR
Increased values were noted in grip strength tests for hind limb in all treated male groups and in females of the high-dose group compared to the controls. The differences reached statistical significance in mid-dose males and high-dose females (p < 0.05), but were considered to be without toxicological significance. Increased vocalization was observed on occasion throughout all the dose groups at the modified Irwin test (functional observation battery). However, as there were no treatment-related differences to the control, and the incidence was similar in all groups and no dose-, or gender-related responses were noted, these variations were considered to be without toxicological significance and within the normal biological variation with respect to behaviour, reactions to different type of stimuli or manipulations. Thus, no toxicologically significant changes in behaviour, general physical condition, or neurobehaviour (including the reactions to different type of stimuli, grip strength or motor activity) were noted on Day 26.
ORGAN WEIGHTS
Test item related organ weight differences were found in liver (males and females) and thyroid (females) weights in the high-dose group. As the increased liver weights in low- and mid-dose animals were of low magnitude (3-10%) without any morphological change or adverse effect in clinical pathology, they were considered as adaptive (Table 1). Thyroid weights were dose-dependently increased in females of the mid-and high-dose group. The increase in the mid-dose group was of low magnitude, with almost all individual weights lying in the control range. Compared to control, the thyroid weights in the high-dose females were significantly elevated by approximately 22% (absolute value) and 19-21% (relative values). 4 of 10 individual values were above the control, but within the physiological range. As the changes were not associated with any morphological change suggesting increased TSH activity or utilization of thyroglobulins, the observations on the thyroid weights were not considered adverse. Absolute kidney weights were slightly increased in all treatment groups of both sexes by approximately 4-9%. Differences were statistically significant in high-dose males. However, as the differences remained minor and were within the physiological range, they were not considered as adverse. No biologically significant differences were observed among the groups in the weights of the remaining evaluated organs compared with the control, although statistically significant variations without trend were noted (i.e thymus). Therefore, in the absence of a dose- or gender-related response, or consistent correlation with histopathological results, these variations were not considered to reflect an adverse effect.
GROSS PATHOLOGY
Multifocal/focal dark red discoloration of the thymus was observed in 1/10 control female and 2/10 high-dose males which was regarded as terminal procedure related. Furthermore, multifocal/focal dark red/brown discolouration of glandular mucosa of the stomach (1/10 mid-dose female), enlarged adrenal (1/10 mid-dose female), unilateral enlargement of the testes (1/10 mid-dose male) were observed. Due to the low incidence and missing dose-response, these findings are regarded as incidental. Unilateral pelvic dilatation of the kidney (2/10 high-dose males) or dilated uterine horns (1/10 control and 2/10 high-dose females) were observed in single animals. As these variations represent common observations in Wistar rats, they are not interpreted as incidental and not adverse due to the incidence. In addition, at external examination, small scars, or scabs on the dorsal neck, thoracic region and on forelimbs were observed with low incidence in control and mid-dose males which were regarded as procedure related.
HISTOPATHOLOGY: NON-NEOPLASTIC
Test item-related findings were observed in the liver of high-dose males and females visible as minimal/mild, periportal/diffuse hepatocellular vacuolation which was found in 3/10 males and females. Minimal centrilobular hepatocellular hypertrophy was observed in 3/10 males and in 2/10 females. As the changes were correlated with increased organ weights, they are considered as treatment-related. No similar observations were made in animals of the lower dose groups. Examination of the skin revealed signs of minimal, multifocal hyperkeratosis/parakeratosis which was observed in 7/10 and 3/10 high-dose males and females, respectively, in 1/10 mid-dose males and in 3/10 control females (skin in the low- and mid-dose animals were not examined, except necropsy findings). In addition, focal purulent inflammation and mixed cellular infiltrate in the skin were observed in 3/10 control females. As these abnormalities occured also in the control group, the changes were considered to be procedure- and not test item-related. Changes observed in various organs including the prostate (only in the control), kidney (1/10 control and 1/10 high-dose male and 1/10 control female), testis (1/10 control and 1/10 mid-dose male, but not in the high-dose group) or thymus (2/10 control and 2/10 high-dose males and 1/10 control female) were regarded as incidental because of the randomly distribution across control and treated groups. Microscopic findings in the uterus (like luminal dilatation and glandular proliferation) seen in 1/10 control and 2/10 high-dose females were related to typical physiological changes observed during oestrus cycle in female rats and are therefore not toxicologically significant and/or associated with test item administration.
OTHER FINDINGS
Oestrus evaluation: All animals showed normal distribution of oestrus phases. - Dose descriptor:
- NOAEL
- Effect level:
- 500 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: based on adverse effects observed on the liver (minimal/mild, periportal/diffuse hepatocellular vacuolation and minimal centrilobular hepatocellular hypertrophy correlated with increased liver weight and increased cholesterol in both sexes)
- Critical effects observed:
- not specified
Reference
Table 1: Body weight [g]
Sex |
Dose group (mg/kg bw/day) |
Treatment day |
||||
0 |
7 |
14 |
21 |
27 |
||
Male |
Control |
319.3 ± 11.5 |
357.8 ± 17.2 |
383.6 ± 20.5 |
414.3 ± 23.4 |
423.1 ± 24.8 |
|
250 |
320.1 ± 10.0 |
361.9 ± 14.0 |
389.8 ± 19.3 |
422.3 ± 23.1 |
433.2 ± 23.6 |
|
500 |
318.0 ± 8.4 |
355.7 ± 9.6 |
381.1 ± 15.1 |
411.1 ± 14.3 |
419.5 ± 17.1 |
|
1000 |
319.9 ± 12.8 |
361.0 ± 15.5 |
389.7 ± 19.2 |
420.0 ± 28.0 |
427.6 ± 29.7 |
Female |
Control |
214.5 ± 11.9 |
228.3 ± 17.7 |
236.8 ± 20.5 |
243.7 ± 17.5 |
246.1 ± 20.7 |
|
250 |
215.0 ± 10.8 |
232.0 ± 14.2 |
244.9 ± 23.9 |
253.4 ± 24.1 |
259.0 ± 26.4 |
|
500 |
216.2 ± 10.7 |
228.1 ± 13.6 |
238.4 ± 14.9 |
255.5 ± 20.7 |
254.8 ± 28.3 |
|
1000 |
210.2 ± 10.8 |
227.0 ± 13.1 |
241.5 ± 11.6 |
253.2 ± 11.7 |
252.2 ± 10.9 |
Table 2: Haematology and Clinical Chemistry: mean values of altered parameters on Day 28.
Sex |
Dose group (mg/kg bw/day) |
Haematology |
Clinical Chemistry |
Urinalysis |
|||||
WBC [K/µL] |
PLT [K/µL] |
PTT [seconds] |
LYMPH [%] |
Chloride [mmol/L] |
Tot. Prot. [g/L] |
Urea [mmol/L] |
Specific Gravity |
||
Male |
Control |
4.533 ± 1.333 |
1062.5 ± 94.2 |
22.01 ± 1.19 |
66.130 ± 6.815 |
103.51 ± 1.20 |
54.78 ± 1.82 |
5.749 ± 0.868 |
1.0091 ± 0.0007 |
250 |
5.271 ± 2.260 |
1035.8 ± 188.4 |
21.18* ± 0.47 |
67.140 ± 3.940 |
101.71** ± 0.94 (DN) |
54.96 ± 1.71 |
5.323 ± 0.474 |
1.0107* ± 0.0021 |
|
500 |
3.370* ± 0.796 (U) |
1157.1* ± 65.6 (U) |
20.97* ± 0.93 (DN) |
65.150 ± 7.770 |
102.89 ± 1.20 |
56.23 ± 1.47 |
6.040 ± 0.599 |
1.0109 ± 0.0028 |
|
1000 |
4.982 ± 1.786 |
1217.9** ± 112.8 (U) |
21.26 ± 0.69 |
71.380 ± 4.818 |
103.29 ± 1.95 |
56.72* ± 2.21 (DN) |
5.522 ± |
1.0125** ± 0.0030 |
|
Female |
Control |
1.818 ± 1.003 |
1144.4 ± 209.4 |
23.03 ± 0.78 |
65.200 ± 10.071 |
103.47 ± 1.41 |
58.01 ± 2.28 |
6.748 ± 0.834 |
1.0143 ± 0.0075 |
250 |
2.517 ± 1.131 |
1119.4 ± 138.3 |
23.08 ± 0.95 |
74.250* ± 6.945 (DN) |
102.83 ± 1.72 |
56.84 ± 1.29 |
5.957 ± 0.985 |
1.0115 ± 0.0037 |
|
500 |
2.181 ± 0.845 |
1153.6 ± 171.4 |
22.99 ± 1.30 |
72.770 ± 9.983 |
102.51 ± 1.36 |
59.25 ± 3.40 |
6.124 ± 1.116 |
1.0146 ± 0.0045 |
|
1000 |
1.614 ± 0.685 |
1072.0 ± 102.0 |
22.20 ± 0.77 |
68.940 ± 6.428 |
102.09 ± 1.68 |
60.33 ± 2.77 |
5.240** ± 0.482 (DN) |
1.0146 ± 0.0056 |
|
*: p < 0.05, **: p < 0.01
DN = Duncan's Multiple Range Test; U = Mann- Whitney U-Test
Table 3: Mean values of cholesterol and creatinine levels on Day 28.
|
Dose group (mg/kg bw/day) |
||||
Control |
250 |
500 |
1000 |
||
Males |
Cholesterol (mmol/L) |
1.64 |
1.56 |
1.75 |
2.04** (DN) |
Cholesterol (% difference) |
|
-5 |
7 |
24 |
|
Creatinine (µmol/L) |
53.3 |
57.7 |
60.1 |
63.2* (DN) |
|
Creatinine (% difference) |
|
8 |
13 |
19 |
|
Females |
Cholesterol (mmol/L) |
1.84 |
1.80 |
2.13 |
2.26** (U) |
Cholesterol (% difference) |
|
-2 |
16 |
23 |
|
Creatinine (µmol/L) |
48.7 |
53.0 |
53.5 |
56.9 |
|
Creatinine (% difference) |
|
9 |
10 |
17 |
|
*: p < 0.05, **: p < 0.01
DN = Duncan's Multiple Range Test; U = Mann- Whitney U-Test
Table 4: Mean weights of liver of males and females on Day 28.
|
Dose group (mg/kg bw/day) |
||||
Control |
250 |
500 |
1000 |
||
Males |
Liver weight (absolute [g]) |
10.8 |
12.0* |
11.4 |
12.2** (DN) |
Difference [%] |
|
11 |
6 |
13 |
|
Body weight relative [%] |
2.7 |
2.9** |
2.9* |
3.0** (DN) |
|
Difference [%] |
|
7 |
7 |
11 |
|
Brain relative [%] |
526 |
578* |
556 |
598** (DN) |
|
Difference [%] |
|
10 |
6 |
14 |
|
Females |
Liver weight (absolute [g]) |
6.64 |
7.32 |
7.66* |
7.87** (DN) |
Difference [%] |
|
10 |
15 |
19 |
|
Body weight relative [%] |
2.9 |
3.0 |
3.2** |
3.3** (DN) |
|
Difference [%] |
|
3 |
10 |
14 |
|
|
Brain relative [%] |
364 |
384 |
414* |
429** |
|
Difference [%] |
|
5 |
14 |
18 |
* = p < 0.05, ** = p < 0.01
DN = Duncan's Multiple Range Test
Table 5: Mean weights of kidneys and thyroids of males and females on Day 28.
Dose group (mg/kg bw/day) |
|||||
Control |
250 |
500 |
1000 |
||
Males |
Kidney weight (absolute [g]) |
2.66 |
2.83 |
2.76 |
2.87* (DN) |
Difference [%] |
|
6 |
4 |
8 |
|
Thyroid weight (absolute [g]) |
21.2 |
21.6 |
19.6 |
20.8 |
|
Difference [%] |
|
2 |
-8 |
-2 |
|
Females |
Kidney weight (absolute [g]) |
1.64 |
1.78 |
1.76 |
1.73 |
Difference [%] |
|
9 |
7 |
6 |
|
Thyroid weight (absolute [g]) |
16.7 |
17.3 |
18.9 |
20.3** (DN) |
|
Difference [%] |
|
4 |
13 |
22 |
|
|
Body weight relative [%] |
7.19 |
7.17 |
7.91 |
8.55** (DN) |
|
Difference [%] |
|
0 |
10 |
19 |
|
Brain relative [%] |
0.92 |
0.91 |
1.02 |
1.11** (DN) |
|
Difference [%] |
|
-1 |
11 |
21 |
* = p < 0.05, ** = p < 0.01
DN = Duncan's Multiple Range Test
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEL
- 500 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rat
- Quality of whole database:
- The available information comprises an adequate and reliable study (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.
Repeated dose toxicity: dermal - local effects
Link to relevant study records
- Endpoint:
- short-term repeated dose toxicity: dermal
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 30 May - 29 Jun 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.3200 (Repeated Dose Dermal Toxicity -21/28 Days)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- ORSZÄGOS GYÖGYSZERESZETI INTEZET, National Institue of Phyrmacy, Budapest, Hungary
- Species:
- rat
- Strain:
- other: Crl:Wistar rats
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany
- Age at study initiation: approx. 9 weeks
- Weight at study initiation: 302-341 g (males), 195-236 g (females)
- Housing: 5 animals of the same sex per cage in type II and/or III polycarbonate cages; individual caging was employed after the onset of treatment to avoid ingestion of the jackets/bandage/test item between the animals housed in the same cage; deep wood sawdust bedding was used (Lignocel® Hygienic Animal Bedding, J. Rettenmaier & Söhne GmbH+Co.KG, Germany).
- Diet: ssniff® SM R/M “Autoclavable Complete diet for Rats and Mice – Breeding and Maintenance” (Ssniff Spezialdiäten GmbH, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 16 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6–24.3
- Humidity (%): 30-65
- Air changes (per hr): 15-20
- Photoperiod (hrs dark/hrs light): 12/12
IN-LIFE DATES: From: 2012-05-31 To: 2012-06-29 - Type of coverage:
- semiocclusive
- Vehicle:
- other: unchanged (the test substance was moistened sufficiently with water (no more than 300 µL))
- Details on exposure:
- TEST SITE
- Area of exposure: clipped flank (area starting approximately at the scapulae (shoulders) to the wing of the ileum (hipbone) and half way down the flank on each side of the animal
- % coverage: 10% of body surface area
- Type of wrap if used: test substance was held in contact with the skin with a porous gauze dressing (less than or equal to 8 plies); the test site was further covered with non-irritating tape to retain the gauze dressing
- Time intervals for shavings or clipplings: approximately 24 hours before the test (repeated as required and as practical depending upon hair growth of individual animals and/or the effects observed)
REMOVAL OF TEST SUBSTANCE
- Washing (if done): test substance was gently wiped from the skin with lukewarm water
- Time after start of exposure: 6 hours
USE OF RESTRAINERS FOR PREVENTING INGESTION: yes (Lomir jackets with inserts and collars as required), but no complete immobilization was used - Duration of treatment / exposure:
- 28 days
- Frequency of treatment:
- daily (7 days/week)
- Remarks:
- Doses / Concentrations:
250, 500 and 1000 mg/kg bw/day
Basis: - No. of animals per sex per dose:
- 10
- Control animals:
- other: control animals were similarly treated with gauze patches and restrainers (Lomir jackets with inserts and collars).
- Details on study design:
- - Route selection rationale: The dermal route was selected to provide information for hazard assessment as skin contact is one of the possible routes of human exposure.
- Dose selection rationale: Based the absence of signs of toxicity following a single dermal application of 2000 mg/kg bw for 24 hours to the shaved flank of male and female Wistar rats, the applied concentrations were selected.
- Animal assignment: according to body weight - Observations and examinations performed and frequency:
- DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: inspection for morbidity and mortality: twice daily, general clinical observation: at least daily, detailed examinations performed outside the home cage: prior to first exposure and and once weekly thereafter
DERMAL IRRITATION: Yes
- Time schedule for examinations: daily, following patch removal
BODY WEIGHT: Yes
- Time schedule for examinations: weekly
FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to treatment and during the last week of exposure (Day 26)
- Dose groups that were examined: prior to treatment: all animals, Day 26: control and high-dose groups (as no effects were observed in the high-dose group, the low- and mid-dose groups were not further examined)
HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to necropsy on Day 28
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: red blood cell count (RBC), haemoglobin concentration (Hgb), haematocrit (Het), mean corpuscular (erythrocyte)
volume (MCV), mean corpuscular (erythrocyte) haemoglobin (MCH), red cell (erythrocyte) volume (RDW), platelet (thrombocyte) count (Plt), mean platelet (thrombocyte) volume (MPV), reticulocyte count (RETIC), white blood cell (leukocyte) count (WBC); coagulation: activated partial thromboplastin time (APTT), prothrombin time (PT)
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to necropsy on Day 28
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: glucose, total bilirubin concentration (T-BIL), urea, cholesterol (Chol), triglycerides (TRIG), creatinine concentration (CREAT), phosphorus concentration (Phos), sodium, potassium, calcium and chloride concentrations, total protein concentration (Tot Prot), albumin (Alb), Alb/glob ratio (A/G), aspartate aminotransferase activity (U/L), alanine aminotransferase activity (U/L), alkaline phosphatase activity (ALKP), gamma glutamyltransferase activity (GGT), bile acids
URINALYSIS: Yes
- Time schedule for collection of urine: prior to necropsy on Day 28
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: leukocyte (LEU), nitrite (NIT), pH, protein (Pro), glucose (GLU), urobilinogen (UBG), bilirubin (BIL), ketones (KET), blood/erythrocytes (BLD/ERY), specific gravity (SG), sediment (SED), volume (VOL)
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during the last week of exposure (on Day 26)
- Dose groups that were examined: all dose groups
- Battery of functions tested: functional observation battery / grip strength / motor activity / other: measurements of landing foot splay, sensory reactivity
OTHER:
Examination of vaginal smears prior to necropsy - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
Cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed macroscopically.
Organs checked: brain, epididymides, heart, kidney, liver, spleen, prostate incl. seminal vesicles with coagulating glands, testes, thymus, uterus incl. cervix, adrenals, ovaries, pituitary and thyroids with parathyroids
HISTOPATHOLOGY: Yes (full histopathology was performed in the control and high-dose group and in all organs with macroscopic abnormalities.
Organs checked: gross findings, larynx, skeletal muscle (quadriceps), treated skin area, liver (all dose groups), skin and subcutis (inguinal), adrenals, lungs with bronchi, small intestine, lymph nodes, spinal cord, aorta, mammary gland, spleen, brain, nose/nasal cavity, sternum with marrow, epididymides, ovaries with oviduct, stomach, eyes with the optic nerves, pharynx, testes, external lachrymal glands, pancreas, thymus, oesophagus, pituitary, thyroid with parathyroids, femur with marrow, prostate, tongue, harderian glands, salivary glands, trachea, heart, sciatic nerve, urinary bladder, kidneys, seminal vesicles with coagulating glands, uterus, large intestine, vagina - Statistics:
- Mean and standard deviations were calculated and statistical analysis was performed using SPSS PC+4.0. The heterogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan’s Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. If the data was not normal distributed, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using Mann-Whitney U-test.
- Clinical signs:
- no effects observed
- Dermal irritation:
- effects observed, treatment-related
- Description (incidence and severity):
- all dose groups: slight skin lesions (scab and scar) were observed due to friction with the restraint (non-adverse)
- Mortality:
- no mortality observed
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- 500 mg/kg bw/day: transient statistically increased body weight in females during Week 3 (non-adverse)
- Food consumption and compound intake (if feeding study):
- no effects observed
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- no effects observed
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- males: all dose groups: lower PTT values; 500 mg/kg bw/day: lower WBC counts and higher platelet counts, 1000 mg/kg bw/day: higher platelet counts (non-adverse); females: 250 mg/kg bw/day: lymphocyte count statistically different (non-adverse)
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- 1000 mg/kg bw/day: increased cholesterol in males and females: all dose groups: increased creatinine (non-adverse)
- Urinalysis findings:
- effects observed, treatment-related
- Description (incidence and severity):
- males, all dose group: slight differences in urine specific gravity (non-adverse)
- Behaviour (functional findings):
- effects observed, treatment-related
- Description (incidence and severity):
- grip strength test: increased values for males from all dose groups and high-dose females (non-adverse)
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- 1000 mg/kg bw/day: increased relative liver weight in males and females, increased weight of thyroid glands in females (non-adverse)
- Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- all dose groups: incidental gross abnormalities (non-adverse)
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- 1000 mg/kg bw/day: minimal centrilobular hepatocellular hypertrophy and minimal/mild diffuse hepatocellular vacuolation in males and females
- Histopathological findings: neoplastic:
- no effects observed
- Details on results:
- CLINICAL SIGNS AND MORTALITY
No mortality or clinical signs of toxicity were observed during the study period. No signs of skin irritation were noted. Occasionally slight skin lesions (scab and scar) were observed with similar low incidence across the groups in males (2/10, 1/10, 1/10 and 0/10) and females (3/10, 0/10, 1/10 and 0/10) in the control, low, mid and high dose groups, respectively. These observations were ascribed to minor effects of friction with the restraint and not related to the test item.
BODY WEIGHT AND WEIGHT GAIN
No treatment-related effect on body weight and weight gain was noted. Due to the transient occurence, the statistically significant higher body weight gain in mid-dose females only during Week 3 (p < 0.05) is not considered as toxicologically significant or related to treatment (Table 1).
FOOD CONSUMPTION
Food consumption was comparable among the groups.
OPHTHALMOSCOPIC EXAMINATION
Opthalmology did not reveal differences between control and test groups.
HAEMATOLOGY
Variations, attaining statistical significance were noted in males in lower WBC counts in the mid-dose group (p < 0.05) and higher platelet counts in the mid- and high-dose groups (p < 0.05 and p < 0.01, respectively). Lower PTT values were noted in all treated males, due to relatively high variations in the control individual values. Furthermore, statistical difference (p < 0.05) was noted for relative lymphocyte count in low-dose females (Table 2). As all parameters fall within the normal physiological and historical control ranges for the age, strain and sex of rats, these effects are not considered as adverse.
CLINICAL CHEMISTRY
High-dose males and females revealed a slight but statistically significant increase in cholesterol concentration around 23-24% , respectively. In mid-dose females, 2/10 animals reached cholesterol values above the control range. As both values still remained within the physiological range, the effect in the mid-dose group is not considered as adverse. Creatinine levels were slightly increased in all dose groups. In the high-dose group, no more than 17-19% difference was noted without any effect on urea concentration in males. As the observed changes in creatinine were of small magnitude, the mean and individual values remained within normal ranges and no consequent effect on urea concentration was observed, the effect on creatinine is not considered as adverse (Table 3). Other minor variations noted in chloride concentration, total protein or urea achieved statistical significance occasionally (Table 2). Due to the absence of consistent patterns or other correlates, these changes were regarded as incidental or individual findings, and are therefore not considered as treatment-related or of toxicological significance.
URINALYSIS
Slight differences were observed in urine specific gravity in males from all dose groups, which attained statistical significance in low- and high-dose males (p < 0.05 and p < 0.01, respectively). As no dose-dependency was present, and due to the lack of any evident renal pathology, these observations were regarded as incidental findings.
No abnormalities were observed in the remaining evaluated parameters.
NEUROBEHAVIOUR
Increased values were noted in grip strength tests for hind limb in all treated male groups and in females of the high-dose group compared to the controls. The differences reached statistical significance in mid-dose males and high-dose females (p < 0.05), but were considered to be without toxicological significance. Increased vocalization was observed on occasion throughout all the dose groups at the modified Irwin test (functional observation battery). However, as there were no treatment-related differences to the control, and the incidence was similar in all groups and no dose-, or gender-related responses were noted, these variations were considered to be without toxicological significance and within the normal biological variation with respect to behaviour, reactions to different type of stimuli or manipulations. Thus, no toxicologically significant changes in behaviour, general physical condition, or neurobehaviour (including the reactions to different type of stimuli, grip strength or motor activity) were noted on Day 26.
ORGAN WEIGHTS
Test item related organ weight differences were found in liver (males and females) and thyroid (females) weights in the high-dose group. As the increased liver weights in low- and mid-dose animals were of low magnitude (3-10%) without any morphological change or adverse effect in clinical pathology, they were considered as adaptive (Table 1). Thyroid weights were dose-dependently increased in females of the mid-and high-dose group. The increase in the mid-dose group was of low magnitude, with almost all individual weights lying in the control range. Compared to control, the thyroid weights in the high-dose females were significantly elevated by approximately 22% (absolute value) and 19-21% (relative values). 4 of 10 individual values were above the control, but within the physiological range. As the changes were not associated with any morphological change suggesting increased TSH activity or utilization of thyroglobulins, the observations on the thyroid weights were not considered adverse. Absolute kidney weights were slightly increased in all treatment groups of both sexes by approximately 4-9%. Differences were statistically significant in high-dose males. However, as the differences remained minor and were within the physiological range, they were not considered as adverse. No biologically significant differences were observed among the groups in the weights of the remaining evaluated organs compared with the control, although statistically significant variations without trend were noted (i.e thymus). Therefore, in the absence of a dose- or gender-related response, or consistent correlation with histopathological results, these variations were not considered to reflect an adverse effect.
GROSS PATHOLOGY
Multifocal/focal dark red discoloration of the thymus was observed in 1/10 control female and 2/10 high-dose males which was regarded as terminal procedure related. Furthermore, multifocal/focal dark red/brown discolouration of glandular mucosa of the stomach (1/10 mid-dose female), enlarged adrenal (1/10 mid-dose female), unilateral enlargement of the testes (1/10 mid-dose male) were observed. Due to the low incidence and missing dose-response, these findings are regarded as incidental. Unilateral pelvic dilatation of the kidney (2/10 high-dose males) or dilated uterine horns (1/10 control and 2/10 high-dose females) were observed in single animals. As these variations represent common observations in Wistar rats, they are not interpreted as incidental and not adverse due to the incidence. In addition, at external examination, small scars, or scabs on the dorsal neck, thoracic region and on forelimbs were observed with low incidence in control and mid-dose males which were regarded as procedure related.
HISTOPATHOLOGY: NON-NEOPLASTIC
Test item-related findings were observed in the liver of high-dose males and females visible as minimal/mild, periportal/diffuse hepatocellular vacuolation which was found in 3/10 males and females. Minimal centrilobular hepatocellular hypertrophy was observed in 3/10 males and in 2/10 females. As the changes were correlated with increased organ weights, they are considered as treatment-related. No similar observations were made in animals of the lower dose groups. Examination of the skin revealed signs of minimal, multifocal hyperkeratosis/parakeratosis which was observed in 7/10 and 3/10 high-dose males and females, respectively, in 1/10 mid-dose males and in 3/10 control females (skin in the low- and mid-dose animals were not examined, except necropsy findings). In addition, focal purulent inflammation and mixed cellular infiltrate in the skin were observed in 3/10 control females. As these abnormalities occured also in the control group, the changes were considered to be procedure- and not test item-related. Changes observed in various organs including the prostate (only in the control), kidney (1/10 control and 1/10 high-dose male and 1/10 control female), testis (1/10 control and 1/10 mid-dose male, but not in the high-dose group) or thymus (2/10 control and 2/10 high-dose males and 1/10 control female) were regarded as incidental because of the randomly distribution across control and treated groups. Microscopic findings in the uterus (like luminal dilatation and glandular proliferation) seen in 1/10 control and 2/10 high-dose females were related to typical physiological changes observed during oestrus cycle in female rats and are therefore not toxicologically significant and/or associated with test item administration.
OTHER FINDINGS
Oestrus evaluation: All animals showed normal distribution of oestrus phases. - Dose descriptor:
- NOAEL
- Effect level:
- 500 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: based on adverse effects observed on the liver (minimal/mild, periportal/diffuse hepatocellular vacuolation and minimal centrilobular hepatocellular hypertrophy correlated with increased liver weight and increased cholesterol in both sexes)
- Critical effects observed:
- not specified
Reference
Table 1: Body weight [g]
Sex |
Dose group (mg/kg bw/day) |
Treatment day |
||||
0 |
7 |
14 |
21 |
27 |
||
Male |
Control |
319.3 ± 11.5 |
357.8 ± 17.2 |
383.6 ± 20.5 |
414.3 ± 23.4 |
423.1 ± 24.8 |
|
250 |
320.1 ± 10.0 |
361.9 ± 14.0 |
389.8 ± 19.3 |
422.3 ± 23.1 |
433.2 ± 23.6 |
|
500 |
318.0 ± 8.4 |
355.7 ± 9.6 |
381.1 ± 15.1 |
411.1 ± 14.3 |
419.5 ± 17.1 |
|
1000 |
319.9 ± 12.8 |
361.0 ± 15.5 |
389.7 ± 19.2 |
420.0 ± 28.0 |
427.6 ± 29.7 |
Female |
Control |
214.5 ± 11.9 |
228.3 ± 17.7 |
236.8 ± 20.5 |
243.7 ± 17.5 |
246.1 ± 20.7 |
|
250 |
215.0 ± 10.8 |
232.0 ± 14.2 |
244.9 ± 23.9 |
253.4 ± 24.1 |
259.0 ± 26.4 |
|
500 |
216.2 ± 10.7 |
228.1 ± 13.6 |
238.4 ± 14.9 |
255.5 ± 20.7 |
254.8 ± 28.3 |
|
1000 |
210.2 ± 10.8 |
227.0 ± 13.1 |
241.5 ± 11.6 |
253.2 ± 11.7 |
252.2 ± 10.9 |
Table 2: Haematology and Clinical Chemistry: mean values of altered parameters on Day 28.
Sex |
Dose group (mg/kg bw/day) |
Haematology |
Clinical Chemistry |
Urinalysis |
|||||
WBC [K/µL] |
PLT [K/µL] |
PTT [seconds] |
LYMPH [%] |
Chloride [mmol/L] |
Tot. Prot. [g/L] |
Urea [mmol/L] |
Specific Gravity |
||
Male |
Control |
4.533 ± 1.333 |
1062.5 ± 94.2 |
22.01 ± 1.19 |
66.130 ± 6.815 |
103.51 ± 1.20 |
54.78 ± 1.82 |
5.749 ± 0.868 |
1.0091 ± 0.0007 |
250 |
5.271 ± 2.260 |
1035.8 ± 188.4 |
21.18* ± 0.47 |
67.140 ± 3.940 |
101.71** ± 0.94 (DN) |
54.96 ± 1.71 |
5.323 ± 0.474 |
1.0107* ± 0.0021 |
|
500 |
3.370* ± 0.796 (U) |
1157.1* ± 65.6 (U) |
20.97* ± 0.93 (DN) |
65.150 ± 7.770 |
102.89 ± 1.20 |
56.23 ± 1.47 |
6.040 ± 0.599 |
1.0109 ± 0.0028 |
|
1000 |
4.982 ± 1.786 |
1217.9** ± 112.8 (U) |
21.26 ± 0.69 |
71.380 ± 4.818 |
103.29 ± 1.95 |
56.72* ± 2.21 (DN) |
5.522 ± |
1.0125** ± 0.0030 |
|
Female |
Control |
1.818 ± 1.003 |
1144.4 ± 209.4 |
23.03 ± 0.78 |
65.200 ± 10.071 |
103.47 ± 1.41 |
58.01 ± 2.28 |
6.748 ± 0.834 |
1.0143 ± 0.0075 |
250 |
2.517 ± 1.131 |
1119.4 ± 138.3 |
23.08 ± 0.95 |
74.250* ± 6.945 (DN) |
102.83 ± 1.72 |
56.84 ± 1.29 |
5.957 ± 0.985 |
1.0115 ± 0.0037 |
|
500 |
2.181 ± 0.845 |
1153.6 ± 171.4 |
22.99 ± 1.30 |
72.770 ± 9.983 |
102.51 ± 1.36 |
59.25 ± 3.40 |
6.124 ± 1.116 |
1.0146 ± 0.0045 |
|
1000 |
1.614 ± 0.685 |
1072.0 ± 102.0 |
22.20 ± 0.77 |
68.940 ± 6.428 |
102.09 ± 1.68 |
60.33 ± 2.77 |
5.240** ± 0.482 (DN) |
1.0146 ± 0.0056 |
|
*: p < 0.05, **: p < 0.01
DN = Duncan's Multiple Range Test; U = Mann- Whitney U-Test
Table 3: Mean values of cholesterol and creatinine levels on Day 28.
|
Dose group (mg/kg bw/day) |
||||
Control |
250 |
500 |
1000 |
||
Males |
Cholesterol (mmol/L) |
1.64 |
1.56 |
1.75 |
2.04** (DN) |
Cholesterol (% difference) |
|
-5 |
7 |
24 |
|
Creatinine (µmol/L) |
53.3 |
57.7 |
60.1 |
63.2* (DN) |
|
Creatinine (% difference) |
|
8 |
13 |
19 |
|
Females |
Cholesterol (mmol/L) |
1.84 |
1.80 |
2.13 |
2.26** (U) |
Cholesterol (% difference) |
|
-2 |
16 |
23 |
|
Creatinine (µmol/L) |
48.7 |
53.0 |
53.5 |
56.9 |
|
Creatinine (% difference) |
|
9 |
10 |
17 |
|
*: p < 0.05, **: p < 0.01
DN = Duncan's Multiple Range Test; U = Mann- Whitney U-Test
Table 4: Mean weights of liver of males and females on Day 28.
|
Dose group (mg/kg bw/day) |
||||
Control |
250 |
500 |
1000 |
||
Males |
Liver weight (absolute [g]) |
10.8 |
12.0* |
11.4 |
12.2** (DN) |
Difference [%] |
|
11 |
6 |
13 |
|
Body weight relative [%] |
2.7 |
2.9** |
2.9* |
3.0** (DN) |
|
Difference [%] |
|
7 |
7 |
11 |
|
Brain relative [%] |
526 |
578* |
556 |
598** (DN) |
|
Difference [%] |
|
10 |
6 |
14 |
|
Females |
Liver weight (absolute [g]) |
6.64 |
7.32 |
7.66* |
7.87** (DN) |
Difference [%] |
|
10 |
15 |
19 |
|
Body weight relative [%] |
2.9 |
3.0 |
3.2** |
3.3** (DN) |
|
Difference [%] |
|
3 |
10 |
14 |
|
|
Brain relative [%] |
364 |
384 |
414* |
429** |
|
Difference [%] |
|
5 |
14 |
18 |
* = p < 0.05, ** = p < 0.01
DN = Duncan's Multiple Range Test
Table 5: Mean weights of kidneys and thyroids of males and females on Day 28.
Dose group (mg/kg bw/day) |
|||||
Control |
250 |
500 |
1000 |
||
Males |
Kidney weight (absolute [g]) |
2.66 |
2.83 |
2.76 |
2.87* (DN) |
Difference [%] |
|
6 |
4 |
8 |
|
Thyroid weight (absolute [g]) |
21.2 |
21.6 |
19.6 |
20.8 |
|
Difference [%] |
|
2 |
-8 |
-2 |
|
Females |
Kidney weight (absolute [g]) |
1.64 |
1.78 |
1.76 |
1.73 |
Difference [%] |
|
9 |
7 |
6 |
|
Thyroid weight (absolute [g]) |
16.7 |
17.3 |
18.9 |
20.3** (DN) |
|
Difference [%] |
|
4 |
13 |
22 |
|
|
Body weight relative [%] |
7.19 |
7.17 |
7.91 |
8.55** (DN) |
|
Difference [%] |
|
0 |
10 |
19 |
|
Brain relative [%] |
0.92 |
0.91 |
1.02 |
1.11** (DN) |
|
Difference [%] |
|
-1 |
11 |
21 |
* = p < 0.05, ** = p < 0.01
DN = Duncan's Multiple Range Test
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed
- Study duration:
- subacute
- Species:
- rat
- Quality of whole database:
- The available information comprises an adequate and reliable study (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.
Additional information
A series of studies were carried out to investigate the effects of Triafamone on rats, mice and dogs following repeated exposure via the oral route over subacute and subchronic periods. All the studies were carried out from 2008 to 2013, were performed according to the current OECD guideline and in compliance with the GLP requirements, with the exception of the 28-day dietary studies.
In the rat 28-day dietary toxicity study (M-344191-01-1), groups of 5 Wistar rats/sex were exposed continuously via the diet to Triafamone at concentrations of 0, 500, 7000 and 12000 ppm (equating to approximately 0, 36, 500 and 852 mg/kg bw/day in males and 0, 38, 551 and 945 mg/kg bw/day in females). The study was designed to investigate the effects of the compound on the enzymatic activity of hepatic microsomal cytochrome P-450 isoenzymes and of phase II enzyme UDP-glucuronosyltransferase (UDPGT). Effects were seen in the liver (increased weight and increased incidence of centrilobular hypertrophy) in both sexes and in the thyroid (follicular cell hypertrophy) in females. The assessment of the hepatic microsomal enzyme activities revealed a slight increase in mean total cytochrome P-450 contents, and an increase in CYP2B and CYP3A as well as of UDPGT activities in both sexes, more pronounced in females.
The No Observable Adverse Effect Level (NOAEL) was 500 ppm in both sexes (equivalent to 36 mg/kg bw/day in males and 38 mg/kg bw /day in males and females, respectively).
In the subchronic study (M-363159-01-1), Triafamone was administered continuously via dietary admixture to separate groups of Wistar rats (10/sex/group) at concentrations of 0 (control group) 250, 1100 and 5000 ppm (equating approximately to 16.4, 69.0, 323 mg/kg bw/day in males and 20.0, 88.5, 395 mg/kg bw/day in females) for at least 90 days. In addition, 10 animals/sex were added to the control and 5000 ppm group to assess the reversibility of any effects observed at the high dose level. There were effects in the liver (increased weight correlated with increased incidence of hepatocellular hypertrophy) and in the thyroid (follicular cell hypertrophy an increased incidence of colloid alteration) from 1100 ppm onwards. In the high dose group, the effects in the liver were reversible whereas mild changes in thyroid morphology were still present after the one month recovery period.
The NOAEL was considered to be 250 ppm in both sexes (equivalent to 16.4 and 20.0 mg/kg bw/day in males and females, respectively).
In a 28-day study (M-312057-01-1), Triafamone was administered continuously via the diet to groups of C57BL/6J mice (5/sex/group) at concentrations of 0 (control), 500, 2000 and 7000 ppm (approximately 83.3, 337, 1155 and 89.2, 357 and 1302 mg/kg bw/day in males and females, respectively). There were no adverse effects in this study and the NOAEL was the highest dose level, i.e. 7000 ppm (1155 and 1302 mg/kg bw/day in males and females, respectively). A second 28-day mouse study (M-398022-01-1) was carried out to specifically investigate the effects of Triafamone on the mouse liver. Triafamone was administered continuously via the diet to groups of C57BL/6J mice (20/sex/group) for at least 28 days at concentrations of 500, 2000 and 7000 ppm, corresponding to 84.0, 326 and 1184 mg/kg bw/day in males and 95.5, 376 and 1372 mg/kg bw/day in females, respectively. A similarly constituted group received untreated diet and acted as a control. All animals were necropsied, brain and liver weighed and the liver from 5 animals/sex/group randomly selected was taken, fixed and examined microscopically. Hepatic microsomes were prepared (pools of 4 animals/sex/group) in order to determine cytochrome P-450 and UDP-glucuronosyltransferase (UDPGT) isoenzymes profile. Results showed that Triafamone is an inducer of P-450 isoenzymes CYP 2B and 3A from doses equivalent to 326 mg/kg bw/day and of UDPGT activity in females at doses equivalent to 1372 mg/kg bw/day. However, the increased activity was not paralleled by histopathology changes.
Therefore, the overall NOAEL was considered to be 2000 ppm (equating to approximately 326 and 376 mg/kg bw/day in males and females, respectively)
In the mouse 90-day dietary toxicity study (M-377075-01-1), Triafamone was administered to C57BL/6J mice (10 animals/sex/dose group) at concentrations of 0, 300, 1500 and 6000 ppm (approximately to mean achieved doses of 0, 50.8, 251, 1001 mg/kg bw/day in males and 0, 58.8, 293, 1170 mg/kg bw/day in females). The only relevant finding was increased liver weight in females at the top dose level. However, as the increase was around 10% and was not accompanied by liver morphological changes, this effect was not considered to be adverse. Therefore, the NOAEL of the 90-day dietary toxicity study in mouse was considered to be 6000 ppm (approximately 1001 and 1170 mg/kg bw/day males and females, respectively).
In a preliminary 28-day study in the Beagle dog (M-363152-01-1), Triafamone was administered by gavage in 0.5% methylcellulose 400 at doses of 0, 100, 300 or 800 mg/kg bw/day. In this study two animals/sex/dose group were tested. The main effects consisted of increased platelet counts in females (relative to animals’ pre-study value) and increased alkaline phosphatase activity in males from 300 mg/kg bw/day onwards. Liver morphological changes (panlobular hepatocellular hypertrophy) were also observed in all animals at the highest dose tested.
In the preliminary 28-day study the test material was administered by gavage, to overcome the problems of inappetence of the diet mixture. In fact dogs did not eat diets containing Triafamone at doses higher than 800 ppm. However, in the 90-day study, it was possible to administer diets containing Triafamone at concentration of 800, 2400 and 7200/4000 ppm (equivalent to 27.2, 74.6 and 155 mg/kg bw/day in males and 30.3, 81.0 and 123 mg/kg bw/day in females, respectively) by administering the higher doses stepwise during the first week of administration.
The two ways of administration, i.e. gavage vs. dietary, had a different impact on the toxicity of Triafamone in dogs, because administration by gavage had no toxic effects at doses up to 100 mg/kg bw/day and administration via the diet provoked hepatotoxicity at doses equivalent to about 30 mg/kg bw/day. Therefore in the 90-day dietary toxicity studies in dogs, no NOAEL was established due to blood biochemistry changes, increased liver weight accompanied by microscopic changes with occurrence of dose-related incidence of single cell necrosis and necrotic foci at all dose levels.
Nevertheless a safe level for hepatotoxicity in dogs was identified in the one year dietary toxicity study. In this study, three groups of 4 males and 4 females received Triafamone at doses of 100, 300 or 800 ppm (equating to an average of 2.8, 8.8, 21.9 mg/kg bw/day in males and 3.0, 9.7, 24.3 mg/kg bw/day in females over the exposure period) for at least 52 weeks. A similarly constituted group of 4 males and 4 females received untreated diet, and acted as a control group. The liver was confirmed to be the target organ with morphological changes (hepatocellular hypertrophy) from 300 ppm (or 8.8 mg/kg bw/day) onwards, but there was no evidence of necrosis up to the highest dose tested of 800 ppm (equivalent to 21.9 and 24.3 mg/kg bw/day).
In conclusion, a dietary level of 100 ppm (equating to 2.8 mg/kg bw/day in males and 3.0 mg/kg bw/day in females) is considered to be the overall NOAEL in male and female Beagle dogs for hepatotoxicity after subchronic exposure or exposure equivalent to 1/10 of the dog lifespan.
Dermal route
Wistar rat the potential toxicological effects after dermal exposure where investigated following repeated dermal exposure for 6 hour a day for 28 days (M-446559-01-1) according to OECD No. 410 (1981) and EPA OPPTS 870.3200 (1998).80 Wistar rats of both sexes were involved in the study. Each experimental group consisted of 10 animals/sex. Test item was applied to the rat skin (shaved flank) by a semi-occluded application at dose of 250, 500 and 1000 mg/kg bw/day each day using special jackets.
During the study, observations for mortality, clinical signs and observations on the skin following the test item application were performed daily, detailed clinical examination, body weight and food consumption measurements were performed at weekly intervals. Ophthalmoscopy and neurological assessment including functional observation battery (FOB) and measurements of the landing foot splay and grip strength were performed during the last week of the treatment. Clinical pathology examinations (haematology, clinical chemistry and urinalysis) were conducted prior to necropsy on Day 28 followed by necropsy with macroscopic examination and selected organ weight measurements. Full histopathology was performed on control and high dose animals and livers of the low and mid dose groups animals.
Treatment-related effects were observed at 1000 mg/kg bw/day and consisted of minimal /mild, periportal /diffuse hepatocellular vacuolation (3/10 males and 3/10 females) and minimal centrilobular hepatocellular hypertrophy (3/10 males and 2/10 females) correlated with increased liver weight (approximately 11-14%) and increased cholesterol in both sexes and slight increase in the thyroid weight in females without morphological changes. No adverse effects or test item related histopathological findings were observed at the low or mid-dose levels. In conclusion, the systemic NOAEL following 28-day dermal toxicity study was 500 mg/kg bw/day and the NOAEL for dermal local effects is 1000 mg/kg bw/day.
Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The selected study is the most adequate and reliable study based on overall assessment of quality, duration and dose descriptor level (refer to the
endpoint discussion for further details).
Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
A 28-day study is available and the results show the same target organs and toxic endpoints observed after oral toxicity.
Justification for selection of repeated dose toxicity dermal - local effects endpoint:
In the 28-day dermal toxicity study there were no skin effects up to the highest dose tested of 1000 mg/kg bw/day.
Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver
Repeated dose toxicity: dermal - systemic effects (target organ) digestive: liver
Justification for classification or non-classification
The available data on repeated dose toxicity of the test substance meet the criteria for classification as STOT-RE Cat. 2 (H373) according to Regulation (EC) 1272/2008 and as Xn, R48/22 Directive 67/548/EEC.
The available data on repeated dose toxicity of the test substance indicate occurrence of hepatotoxic effects (single cell necrosis) in the dog 90-day dietary study and meet the criteria for classification. The effects were observed at dose levels starting from 27.2 mg/kg bw/day in 1/4 male and 1/4 female at doses equivalent up to 27.2 mg/kg bw/day. However, there was no hepatocellular necrosis but slight hepatocellular hypertrophy in the 1-year dog following dietary level up to 24.3 mg/bw bw/day. Therefore, classification for STOT-RE Cat. 2 does apply because liver effects justifying classification are occurring at doses ≥ 10 mg/kg bw/day and ≤ 100 mg/kg bw/day.
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