Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Remarks:
in accordance with the GLP provisions of the ‹Chemicals Act" (Chemikaliengesetz; Bundesgesetzblatt 1994, Teil 1, 29.07.94; FR Germany) and with the "OECD Principles of Good Laboratory Practice" (Paris, 1981).
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): s-MOIPA (test substance no.: 98/269-1)
- Physical state: homogeneous fluid/colorless-yellowish clear
- Purity test date: 94.1%
- Lot/batch No.: 7/11/97
- Storage condition of test material: the test material was stored at room temperature.
- Stability under test conditions: proven by reanalysis after the in life phase of the study; the analyses were carried out at Covance Laboratories, Madison, WI, USA (Characterization) and the Analytical Department of BASF Aktiengesellschaft (Reanalysis).

Test animals

Species:
rat
Strain:
other: Wistar Chbb:THOM (SPF)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: were supplied by Boehringer Ingelheim Pharma KG, Biberach/Riss, FRG. OnIy animals free from clinical signs of disease were used tor the study.
- Age at study initiation: 42 days
- Weight at study initiation: 179 -217 g (group mean: 194 g) for male and 136 - 159 g (group mean: 148 g) for female animals
- Fasting period before study: not specified
- Housing: housed singly in type DK 111 stainless steel wire mesh cages supplied by Becker & Co., Castrop-Rauxel, FRG (floor area about 800 cm2). Underneath the cages, waste trays were fixed containing absorbent material (type 3/4 dustfree embedding, supplied by SSNIFF, Soest, FRG). Motor activity measurements were conducted in Polycarbonate cages with wire covers from Ehret, Emmendingen, FRG (floor area about 800 cm2 ) and small amounts of absorbent material (see above). The animals were housed in a fully air-conditioned room.
- Diet (e.g. ad libitum): Kliba maintenance diet rat/mouse/hamster, meal (Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water (e.g. ad libitum): tap water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12 hours light from 06.00 am - 06.00 pm, 12 hours dark from 06.00 pm - 06.00 am


IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: doubly distilled water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The appropriate amount of test substance was weighed in, then doubly distilled water was filled up to the desired volume and subsequently mixed using a magnetic stirrer. The test substance preparations were prepared at least once a week (see Table 1 for test groups).

The test substance was administered daily by gavage using 3 m1/5 ml-syringes for about 4 weeks. Control animals received the vehicle, only. At the end of the administration period all surviving animals were sacrificed after a fasting period (withdrawal of food) tor at least 16 hours.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The Analyses were carried out at the Bioanalytical Laboratory .The stability of the test substance in doubly distilled water was demonstrated for a period of 7 days at room temperature prior to the study. Analyses of test substance concentrations were performed for all test solutions at the start of the administration period. As the test substance was administered as a solution, no homogeneity analyses were necessary. The analyses confirmed the correctness of the concentrations. The mean values were 94 - 106 % of the target concentrations.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 30, 100 and 300 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a test study [BASF 1998], S-MOIPA was administered to groups of 3 male and 3 female Wistar rats by gavage at dose levels of 0 (group 0 ), 50 (group 1), 100 (group 2) and 200 (group 3) mg/kg body weight/day (mg/kg bw/d). Clinical signs, food consumption, body weights, and macroscopic findings were recorded. No signs of toxicity were seen after 2 weeks of treatment. Therefore, group 0 was treated for another 2 weeks with 400 mg/kg bw/d. In groups 1 and 2 treatment was stopped and these groups served as control. Group 3 was necropsied. During these additional 2 weeks, food and water consumption were decreased in males and females of group 0 (400 mg/kg bw/d). As a consequence, body weight was impaired, resulting in reduced values of -9.4% and -6.8%, respectively. Abnormal clinical signs such as red discharge of the nose, respiratory sounds, and/or salivation were observed. One female animal died after being treated for 11 days with 400 mg/kg bw/d.
Based upon the above mentioned findings, the following dose levels were selected for the present study: 300 mg/kg bw/day (as high dose level with expected toxic effects), 100 mg/kg bw/day (as mid dose) and 30 mg/kg bw/day (as low dose).
The oral route was selected since this was proven to be suitable for the detection of a toxicological hazard.
Positive control:
none

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule for open field observation (OFO): detailed clinical observations (open field observations) were carried out in all males/females prior to the test substance administration (day -3), and on day 7, 14, and 21 each time from about 10.00 a.m. onwards. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 x 50 cm with sides of 25 cm high). Following parameters were examined: behavior when removed from cage, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmus, feces, appearance/consistency), urine and pupil size.

- Time schedule: the animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) from Mondays to Fridays and once a day (in the morning) on Saturdays, Sundays and public holidays. Additionally, further general clinical observations were carried out daily just before treatment, less than 1 hour after treatment, and between 3 and 4 hours after treatment.

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

FOOD CONSUMPTION: Yes
- Time schedule for examinations: food consumption was determined weekly and calculated as mean food consumption in grams per animal and day.

FOOD EFFICIENCY: Yes
- 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

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

OPHTHALMOSCOPIC EXAMINATION: Yes / No / No data
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 28
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: all
- Parameters examined: Leukocytes, erythrocytes, hemoglobin, haematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets and differential blood count. Furthermore differential blood smears were prepared and stained according to Wright without being evaluated.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 28
- Animals fasted: Yes
- How many animals: all
- Parameters examined: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-y-glutamyltransferase, sodium, potassium, chioride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, and magnesium.

URINALYSIS: Yes
- Time schedule for collection of urine: day 23
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters examined: volume, color, turbidity, nitrite, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity and sediment.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: functional observational batteries and motor activity measurements were performed on day 24 (males) and on day 25 (females). Functional observational batteries were performed each time from about 10 a.m. and motor activity measurements were conducted each time from about 2 p.m. onwards.
MA was measured an the same day as FOB was performed.
- Dose groups that were examined: all
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; the animals were sacrificed by decapitation under CO2 anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology. The female rat that died intercurrently was necropsied and assessed as soon as possible after death to avoid autolysis. The following weight parameters from all animals sacrificed at scheduled dates were determined: anesthetized animals, liver, kidneys, adrenal glands, testes, epididymides, ovaries, brain, thymus, heart and spleen.
HISTOPATHOLOGY: Yes; the following organs or tissues were fixed in 4% formaldehyde solution: all gross lesions, brain, pituitary gland, thyroid glands (with parathyroid glands), thymus, trachea, lungs, heart, liver, spleen, kidneys, adrenal glands, testes/ovaries, uterus/vagina, accessory genital organs (epididymides, prostate gland, seminal vesicles), stomach (glandular and non-glandular), duodenum, jejunum, ileum, cecum, colon, rectum, urinary bladder, lymph nodes (mandibular and mesenteric), sciatic nerve, bone marrow (femur), eyes, spinal cord (cervical, thoracic and lumbar cord). After the organs were fixed, histotechnical processing and examination by light microscopy were performed.
Other examinations:
not applicable
Statistics:
- Food consumption, body weight, body weight change, food efficiency: parametric one-way analysis using the F- test (ANOVA) (two-sided). If the resulting p-value was equal or less 0.05, a comparison of each group with the control group control using the DUNNETT's test
(two-sided) was performed tor the hypothesis of equal means.
- Feces, rearing, grip strength forelimbs, grip strength hind limbs, landing foot-splay test, motor activity: Non-parametric one- way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using MANN-WHITNEY U-test (two-sided) tor the equal medians
- Clinical pathology parameters excepting differential blood count: non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided).If the resulting p-value was equal or less 0.05, a pairwise comparison of each dose group with the control group was performed using MANNWHITNEY U-test (two-sided) for the equal medians
- Urinalysis, except volume color, turbidity and specific gravity: pairwise comparison of each dose group with the control group using FISHER's exact test for the hypothesis of equal proportions.
- Pathology weight parameters: non-parametric one-way parameters analysis using KRUSKAL- WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using the WILCOXON test for the hypothesis of equal medians

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
- General clinical observations: in the high dose group, abnormal clinical signs were observed such as reddish smeared snout region or red smeared nose, urine smeared anogenital region, salivation after treatment, respiratory sounds, abdominal respiration, labored respiration, and/or piloerection were observed on different days of the study. No abnormal signs were seen in the mid and low dose group.
- Mortality: one high dose female died during the administration period (day 12).
- Open field observations; the following deviations from ‹zero values" were observed
Animal did not walk during observation period; no defecation during the observation period; no urination during observation period; Urine staining of anogenital region, gasping/respiration sounds, polyurea and piloerection. As all findings were equally distributed between treated and control animals, therefore, the variation were clearly incidental in nature.

BODY WEIGHT AND WEIGHT GAIN
No statistically significant or biologically relevant findings were observed (see Table 3).

FOOD CONSUMPTION
No statistically significant or biologically relevant findings were observed (see Table 4).

FOOD EFFICIENCY
Food efficiency was statistically significantly increased in high dose females on day 21. Due to the isolated occurrence, this was assessed as being incidental.

HAEMATOLOGY
Compound-related differences in hematology parameters were not evident at any concentration tested in either males or females.

CLINICAL CHEMISTRY
There are no treatment-related changes in the clinical chemistry parameters measured.

URINALYSIS
No treatment-related changes were found in the urinalyses of either sex.

NEUROBEHAVIOUR
- Sensorimotor tests/Reflexes [Approach response (no reaction), touch response (no reaction)]: as all findings were equally distributed between treated and control animals, this was clearly incidental in nature.
- Regarding the overall motor no statistically significant deviations were seen in treated males or females. The comparison of the single intervals with the control group resulted in a statistically significantly increased value of interval 3 in males of group 1, decreased value of interval 3 in males of group 2, and decreased values of interval 7 in all females of group 1-3. Due to the isolated occurrence and the lack of a dose-response relationship, this was assessed as being incidental and biologically not relevant.
ORGAN WEIGHTS
- Absolute weights: the mean weight of the spleen was slightly although significantly increased in females of group 1. This was regarded incidental.
In females of group 3, the mean liver weight was increased (+ 17.3%). However, due to the premature death of one female rat of dose group 3 (animal No. 36), no significance is indicated for the mean liver weight, as the number of animals (n = 4) was too small, making the statistical test used too insensitive. The other mean absolute weight parameters did not show significant differences when compared with the control group.
- Relative weights (related to terminal body weight)
In females of group 3, the mean liver weight was increased (+ 12.6%). However, due to the premature death of one female rat of dose group 3 (animal No. 36), no significance is indicated for the mean relative liver weight, as the number of animals (n = 4) was too small, making the statistical test used too insensitive. The other mean relative weight parameters (when related to terminal body weight) did not show significant differences when compared with the control group.

GROSS PATHOLOGY
Only a few gross lesions were noted in the testes (severe organ size reduction) and epididymides (moderate organ size reduction) of a control male. These gross lesions are of spontaneous origin and have developed unrelated to treatment.
One top dose female (animal No. 36) died 12 days after start of treatment. Major gross lesions were dark red discoloration of the lungs and gray discoloration of the thyroid glands.

HISTOPATHOLOGY: NON-NEOPLASTIC
With the exception of the gray discoloration of the thyroid glands, the few other gross lesions were all correlated with a meaningful microscopic finding. The grossly recorded severe (testes) or moderate (epididymides ) organ size reduction noted in a control male resulted in an extreme diffuse tubular atrophy in the testes (Sertoli cells only) and aspermia in the epididymides, respectively. As these findings occurred in a control animal, they were regarded to have developed spontaneously and unrelated to treatment.
Female animal No. 36 of dose group 3, died 12 days after start of treatment. Major microscopic findings were slight (grade 2) to severe (grade 4) congestion in the large parenchyma (lungs, liver, kidneys, heart, spleen, thymus, brain) and several smaller organs (mandibular lymph nodes, adrenal cortexlmedulla, thyroid glands). The dark red discoloration of the lungs correlated with a severe pulmonary congestion. This finding was given a grade 4 of severity (severe), and it was associated with a moderate (grade 3) edema in the perivascular tissue. The edematous fluid was loosely intermingled with 1) pseudo-eosinophils (granulocytes). These findings may be related to treatment. The gray discoloration of the thyroid glands did not really match the noted slight congestion. Although post mortem autolysis may have partly obscured the histopathologic evaluation of this animal in general and the thyroid glands (and several other organs like e. g. the gastro-intestinal tract) in special, a relationship of the few microscopic findings noted in animal No. 36 (except findings in the lung) to treatment seems unlikely.
Histopathology did not reveal a microscopic finding in the liver that may account for the increased mean absolute and relative liver weights in females of group 3. In addition, no microscopic findings were recorded in the liver of female rats of dose groups 1 and 2 differing biologically remarkably from the findings recorded in the female animals of the control group, by this excluding any possible target organ involvement of the liver in female rats.
All other microscopic findings recorded were either single observations, or they occurred in control animals only, or they were recorded at comparable incidence and graded severity in control and high dose males and/or females.
After a 4-week application period, there was no indication of a morphologic affection of the organs of the central or peripheral nervous system, the reproductive system, and the immune system.

OTHER FINDINGS
There are statistically significant intergroup differences in the results of clinical pathology testing. These deviations are marginal, incidental or inconsistent, when compared with the other sex, or lack dose-response relationship. Accordingly, these findings are considered to be of no toxicological significance.

Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 100 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: one animal died at 300 mg/kg bw dose level (for additional adverse effects, see below)

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 3: Mean body weight

 

Dose groups (mg/kg)

Mean body weight (g)

Day 0

Day 7

Day 14

Day 21

Day 27

Males

Females

Males

Females

Males

Females

Males

Females

Males

Females

0

195.9±14.0

148.9±5.4

253.0±20.1

173.6±5.2

302.2±21.7

194.9±5.1

336.8±23.1

205.2±7.2

356.9±25.2

213.2±8.9

30

195.9±10.3

148.3±7.8

250.7±16.5

173.6±10.4

302.2±17.0

196.6±12.3

336.0±15.5

211.0±13.3

354.2±18.7

219.9±13.0

100

193.9±4.6

147.3±4.2

243.0±9.5

170.5±6.7

293.0±9.5

193.9±7.8

328.9±15.3

204.7±11.7

341.4±14.7

215.1±12.7

300

191.2±6.3

149.4±7.3

242.4±10.3

170.6±6.6

288.0±16.1

191.3±3.6

321.9±20.4

213.8±5.4

342.2±23.8

222.6±4.6

Table 3: Mean food consumption

 

Dose groups (mg/kg)

Mean food consumption (g/day)

Day 7

Day 14

Day 21

Day 27

Males

Females

Males

Females

Males

Females

Males

Females

0

26.6±1.7

18.3±0.8

27.5±2.0

19.3±1.3

29.3±1.8

18.9±1.4

27.2±1.1

17.3±1.0

30

26.2±2.0

18.6±1.6

25.9±3.6

20.0±1.2

28.7±1.1

19.7±1.4

26.1±1.9

17.8±1.4

100

25.1±0.8

17.8±1.1

27.9±1.4

19.1±1.0

28.8±1.9

18.7±1.9

25.1±0.9

17.5±1.3

300

25.2±1.9

17.5±0.7

27.4±3.0

18.2±1.0

28.0±2.3

20.3±0.9

25.7±2.3

18.8±0.7

 

Applicant's summary and conclusion