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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
1998
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Version / remarks:
1998
Qualifier:
according to
Guideline:
other: JMAFF, The guidelines related to the study reports for the registration application of pesticide, No. 12-Nousan-8147, 2-1-9: 90-day repeated dose oral toxicity tests
GLP compliance:
yes (incl. certificate)
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- State of aggregation: liquid
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, protected from daylight

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Age at study initiation: 36 ± 1 days
- Fasting period before study: no
- Housing: together (5 animals per cage/ sex) in H-Temp polysulfonate cages type 2000P
- Diet: ground Kliba maintenance diet mouse/rat “GLP”, ad libitum
- Water: ad libitum, from water bottles
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 30-70 %
- Air changes: 15 per minute
- Photoperiod: 12/12 hrs dark / hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG 400
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The appropriate amount of test substance was weighed out depending on the desired concentration. Then, Polyethylene glycol 400 was filled up to the desired volume, subsequently mixed with a magnetic stirrer. The test-substance preparations were produced daily, protected from light. The application occurred under indirect light and the test substance preparations were kept homogeneous by stirring with a magnetic stirrer. The administration volume was 5 mL/kg body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The various analyses confirmed
• the stability of the test-substance preparations for a period of 48 hours, protected from light, at room temperature,
• the homogeneous distribution of the test substance in the vehicle,
• the correctness of the prepared concentrations.
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
All animals were checked daily for any abnormal clinically signs before the administration as well as within 2 hours and within 5 hours after the administration (3 times a day). Abnormalities and changes were documented for each animal.

DETAILED CLINICAL OBSERVATIONS: Yes
Detailed clinical observations (DCO) were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 × 37.5 cm with sides of 25 cm height). The following parameters were examined:
Abnormal behavior when handled, Fur, Skin, Posture, Salivation, Respiration, Activity/ arousal level, Tremors, ,Convulsions, Abnormal movements, Gait abnormalities, Lacrimation, Palpebral closure, Exophthalmos, Assessment of the feces discharged during the examination (appearance/ consistency), Assessment of the urine discharged during the examination, Pupil size

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

FOOD CONSUMPTION
Food consumption was determined weekly (as representative value over 7 days) for each cage. The average food consumption/cage was used to estimate the mean food consumption in grams per animal and day.

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

OPHTHALMOSCOPIC EXAMINATION: Yes
The eyes of all animals were examined prior to the start of the administration period. At the end of the administration period, i.e. study day 91, the eyes of animals in test groups 0 (control) and 3 (1000 mg/kg bw/d) were examined for any changes using an ophthalmoscope (HEINE OPTOTECHNIK, Herrsching, Germany) after application of a mydriatic agent (Mydrum, Chauvin ankerpharm GmbH, Rudolstadt, Germany).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in the morning
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters examined: Leukocyte count, Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Platelet count, Differential blood count, Reticulocytes, Prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in the morning
- Animals fasted: Yes
- How many animals: all
- Parameters examined: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, γ-Glutamyltransferase, Sodium, Potassium, Chloride, Inorganic phosphate, calcium, Urea, creatinine, glucose, Total bilirubine, total proteine, albumine, globulines, triglycerides, cholesterol

URINALYSIS: Yes
- Time schedule for collection of urine: overnight
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: pH, Protein, Glucose, Ketones, Urobilinogene, Blood, Specific gravity, Sediment, Color, turbidity, Volume

NEUROBEHAVIOURAL EXAMINATION: Yes
A functional observational battery (FOB) was performed in all animals at the end of the administration period starting at about 10:00 h. At least one hour before the start of the FOB the animals were transferred to single-animal polycarbonate cages. Drinking water was provided ad libitum, but no food was offered during the measurements. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensory motor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.
- Battery of functions tested: Home cage observations, Open field observations, Sensory motor tests/ reflexes, Motor activity assessment
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
The animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (terminal body weight)
2. Adrenal glands
3. Brain
4. Epididymides
5. Heart
6. Kidneys
7. Liver
8. Ovaries
9. Spleen
10. Testes
11. Thymus
12. Thyroid glands
13. Uterus with cervix

HISTOPATHOLOGY: Yes
See table below for details
Statistics:
- DUNNETT's test (two-sided): Body weight, body weight change
- KRUSKALWALLIS test (two-sided): rearing, grip strengthforelimbs, grip strength hindlimbs, foot-splay test, motor activity, Blood parameters, Urine pH, volume and specific gravity, organ weight parameters
- WILCOXON-test (one-sided): Urinalysis parameters (apart from pH, urine volume, specific gravity, color and turbidity)

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Salivation after treatment from slight to severe was observed in all male and female animals of test group 3 (1000 mg/kg bw/d) as well as salivation after treatment from slight to moderate in all male and 5 of 10 female animals of test group 2 (300 mg/kg bw/d). From the temporary, short appearance immediately after dosing it was considered that salivation was induced by a bad taste of the test substance or local affection of the upper digestive tract. No abnormal clinical findings were observed for male and female animals in test group 1 (100 mg/kg bw/d).
Mortality:
no mortality observed
Description (incidence):
No animal died prematurely in the present study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related changes of mean body weights and mean body weight change values in both sexes were observed. For male and female animals of test group 3 (1000 mg/kg bw/d) mean body weight change value was slightly significantly lower on study day 7 (-11% in males and -28% in females, in females not statistically significant). The decreased body weight gain in the first days of administration period of both sexes were assessed as treatment-related but not adverse because of their transient occurrence. For female animals of test group 3 (1000 mg/kg bw/d) mean body weight change value was slightly significantly higher on study day 28 (+15%). This single and isolated finding was assessed as spontaneous in nature and not related to treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test substance-related findings were observed.
Food efficiency:
no effects observed
Description (incidence and severity):
No test substance-related findings were observed.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No overt changes with respect to water consumption were observed visually for animals treated with the test substance.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
All apparent findings were assessed as being incidental in nature since they occurred in individual animals only and did not show a dose-response relationship.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among hematological parameters were observed. At the end of the administration period in males of test group 2 (300 mg/kg bw/d) hemoglobin and hematocrit values were higher compared to controls. However, the changes were not dose-dependent and therefore they were regarded as incidental and not treatment-related. In males of test group 3 (1000 mg/kg bw/d) absolute and relative neutrophil cell counts were decreased whereas relative lymphocyte counts were increased. The relative cell counts were already changed in males of test group 2 (300 mg/kg bw/d). All cell counts were within historical control ranges. Therefore, these changes were regarded as incidental and not treatment-related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related, adverse changes among clinical chemistry parameters were observed.
At the end of the administration period in females of test groups 1 and 3 (100 and 1000 mg/kg bw/d) inorganic phosphate levels were higher compared to controls. However, the means were within (test group 1) or marginally above (test group 3) the historical control range (inorganic phosphate 1.14-1.55 mmol/L). The parameter was not changed dosedependently regarding test group 1 and in females of test group 3 this was the only changed clinical pathology parameter. Therefore, this alteration was regarded as incidental in test group 1 and maybe treatment-related but not adverse in test group 3 (ECETOC Technical Report No. 85, 2002).
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related, adverse changes among urinalysis parameters were observed.
In males of test group 3 (1000 mg/kg bw/d) urine pH value was decreased and more triple phosphate crystals were found in the urine sediment. These findings without any other change in the renal system was regarded as maybe treatment-related, but not adverse.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Deviations from "zero values" were obtained in several animals. However, as most findings were equally distributed between test-substance treated groups and controls, were without a dose-response relationship or occurred in single animals only, these observations were considered to have been incidental. The following examinations were performed during FOB and have to be assessed individually:
Home cage observations: No test substance-related effects were observed.
Open field observations: No test substance-related effects were observed.
Sensorimotor tests/reflexes: No test substance-related effects were observed.
Quantitative parameters: No test substance-related effects were observed.
For the males of test group 2 (300 mg/kg bw/d) a decreased number of rearing was determined. Without a dose-dependency this finding was considered as incidental and not treatment-related.
Motor activity:
Comparing the single intervals with the control group, interval 3 in males of test group 1 (100 mg/kg bw/d) was significantly increased. This single isolated finding was considered as spontaneous in nature and not treatment-related. Furthermore, interval 12 in females of test group 1 and 3 (100 and 1000 mg/kg bw/d) was significantly decreased in comparison with the corresponding single intervals of control. However, the control group in this interval showed a relative high number of beam breaks. Therefore, these isolated findings in interval
12, without a dose dependency, were considered as incidental in nature and not treatmentrelated. No significant changes were observed in the respective sum of all intervals.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Absolute organ weights:
When compared with control group 0 (=100%), the mean absolute kidney weights were significantly changed in the highest female test group (112%). All other mean absolute weight parameters did not show significant differences when compared to the control group 0.
Relative organ weights:
When compared with control group 0 (=100%), the mean relative kidney weights were significantly changed in the lowest and highest female test groups (96% and 111%,respectively). In addition, the mean relative spleen weights were changed in the lowest female dose group (89%)
All other mean relative weight parameters did not show significant differences when compared to the control group 0.
For the increase of absolute and relative kidney weight in females of test group 3 (1000 mg/kg bw/d) a treatment-related effect seems unlikely due to a missing
histopathological correlate. The decrease in relative spleen weight of females of test group 1 (100 mg/kg bw/d) was regarded to be incidental due to the missing dose response relationship.
Gross pathological findings:
no effects observed
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):
In the kidneys of two males of test group 3 (1000 mg/kg bw/d) unilaterally a minimal focal tubular necrosis was observed. Due to the minimal severity, the fact that only two animals were affected and it was noted only unilaterally, this finding was regarded to be incidental and not related to treatment. All other 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:
no effects observed
Other effects:
no effects observed

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no relevant effects recorded

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
The oral administration of the test item by gavage to male and female Wistar rats for 3 months caused no signs of systemic toxicity up to the highest dose level tested (1000 mg/kg bw/d). Therefore, under the conditions of the present study the no observed adverse effect level (NOAEL) was ≥1000 mg/kg bw/d for male and female Wistar rats.
Executive summary:

The test article was administered orally by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0 (test group 0), 100 (test group 1), 300 (test group 2) and 1000 mg/kg body weight/day (mg/kg bw/d; test group 3) over a period of 3 months. Polyethylene glycol 400 served as vehicle. Food consumption and body weight were determined weekly. The animals were examined for signs of toxicity or mortality at least once a day. In addition, the rats were daily examined for any clinically abnormal signs before and within 2 hours as well as within 5 hours after treatment. Furthermore, detailed clinical examinations in an open field were conducted prior to the start of the administration period and weekly thereafter. Ophthalmological examinations were performed before the beginning and at the end of the administration period. In addition, a functional observational battery (FOB) as well as measurement of motor activity (MA) were carried out at the end of the administration period. Clinicochemical and hematological examinations as well as urinalyses were performed towards the end of the administration period. After the administration period all animals were sacrificed and assessed by gross pathology. Organ weights were determined followed by histopathological examinations. No treatment-related, adverse effects were observed at any of the dose levels in any of the parameters examined. In conclusion, the oral administration of the test article by gavage to male and female Wistar rats for 3 months caused no signs of systemic toxicity up to the highest dose level tested (1000 mg/kg bw/d). Therefore, under the conditions of the present study the no observed adverse effect level (NOAEL) was 1000 mg/kg bw/d for male and female Wistar rats.