Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

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:
From 2012-05-09 to 2012-06-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with current test methods and in compliance with GLP regulations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): METH-E (Methyl-(endo)-5-norbornene-2,3-dicarboxylic anhydride)
- Physical state: Liquid
- Analytical purity: 99.6%
- Lot/batch No.: T120211293
- Expiration date of the lot/batch: 2012-10-20
- Storage condition of test material: Cool, dry, well ventilated place, protected from humidity

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt., Hungary
- Age at study initiation: 68 - 74 days (males and females)
- Weight at study initiation: 287 - 345 g (males); 159 - 196 g (females)
- Fasting period before study: No
- Housing: Before mating - 2 animals of the same sex/cage; During mating - 1 male and 1 female/cage; Pregnant females – individually; Males after mating - 2 animals/cage
- Diet (e.g. ad libitum): Ssniff SM R/M-Z+H complete rodent diet, ad libitum
- Water (e.g. ad libitum): Municipal supply, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25 deg C
- Humidity (%): 30 - 70% RH
- Air changes (per hr): 8 - 12
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 2012-05-09 To: 2012-06-25

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
vegetable oil
Remarks:
(sunflower)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Substance unstable in aqueous media
- Concentration in vehicle: 1.4, 4.0 and 10.0 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Performed twice during the study. Concentration of the test item in the dosing formulations varied in the range of 95% to 105% relative to nominal values.
Duration of treatment / exposure:
Males - 42 days; starting 2 weeks before mating
Females - 41 - 47 days, depending on date of mating; Those with living pups dosed for 14 days pre-mating, during mating period, through gestation and up to lactation days 3-10. Non-pregnant animals were treated up to and including the day before necropsy (for 42 days).
Frequency of treatment:
Daily, 7 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 7, 20 and 50 mg/kg body weight
Basis:

No. of animals per sex per dose:
12 males / 12 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on outcome of 14-day preliminary study
- Rationale for animal assignment: Random
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily, after treatment
- Cage side observations included: General clinical observations including behavioural changes.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Males - weekly; Females - weekly with additional measurements on gestation days 10 and 17 and post-partum days 0 and 4

FOOD CONSUMPTION AND COMPOUND INTAKE):
- Food consumption determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: Males - weekly; Females - weekly with additional measurements on post-partum days 0 and 4

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On termination
- Anaesthetic used for blood collection: Yes - Isofluran
- Animals fasted: Yes - Overnight
- How many animals: 5 males / 5 females / treatment group
- Parameters examined: Leucocyte count, erythrocyte count, haemoglobin concentration, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelet count, reticulocyte count, differential leucocyte count, prothrombin time, activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On termination
- Anaesthetic used for blood collection: Yes - Isofluran
- Animals fasted: Yes - Overnight
- How many animals: 5 males / 5 females / treatment group
- Parameters examined: Alanine aminotransferase, aspartate aminotransferase, gamma glutamyl transferase,, alkaline phosphatase, total bilirubin, creatinine, urea, glucose, cholesterol, bile acids, total protein, albumin, globulin, albumin/globulin ratio, inorganic phosphate, calcium, sodium, potassium, chloride,

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Last week of treatment
- Dose groups that were examined: 5 males / 5 females from each treatment group
- Battery of functions tested: sensory activity / grip strength / motor activity
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Examination of external appearance, appearance of the tissues and organs examined after opening of the cranial, thoracic and abdominal cavities. Abnormalities recorded with details of location, color, shape and size. Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea was recorded.
Organ weights: All males - testes, epididymides, brain. 5 males/5 females/group - Adrenal glands, Brain, Heart, Kidneys, Liver, Spleen, Thymus
Tissues fixed and preserved: (from all animals) - Uterus with cervix, vagina, testes, epididymides (total and cauda), prostate, seminal vesicles with coagulating glands, ovaries, pituitary and all organs showing macroscopic lesions. (from 5 males/5 females/group) – Abnormalities, Adrenal glands, Aorta, Bone marrow (from femur), Brain (cerebrum, cerebellum, pons and medulla oblongata), Caecum, Colon, Duodenum, Epididymides, Eyes, Mammary area, Heart, Ileum, Jejunum (including Peyer’s patches), Kidneys, Liver, Lungs (including mainstem bronchi), Lymph nodes - submandibular, Lymph nodes - mesenteric, Oesophagus, Ovaries, Pancreas, Pituitary gland, Prostate gland, Rectum, Salivary glands, Sciatic nerve, Seminal vesicles with coagulating gland, Skeletal muscle, Skin, Spinal cord (cervical, mid-thoracic, lumbar), Spleen, Sternum, Stomach, Testes, Thymus, Thyroid, Trachea, Urinary bladder, Uterus – cervix, Vagina

HISTOPATHOLOGY: Yes
Tissues examined - Ovaries, uterus, vagina, pituitary, testes and epididymides (with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure) of the animals in the control and high dose groups and non-pregnant females and corresponding cohabited males. Examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma. Full examinations undertaken on the preserved organs and tissues of the randomly selected animals in the control and high dose groups and in dead animals. Examination of kidneys was extended to 5 animals/sex from the low- and mid-dose groups.

Statistics:
Homogeneity 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 (ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to assess the significance of inter-group differences.
Where the result of the Bartlett’s test was significant the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test.
Chi2 test was performed if feasible.
The frequency of clinical signs, pathology and histopathology findings were calculated.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Mortality in 2 females dosed at 50 mg/kg/day
Mortality:
mortality observed, treatment-related
Description (incidence):
Mortality in 2 females dosed at 50 mg/kg/day
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduced in females dosed at 50 and 20 mg/kg/day
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Effects in females treated at 50 and 20 mg/kg/day
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Kidney weight elevated in females dosed at 50 mg/kg/day
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Kidneys, males and females dosed at 50 mg/kg/day
Details on results:
CLINICAL SIGNS AND MORTALITY
One female dosed at 50 mg/kg/day found dead on postpartum day 4 (Day 42). Piloerection, decreased activity, pale skin and mucous membranes, narrow eye orifices, reddish fur around the eyes and body weight loss were observed between lactation days 2 and 3. Histological investigation revealed serious catarrhal-purulent pyelitis and groups of bacteria in the kidneys to be the probable cause of death. This finding was considered as an individual disease (probably ascending bacterial infection via the urinary tract.)
A second female of the same treatment group was euthanized in moribund conditions on gestation day 21 (Day 45). Piloerection, prone position, marked activity decrease, body tone decrease and dyspnoea as well as paleness were observed. Acute alveolar emphysema and haemorrhages were detected in the lungs at necropsy. Slight focal tubular basophilia accompanied with flattened tubular epithelial cells was observed in the kidneys
There were treatment related clinical signs in male and female animals at any dose level at the weekly detailed observations during the entire treatment period. Brownish fur around the right eye of one male rat (20 mg/kg /day) was noted at the weekly observation on Days 0, 7, 13, 20, 27 and 41. Alopecia in female animals was noted in one control animal and in a high dose (50 mg/kg/day) animal.

BODY WEIGHT AND WEIGHT GAIN
The mean body weight and body weight gain of treated males were comparable to control values. A statistically significant higher mean body weight gain was apparent at 7 mg/kg/day during the last week of treatment but was considered to be not toxicologically relevant.
In the females, the mean body weight gain was significantly less at all treatment levels (50, 20 and 7 mg/kg/day) with respect to control between gestation days 14 and 21. The reduced body weight gain resulted in a slightly reduced mean body weight at 50 mg/kg/day on gestation day 21, as well as a reduced mean total body weight gain at 50 and 20 mg/kg/day. The reduced mean body weight gain at 20 and 7 mg/kg/day was not considered to be toxicologically significant because it was of low magnitude and there was no significant difference with respect to controls in the mean body weight on gestation day 21. There were no statistically significant differences in the mean body weight and body weight gain of female animals between control and dosed groups during the pre-mating and lactation periods. However the body weight decreased to a higher degree and in more animals at 50 mg/kg/day than in control animals during the lactation period.

FOOD CONSUMPTION AND COMPOUND INTAKE
The mean daily food consumption of male animals was similar in the control and treated groups during the entire treatment period. A statistically significant less mean food consumption noted in animals treated at 50 mg/kg/day on the first week of treatment period was not considered to be biologically significant.
The mean daily food consumption was comparable in the control and treated female animals (50, 20 and 7 mg/kg/day) groups during the pre-mating period. The mean daily food consumption was slightly but statistically significantly less in female animals treated at 50 mg/kg/day during the last week of gestation and at 50 and 20 mg/kg/day between lactation days 0 and 4 with respect to control.

HAEMATOLOGY
There were no test item related changes in the examined haematological parameters in male and female animals at any dose level. Sporadic statistically significant differences were observed for some parameters with respect to controls: mean prothrombin time at 20 mg/kg/day (male), haemoglobin concentration and haematocrit at 50 and 20 mg/kg/day (female), white blood cell count at 7 mg/kg/day (female) group and percentage of monocytes at 50 mg/kg/day (female). These statistical significant differences were not considered toxicologically relevant. The haemoglobin concentration and haematocrit values were well within the historical control range. Slight changes in white blood cell count and prothrombin time occurred in the lower doses only and values were within the historical control range. The mean percentage of monocytes in female animals dosed at 50 mg/kg/day was slightly over the historical control range due to the high values of two animals.

CLINICAL CHEMISTRY
There were no treatment related significant differences in the examined clinical chemistry parameters with respect to controls at any dose level investigated. Those statistically significant differences that were observed between the control and treated groups were judged to be of little or no biological significance. Slight changes in glucose and chloride concentrations were found in male animals only at 7 mg/kg/day, but not in the higher dose treated groups. The differences in sodium levels in each dosed groups (50, 20 and 7 mg/kg/day) with respect to controls were small and independent from dose. In female animals treated at 50 or 20 mg/kg/day, albumin and total protein concentrations were slightly less than in the control but were within the normal (historical control) values and were not related to doses.

NEUROBEHAVIOUR
Functional observations did not demonstrate any treatment related changes. The behaviour, physical condition and reactions to different type of stimuli of animals selected for examination were considered to be normal in all groups. A recorded more pronounced startle reaction was considered to be an individual variation and without any toxicological significance because it was noted for one control (1/5) and for one 20 mg/kg/day treated female (1/5) animal.

ORGAN WEIGHTS
Slightly higher weights of kidneys (absolute and relative to body and brain weights) were noted and considered may be indicative of an influence of treatment in female animals at 50 mg/kg/day. Other statistically significant differences, relative to controls, were only noted at 7 mg/kg/day. A higher mean absolute liver weight and a reduced kidney weight relative to body weight were observed in male and female animals, respectively. These differences were small and were not considered toxicologically relevant.

GROSS PATHOLOGY
Gross pathology examination did not reveal treatment related macroscopic findings in the organs or tissues of animals subjected to necropsy.
Dead or moribund animals - Pale skin and mucous membranes were observed in the animal treated at 50 mg/kg/day and subjected to necropsy after euthanasia in moribund condition on gestation day 21 (Day 45). There were no macroscopic findings in the visceral organs and tissues. Uneven surface of kidneys, enlarged adrenal glands, small thymus, dark red liver and red colored fur around the eyes were noted for an underweight dead dam dosed at 50 mg/kg/day on lactation day 4 (Day 42).
Surviving animals - Pulmonary haemorrhages (1/12 at 7 mg/kg/day), pyelectasia (1/12 control, 1/12 at 7 mg/kg/day) and pale kidneys (1/12 at 7 mg/kg/day) were observed in male animals during the gross pathology examination. In females, one side pyelectasia (2/10 at 50 mg/kg/day), haemorrhages in cervical lymph nodes (1/10 at 7 mg/kg/day) and alopecia (1/9 control and 1/10 at 50 mg/kg/day) were observed. Alopecia was an individual change occurring in a single female of control (1/9) and high dose (1/10) groups. In non-pregnant female animals, one side hydronephrosis (1/3 control) and hydrometra (2/2 at 7 mg/kg/day) were observed. Hydrometra (indicative of the sexual cycle of female animals) and hydronephrosis are frequent observations in experimental rats and were only present in low dose treated and control animals, therefore were regarded as having no toxicological meaning. In the pericardium of one the non-pregnant females treated at 7 mg/kg/day, a sac-like formation was detected with thickened wall and with liquid and granulated material, this regarded as an individual alteration

HISTOPATHOLOGY: NON-NEOPLASTIC
Dead or moribund animals - Histological examination revealed serious catarrhal-purulent pyelitis (both sides) and groups of bacteria in the kidneys in the one animal treated at 50 mg/kg/day and was regarded as the probable cause of death. This finding was considered to be an individual disease (probably ascending bacterial infection via the urinary tract.) In the second animal, also treated at 50 mg/kg/day, acute alveolar emphysema and haemorrhages were detected in the lungs. In the kidneys of this animal, mild focal tubular basophilia accompanied with flattened tubular epithel cells were observed.
Surviving animals - Focal or multifocal tubular basophilia accompanied with flattened tubular epithelial cells were found in the kidneys of male animals (5/12) and females (5/10) as well as slight inter-tubular lymphocytic infiltration (females 2/10) in the cortical region affecting the proximal convoluted tubules. The tubular basophilia accompanied with slight inter-tubular lymphocytic infiltration in the cortical region affecting the proximal convoluted tubules reflected a tubular damage and decreased functional activity of these tubules. The above mentioned lesions were not detectable in the kidneys of male and female animals treated at lower levels of 20 or 7 mg/kg/day and were considered to be treatment related. Focal alveolar emphysema (control male: 2/5, control female: 1/5; 50 mg/kg/day female: 1/5) and haemorrhage (control male: 1/5, 20 mg/kg/day male: 1/1) in the lungs were observed sporadically in minimal or mild degree and were considered to be a consequence of hypoxia, dyspnoea and circulatory disturbance developed during exsanguinations on termination.

Effect levels

Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In a repeat-dose toxicity combined with a screening study of reproductive toxicity the substance caused changes in body weight and food consumption in females during gestation and lactation, and changes in organ pathology (kidneys) in both males and females following oral administration at 50 mg/kg/day. The No Observed (Adverse) Effect Levels (NO(A)EL) was regarded as being 20 mg/kg body weight/day.
Executive summary:

In a repeat-dose toxicity combined with a screening study of reproductive toxicity conducted in accordance with OECD test methods, the substance caused changes in body weight and food consumption in females during gestation and lactation, and changes in organ pathology (kidneys) in both males and females following an oral administration at 50 mg/kg/day. The No Observed (Adverse) Effect Levels (NO(A)EL) was regarded as being 20 mg/kg body weight/day.