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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:
27 November 2018 - 19 February 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
25 June 2018
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Purity > 99 %
- Batch No.of test material:
180006PO40
- Expiration date of the batch:
Stable until 04 July 2019
- Storage condition of test material:
Avoid temperatures >45 °C; ambient (room temperature)
- Stability under test conditions:
The stability of the test substance under storage conditions over the test period was guaranteed.

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on species / strain selection:
The rat is a frequently used laboratory animal, and there is comprehensive experience with this animal species and the Wistar strain. Moreover, the rat has been proposed as a suitable animal species by the OECD and the EPA for this type of study.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 42 ± 1 days
- Weight at study initiation: male: 150.8 - 171.8 g; female: 116.4 - 144.6 g
- Fasting period before study: no
- Housing: group housing (5 animals/cage), polysulfonate cages (floor area about 2065 cm^2), dust-free wooden bedding, wooden gnawing blocks and large play tunnels
- Diet: ad libitum, mouse and rat maintenance diet “GLP” (Granovit AG, Kaiseraugst, Switzerland)
- Water: ad libitum, tap water in water bottles
- Acclimation period: 8 days

DETAILS OF FOOD AND WATER QUALITY:
The supplier assayed the food used in the study for chemical and microbiological contaminants. On the basis of duration of use and the analytical findings with respect to chemical and microbiological contaminants, the diet was found to be suitable.
The drinking water is regularly assayed for chemical contaminants by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring Department of BASF SE as well as for the presence of microorganisms by a contract laboratory. On the basis of the analytical findings, the drinking water was found to be suitable. German “Trinkwasserverordnung” (Drinking Water Regulation) served as a guideline for maximum tolerable contaminants.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 45 - 65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route was selected since this was proven to be suitable for the detection of a toxicological hazard.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was applied as a suspension. To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, corn oil was filled up to the desired volume and homogenized with a magnetic stirrer.
- Rate of preparation: once per week

VEHICLE
- Concentration in vehicle: 60 mg/kg bw: 1.5 g/100 mL; 180 mg/kg bw: 4.5 g/100 mL; 540 mg/kg bw: 13.5 g/100 mL
- Amount of vehicle: 4 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneous distribution of the test substance in preparations was performed in the highest and lowest concentration. Additionally, concentration control was performed in all concentrations at the beginning and towards the end of the administration period.
The test substance was determined to be homogeneously distributed in the vehicle and the determined concentrations corresponded to 99 - 103 % of the nominal concentrations.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
60 mg/kg bw/day (nominal)
Dose / conc.:
180 mg/kg bw/day (nominal)
Dose / conc.:
540 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The selection of dose levels based mainly on the results of a test study in female Wistar rats conducted at dose levels of 0, 600 and 1000 mg/kg bw/d. In this study, a NOAEL was not established due to erosions and ulcerations in the stomach of different animals at both dose levels of 600 and 1000 mg/kg bw/d as well as clinical findings like poor general condition, piloerection and body weight loss after 1 week of treatment.
In a subsequently performed OECD 422 study male and female parental animals were treated at dose levels of 50, 150 and 500 mg/kg bw/d. In this study, neither signs of general systemic toxicity nor of local irritation in the stomach were observed.
Considering dose levels of both previous studies the dose levels of this study were selected.

- Fasting period before blood sampling for clinical biochemistry: at least 16 hours
Positive control:
No positive control was used.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily, before administration and within 2 and 5 hours after administration

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the administration period and thereafter at weekly intervals
- Parameters checked in table No.1 were examined.

BODY WEIGHT: Yes
- Time schedule for examinations: prior to the administration period, on day 0 and afterwards weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption was determined weekly and calculated as mean food consumption in grams per animal and day.

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: No

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
- Time schedule for examinations: prior to the start of the administration period on day -1 / 0 and on study day 91
- Dose groups that were examined: day -1/0: all animals; day 91: control and high-dose animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before sacrifice
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, at least 16 hours
- How many animals: all
- Parameters checked in table No.5 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before sacrifice
- Animals fasted: Yes, at least 16 hours
- How many animals: all
- Parameters checked in table No.6 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, during urine collection (overnight)
- Parameters checked in table No.7 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the administration period starting
- Dose groups that were examined: all (also all animals)
- Battery of functions tested: Home cage and open field observations, sensory activity (incl. grip strength) and motor activity
- Parameters checked in table No.2-4 were examined.
- Details on motor activity assessement: The examinations were performed using the TSE Labmaster System (TSE Systems GmbH, Bad Homburg, Germany). The number of beam interrupts was counted over 12 intervals for 5 minutes per interval. The sequence in which the rats were placed in the cages was selected at random. The measurement period began when the 1st beam was interrupted and finished exactly 1 hour later. No food or water was offered to the rats during these measurements and the measurement room was darkened.

IMMUNOLOGY: No

OTHER:
- Thyroid hormones (total triiodothyronine (T3), total thyroxine (T4), thyroid stimulating hormone (TSH)) were determined using the blood samples collected.
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. Organ weights were determined (table No. 8). All paired organs were weighed together (left and right).

HISTOPATHOLOGY: Yes
Organs or tissues that were fixed in 4 % neutral buffered formaldehyde solution or in modified Davidson’s solution are listed in table No. 9. The assessement was done for all organs and animals for control and high dose groups. For low and median dose groups assessment of gross lesions was done in animals affected.
Statistics:
Please refer to table No. 10 for details on statistical methods.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No test substance-related, adverse effects were obtained in any test group (60, 180 and 540 mg/kg bw/d).

Salivation shortly after treatment was observed in all male and female animals treated with 180 mg/kg bw/d and 540 mg/kg bw/d as well as in 9 male and 7 female animals of test group 1 (60 mg/kg bw/d) on several days of the application period.
From the temporary, short appearance immediately after dosing it was concluded that the finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. The effect was related to the test substance but assessed as being non-adverse as no lesions in the upper digestive tract were observed in male and female animals during pathological examinations.
One female animal of the control group showed respiration sounds on study day 51. Since the animal belonged to the control group, a relation to the test substance was excluded.
Mortality:
no mortality observed
Description (incidence):
No animal died prematurely.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test substance-related, adverse effects on body weight development were obtained in any test group (60, 180 and 540 mg/kg bw/d).
Mean body weights and body weight change values of male and female animals did not show any significant deviations to the control.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test substance-related, adverse changes with regard to food consumption were observed.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No test substance-related, adverse changes with regard to water consumption were observed.
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related findings were observed.
All other apparent findings were also assessed as being incidental in nature since they occurred in control as well as in treated animals 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, platelet counts were significantly decreased in males of test group 3 (540 mg/kg bw/d; 650 giga/L, -12 % compared to control group, p ≤ 0.01), but the mean was within the historical control range (males, platelets 592-768 giga/L).
In males of test groups 1 and 3 (60 and 540 mg/kg bw/d), relative basophil counts were significantly increased (60 mg/kg bw/d 78.6 % (mean 0.2 %) and 540 mg/kg bw/d 171.4 % (mean 0.4 %) compared to control group, p ≤ 0.05), and in females of test group 3 relative neutrophil counts were significantly decreased (-22.5 % (mean 15.4 %), p ≤ 0.01). However, all values were within historical control ranges (males, relative basophils 0.1-0.4 %; females, relative neutrophils 14.6-24.9 %).
Therefore, all mentioned alterations were regarded as incidental and not treatment-related.
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, glucose levels were significantly decreased in males of test groups 2 and 3 (180 mg/kg bw/d: -13.44 % (mean 5.62 mmol/L) p ≤ 0.01, 540 mg/kg bw/d: -11.82 % (mean: 5.72 mmol/L) p ≤ 0.05), but the means were within the historical control range (males, glucose 5.46-6.98 mmol/L).
In males of test group 1 (60 mg/kg bw/d), inorganic phosphate levels were significantly decreased, but the change was not dose-dependent.
Therefore, all mentioned alterations were regarded as incidental and not treatment-related.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No treatment-related changes among urinalysis parameters were observed.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, were without a dose response relationship or occurred in single rats only, these observations were considered to have been incidental.
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.

Regarding the overall motor activity as well as single intervals, no test substance-related deviations to the control animals were noted for male and female animals of test groups 1-3 (60, 180 and 540 mg/kg bw/d).
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Absolute weights
When compared with control group 0 (2.18 g, set to 100 %), the mean absolute brain weights were significantly reduced in all test groups (60 mg/kg bw/d: -4.2 % (2.09 g), 180 mg/kg bw/d: -3.5 % (2.11 g), 540 mg/kg bw/d: -4.7 % (2.08 g); all p ≤ 0.05). All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

Relative organ weights
All mean relative weight parameters did not show significant differences when compared to the control group 0.

The significant, but only minimally decreased mean absolute brain weights in male animals of test groups 1 to 3 (60, 180 and 540 mg/kg bw/d) did not show a dose-response relationship. In addition, no significance was observable for the relative weights and all absolute values were within the historical control data (2.015 - 2.199 g). Thus, the changes were regarded not to be related to treatment.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All findings occurred individually. 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
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormones:
In males and females of test groups 1, 2 and 3 (60, 180 and 540 mg/kg bw/d) no treatment related alterations of T3, T4 and TSH levels were observed.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
540 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No test substance related adverse effects were observed.

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Discussion

The test substance was administered orally by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0 (test group 0), 60 (test group 1), 180 (test group 2) and 540 mg/kg bw/d (test group 3) over a period of 3 months.

Regarding clinical examinations, signs of general systemic toxicity were not observed in male or female parental animals of test groups 1-3 (60, 180 and 540 mg/kg bw/d) during the entire study period.

Salivation after treatment was seen in all animals of test groups 2 and 3 (180 and 540 mg/kg bw/d) and sporadically in a few animals of test group 1 (60 mg/kg bw/d). From the temporary, short appearance immediately after dosing it is likely, that this finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. This finding was not considered to be an adverse and toxicologically relevant effect.

Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose level of the compound of 540 mg/kg bw/d.

Regarding pathology, 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.

 

CONCLUSION

Under the conditions of this study the oral administration of the test substance by gavage to male and female Wistar rats over a period of 3 months did not reveal adverse signs of toxicity even at a dose level of 540 mg/kg bw/d.

Thus, the no observed adverse effect level (NOAEL) for was set to 540 mg/kg bw/d for male and female Wistar rats.

Applicant's summary and conclusion