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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 Sep 2014 - 26 May 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to guideline
Guideline:
other: EPA, Health Effects Test Guidelines; OPPTS 870.3650
GLP compliance:
yes (incl. QA statement)
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Etocrilene
EC Number:
226-029-0
EC Name:
Etocrilene
Cas Number:
5232-99-5
Molecular formula:
C18H15NO2
IUPAC Name:
etocrilene
Test material form:
solid
Details on test material:
- Physical state: solid/white

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 11 - 13 weeks
- Weight at study initiation: Male animals: 328.2 g - 368.9 g; Female animals: 190.2 g - 215.9 g
- Housing: individually in Polycarbonate cages type III; during overnight matings, male and female mating partners were housed together in Polycarbonate cages type III. Pregnant animals and their litters were housed together until PND 4.
- Diet: ground Kliba maintenance diet mouse/rat “GLP” meal, ad libitum
- Water: supplied from water bottles (ad libitum).
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24°C
- Humidity (%): 30-70
- Air changes (per hr): 115
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1%
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
For the preparation of the administration suspensions the test substance was weighed in a calibrated beaker depending on the dose group, topped up with 1% Carboxymethylcellulose suspension in drinking water and intensely mixed with a homogenizer. During administration, the preparations were kept homogeneous with a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 1.00, 3.00 and 10.00 g/100ml
- Amount of vehicle (if gavage): 10 ml/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical verifications of the stability of the test substance in 1% Carboxymethylcellulose suspension in drinking water for a period of 96 hours at room temperature were carried out prior to the start of the study. Samples of the test substance preparations were sent to the analytical laboratory once during the study period for verification of the concentration. The samples, which were taken for the concentration control analysis at the beginning of the administration period, were also used to verify the homogeneity. Three samples (one from the top, middle and bottom in each case) were taken from the beaker with a magnetic stirrer running. Of each sample, one additional reserve sample was retained. Details of the sampling schedule were recorded with the raw data.
The various analyses:
• Demonstrated the stability of the test substance in 1% Carboxymethylcellulose suspension in drinking water over a period of 96 hours at room temperature
• Confirmed the homogeneous distribution of the test substance in 1% Carboxymethylcellulose suspension in drinking water
• Verified correct concentrations of the test substance preparations.
Duration of treatment / exposure:
The duration of treatment covered a 2-week pre-mating and a mating period in both sexes, approximately 4 days post-mating in males, and the entire gestation period as well as approximately 4 weeks of the lactation period.
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300, 1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Each of the male and female animals was mated overnight in a 1:1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same dose group.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A check for moribund or dead animals was made twice daily on working days or once daily (Saturday, Sunday or on public holidays). A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations (DCO) were performed in all animals once prior to the first administration (day 0) and at weekly intervals during the administration period. The examinations started in the morning. The findings were ranked according to the degree of severity, if applicable.

BODY WEIGHT: Yes
In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice. The following exceptions are notable for the female animals:
• During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day of parturition (PND 0) and on PND 4.
Females without positive evidence of sperm, without litter or waiting for necropsy, were weighed weekly. These body weight data were solely used for the calculations of the dose volume.

FOOD CONSUMPTION
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
• Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female F0 animals).
• Food consumption of the F0 females with evidence of sperm was determined on gestation days (GD) 0 - 7, 7 - 14, and 14 - 20.
• Food consumption of F0 females which gave birth to a litter was determined on PND 1 - 4.
Food consumption was not determined in females without positive evidence of sperm during the mating and the gestation period and in females without litter during the lactation period.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: study day 32 (males) and 56 (females).
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5 male and 5 female animals (with litter) per group
- Parameters examined: Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume, (MCV),
Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes (RET), Prothrombin time (Hepato Quick’s test) (HQT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: study day 32 (males) and 56 (females).
- Animals fasted: Yes
- How many animals: 5 male and 5 female animals (with litter) per group
- Parameters 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), Bile acids (TBA)

URINALYSIS: Yes
- Time schedule for collection of urine: Urinalysis was carried out on urine from 5 male and 5 female animals (with litter) per group on study day 30 (males) and 52 (females).
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: pH, Protein (PRO), Glucose (GLU), Ketones (KET), Urobilinogen (UBG), Bilirubin (BIL), Blood, Specific gravity, Sediment, Color, turbidity (COL, TURB), Volume (VOL)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: On study day 28, a functional observational battery and motor activity measurement were carried out in five male animals per group. On study day 50, a functional observational battery and motor activity measurement were carried out in five female animals (with litter) per group.
- Battery of functions tested: Home cage observations, Open field observations, Sensory motor tests/Reflexes, Motor activity measurement
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.

ORGAN WEIGHTS
The following weights were determined in all animals sacrificed on schedule: Anesthetized animals, Epididymides, Testes
The following weights were determined in 5 animals/sex and test group (females with litters, same animals as used for clinical pathology examinations): Adrenal glands, Brain, Heart, Kidneys, Liver, Spleen, Thymus

HISTOPATHOLOGY: Yes
see table below for details. Special attention was given on the stages of spermatogenesis in the testes.
Statistics:
• DUNNETT-test (twosided): Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), gestation days.
• FISHER'S EXACT test (one-sided): Male and female mating indices, male and female fertility indices, females mated, females delivering, gestation index (females with liveborn pups), females with stillborn pups, females with all stillborn pups.
• WILCOXON test (one-sided) with BONFERRONI-HOLM adjustment: Mating days until day 0 pc, %postimplantation loss, pups stillborn, %perinatal loss, Implantation sites, pups delivered, pups liveborn, live pups day x, viability Index, Urinalysis parameters (apart from pH, urine volume, specific gravity, color and turbidity.
• WILCOXON test (twosided): % live male day x, %live female day x
• KRUSKAL-WALLIS test (two-sided): Rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity, Blood parameters, Urine pH, volume, specific gravity, color and turbidity, Weight parameters (pathology)

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There were no test substance-related or spontaneous mortalities in any of the groups. No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any male or female F0 generation parental animal during the entire study period.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
While in the mean body weights of the high-dose parental males were without statistical significance nearly comparable to the concurrent control values, the body weight change of this group was statistically significantly below the concurrent control values during premating days 0 - 7 and 0 - 13 (about 27% and 29%, respectively).
The mean body weights of the high-dose parental females were statistically significantly below the concurrent control values on premating day 13 (about 4%), during GD 7 - 14 (up to 6%) and on PND 4 (about 6%). The body weight change of the high-dose parental females was statistically significantly below the concurrent control values during premating days 7 - 13 and 0 - 13 (about 69% and 51%, respectively).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption of the high-dose F0 females (1000 mg/kg bw/d) was statistically significantly below the concurrent control values during GD 7 - 14 and GD 0 - 20 (about 8%, respectively). This was assessed as treatment related. The food consumption of the high-dose F0 females (1000 mg/kg bw/d) during the premating and lactation period, as well as the food consumption of all test substance-treated F0 male animals (100, 300 and 1000 mg/kg bw/d) and the mid- and low-dose F0 females was comparable to the concurrent control values throughout the entire study period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period in females of test groups 1 and 3 (100 and 1000 mg/kg bw/d), platelet counts were lower compared to controls. In test group 1 the mean was not dose-dependently altered and therefore the change was incidental and not treatmentrelated. In test group 3 the median platelet count was higher compared to test group 1, but the mean was lower. Mean and median platelet counts in females of test group 3 (1000 mg/kg b w/d) were below the historical control range. Therefore, this decrease has to be regarded as adverse.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among clinical chemistry parameters were observed.
At the end of the administration period in males of test groups 1 and 2 (100 and 300 mg/kg bw/d), aspartate aminotransferase (AST) activities were lower compared to controls. In males of test groups 2 and 3 (300 and 1000 mg/kg bw/d) glucose levels were increased and in females of test group 2 (300 mg/kg bw/d) urea levels were decreased. All mentioned parameters were not dose-dependently changed and therefore the alterations were regarded as incidental and not treatment-related.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among urinalysis parameters were observed.
In females of test group 2 (300 mg/kg bw/d) urine volume was decreased and urine specific gravity was increased (not statistically significantly). These alterations were not dosedependent and therefore they were regarded as incidental and not treatment-related.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation. The open field observations did not reveal any test substance-related findings in male and female animals of all test groups. There were no test substance-related findings in male and female animals of all test groups. No test substance-related impaired parameters were observed in male and female animals of all test groups. Comparing single intervals of treated animals with the control group, no significant deviations were measured during motor activity merasurement with the exception of the decreased mean value at interval 2 in male animals of test group 3 (1000 mg/kg bw/d). As the overall motor activity measurement in male animals of test group 3 (1000 mg/kg bw/d) was not statistically significant impaired and in females in the comparable intervals increased values (without statistical significance) were seen, the change was assessed as being spontaneous and not related to treatment.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
When compared to control group 0 (set to 100%), the mean absolute kidney weights were significantly decreased in mid dose females. The significant kidneys weight decrease was regarded as incidental. All other mean absolute weight parameters did not show significant differences when compared to the control group 0.
When compared to control group 0 (set to 100%), the mean relative weights of the brain (mid dose females and high dose male), epididymides (high dose males) and kidneys (mid and high dose males and high dose females) were statistically significantly increased. The significant weight increase of the brain, epididymides and kidneys in males of test group 3 (1000 mg/kg bw/d) were secondary to the decreased terminal body weight. They were within the historical control values and showed no histopathological correlate. The significant increased weight of the kidneys in females was within the historical values and showed no histopathological correlate. The significant weight increases observed in males and females of test group 2 (300 mg/kg bw/d) showed no dose-dependency and were regarded as incidental. All other mean relative weight parameters did not show significant differences when compared to the control group 0.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Histopathological findings: neoplastic:
not examined

Effect levels

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw (total dose)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
haematology

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Under the conditions of the present OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats, the NOAEL (no observed adverse effect level) for general, systemic toxicity of Etocrilene was 300 mg/kg bw/d based on decreased food consumption and decreased body weight parameters as well as clinical chemistry changes at 1000 mg/kg bw/d. The NOAEL for reproductive performance and fertility was 1000 mg/kg bw/d for the F0 parental rats. The NOAEL for developmental toxicity in the F1 offspring was 1000 mg/kg bw/d, the highest tested dose.
Executive summary:

The test article was administered daily as an aqueous preparation to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at doses of 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/d). Control animals (10 male and 10 female Wistar rats) were dosed daily with the vehicle only (1% Carboxymethylcellulose suspension in drinking water). The duration of treatment covered a 2-week pre-mating and a mating period in both sexes, approximately 4 days post-mating in males, and the entire gestation period as well as approximately 4 weeks of the lactation period. After 2 weeks of premating treatment the F0 animals were mated to produce F1 generation pups. Mating pairs were from the same test group. Mating was discontinued as soon as sperm were detected in the vaginal smear. F0 animals were examined for their reproductive performance including determination of the number of implantation sites and the calculation of postimplantation loss for all F0 females. A detailed clinical observation (DCO) was performed in all animals before initial test substance administration and, as a rule, thereafter at weekly intervals. Food consumption of the F0 parents was determined once weekly during premating. In dams food consumption was determined for gestation days 0 - 7, 7 - 14, 14 - 20 and lactation days 1 - 4. Body weights of F0 parents were determined once a week, in males throughout the study and in females during premating. During gestation and lactation period, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, on the day of parturition (postnatal day [PND] 0) and on PND 4. The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1 and on PND 4. Their viability was recorded. At necropsy on PND 4, all pups were sacrificed with CO2, under isoflurane anesthesia, and examined macroscopically for external and visceral findings. Clinico-chemical and hematological examinations as well as urinalyses were performed in 5 animals per sex and group towards the end of the administration period. At the end of the administration period a functional observational battery was performed and motor activity was measured in 5 parental males and females per group. All F0 parental animals were sacrificed by decapitation, under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

Parental animals of the high dose group (1000 mg/kg bw/d) showed slightly decreased food consumption in the females during gestation (up to about 8% below control). In addition, decreased body weights was recorded in the females on premating day 13 (about 4% below control), during GD 7 - 14 (up to 6% below control) and on PND 4 (about 6% below control). Decreased body weight change in the males and females during premating (up to 29% and 69% below control, respectively were also recorded. Furthermore, decreased platelet counts in females was evident and significantly decreased terminal body weight in males (about 6% below control). No test substance related findings were noted in parental animals of the mid and low dose group and in all F1 animals. Under the conditions of the present OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats, the NOAEL (no observed adverse effect level) for general, systemic toxicity of Etocrilene was 300 mg/kg bw/d based on decreased food consumption and decreased body weight parameters as well as clinical chemistry changes at 1000 mg/kg bw/d. The NOAEL for reproductive performance and fertility was 1000 mg/kg bw/d for the F0 parental rats. The NOAEL for developmental toxicity in the F1 offspring was 1000 mg/kg bw/d, the highest tested dose.