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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

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. 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
- Age at at the beginning of the administration period: 6 weeks
- Fasting period before study: no
- Housing: 5 animals per cage) in polysulfonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany (floor area about 2065 cm2).
- Diet: ad libitum; ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland
- Water: ad libitum
- Acclimation period: 7 days

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was applied as a solution. 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 subsequently homogenized with a magnetic stirrer. All test item formulations were prepared once weekly.

VEHICLE
- Concentration in vehicle: 0.5, 2.5, 12.5 g/100 ml for low, mid and high dose group respectively.
- Amount of vehicle (if gavage): 4 ml/ kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of Anisaldehyde in corn oil at room temperature for a period of 7 days was proven before the start of the administration period.
Concentration control was performed in all concentrations at the beginning and towards the end of the administration period.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
20 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Positive control:
not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
Mortality: A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.
Clinical observations: 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.
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 high). The following parameters were examined:
1. Abnormal behavior when handled
2. Fur
3. Skin
4. Posture
5. Salivation
6. Respiration
7. Activity/arousal level
8. Tremors
9. Convulsions
10. Abnormal movements
11. Impairment of gait
12. Lacrimation
13. Palpebral closure
14. Exophthalmus
15. Feces (appearance/ consistency)
16. Urine
17. Pupil size

BODY WEIGHT: Yes
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 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 day 0 was calculated as body weight change.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption was determined weekly and calculated as 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
Prior to the start of the administration period on day -1 the eyes of all animals and on study day 90 the eyes of the control and high-dose animals were examined for any changes using an ophthalmoscope after administration of a mydriatic agent.

HAEMATOLOGY: Yes
In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane.
The following parameters were determined in blood with EDTA K3 as anticoagulant using a particle counter:
- Leukocyte count
- Erythrocyte count
- Hemoglobin
- Hematocrit
- Mean corpuscular volume
- Mean corpuscular hemoglobin
- Mean corpuscular hemoglobin concentration
- Platelet count
- Differential blood count
- Reticulocytes
Clotting tests were carried out using a ball coagulometer: Prothrombin time (Hepato Quick’s test)

CLINICAL CHEMISTRY: Yes
In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane.
The following parameters were determined:
- Alanine aminotransferase
- Aspartate aminotransferase
- Alkaline phosphatase
- gamma-Glutamyltransferase
- Sodium
- Potassium
- Chloride
- Inorganic phosphate
- Calcium
- Urea
- Creatinine
- Glucose
- Total bilirubin
- Total protein
- Albumin
- Globulins
- Triglycerides
- Cholesterol

URINALYSIS: Yes
On the afternoon preceding the day fixed for urinalysis, the animals were transferred individually into metabolism cages (no food or drinking water provided). Urine was sampled overnight. On the following day, the samples were examined in a randomized sequence.
The following parameters were determined:
- Protein
- Glucose
- Ketones
- Urobilinogen
- Bilirubin
- Blood
- Specific gravity
- Sediment
- Color, turbidity
- Volume
- pH

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 rats were transferred to single-animal polycarbonate cages type III and small amounts of bedding material. The FOB started with passive observations without disturbing the rats, 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.
Home cage observations: The animals were observed in their closed home cages; during this period, any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the rats. Attention was paid to:
1. Posture
2. Tremors
3. Convulsions
4. Abnormal movements
5. Gait
6. Other findings

Open field observations: The animals were transferred to a standard arena (50 × 50 cm with sides of 25 cm height) and observed for at least 2 minutes. The following parameters were examined:
1. Behavior on removal from the cage
2. Fur
3. Skin
4. Salivation
5. Nasal discharge
6. Lacrimation
7. Eyes/ pupil size
8. Posture
9. Palpebral closure
10. Respiration
11. Tremors
12. Convulsions
13. Abnormal movements/ stereotypes
14. Gait
15. Activity/ arousal level
16. Feces excreted within 2 minutes (appearance/ consistency)
17. Urine excreted within 2 minutes (amount/ color)
18. Rearing within 2 minutes
19. Other findings

Sensory motor tests/ reflexes: The animals were then removed from the open field and subjected to following sensory motor or reflex tests:
1. Reaction to an object being moved towards the face (approach response)
2. Touch sensitivity (touch response)
3. Vision (visual placing response)
4. Pupillary reflex
5. Pinna reflex
6. Audition (auditory startle response)
7. Coordination of movements (righting response)
8. Behavior during handling
9. Vocalization
10. Pain perception (tail pinch)
11. Grip strength of forelimbs
12. Grip strength of hindlimbs
13. Landing foot-splay test
14. Other findings

Motor activity assessment
Motor activity (MA) was also measured from 14:00 h onwards on the same day as the FOB was performed. The examinations were performed using the TSE Labmaster System. For this purpose, the rats were placed in new clean polycarbonate cages. For motor activity (MA) measurements, the animals were housed individually in polycarbonate cages type III with a small amount of bedding for the duration of the measurement. Eighteen beams were allocated per cage. 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. On account of the time needed to place the rats in the cages, the starting time was "staggered" for each animal. 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 after the transfer of the last rat.



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
The following weights were determined in all animals sacrificed on schedule:

1. Anesthetized animals (terminal body weight)
2. Adrenal glands
3. Brain
4. Cauda epididymis
5. Epididymides
6. Heart
7. Kidneys
8. Liver
9. Ovaries
10. Pituitary gland
11. Prostate
12. Seminal vesicles with coagulating glands
13. Spleen
14. Testes
15. Thymus
16. Thyroid glands
17. Uterus with cervix

HISTOPATHOLOGY: Yes (see table)
Organs/tissues were fixed in 4% neutral-buffered formaldehyde solution. The left testis and left epididymis of all animals sacrificed at scheduled dates were fixed in modified Davidson’s solution, whereas the right testis and epididymis were used for sperm parameters.
Histopathological examination was performed in:
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Epididymis, left
12. Esophagus
13. Eyes with optic nerve
14. Female mammary gland
15. Heart
16. Ileum
17. Jejunum
18. Kidneys
19. Liver
20. Lung
21. Lymph nodes (mesenteric and axillary lymph nodes)
22. Ovaries
23. Pancreas
24. Parathyroid glands
25. Peyer’s patches
26. Pituitary gland
27. Prostate
28. Rectum
29. Salivary glands (mandibular and sublingual glands)
30. Sciatic nerve
31. Seminal vesicles
32. Skin
33. Spinal cord (cervical, thoracic and lumbar cord)
34. Spleen
35. Stomach (forestomach and glandular stomach)
36. Testis, left
37. Thymus
38. Thyroid glands
39. Trachea
40. Urinary bladder
41. Uterus
42. Vagina
Other examinations:
Estrous cycle determination: Estrous cycle length and normality were evaluated daily for all female animals for a minimum of 3 weeks prior to necropsy.

Sperm parameters: After the organ weight determination, the following parameters were determined in the right testis or right epididymis of all male on schedule:
- Sperm motility
- Sperm morphology
- Sperm head count (cauda epididymis)
- Sperm head count (testis)


Statistics:
see "Any other information on materials and methods incl. tables".

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Not considered adverse
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
effects observed, non-treatment-related
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
Mortality
No animal died prematurely in the present study.

Clinical examinations and detailed clinical observations
No test substance-related, adverse effects were obtained in any test group.
Salivation shortly after treatment was observed in all male and female animals of test group 3 (500 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.

Food consumption
No test substance-related, adverse changes with regard to food consumption were observed.

Water consumption
No test substance-related, adverse changes with regard to water consumption were observed.

Body weight data
No test substance-related, adverse effects on body weight development were obtained in any test group. Mean body weights and body weight change values of male and female animals did not show any significant deviations to the control. However, a slight decrease in male mean body weights (-5.3% deviation vs controls on study day 91) and body weight gains (-8.4% deviation vs controls in the period of day 0 - day 91) has been observed in the high dose group (500 mg/kg bw/d) during the course of the study.

Functional observational battery
- Quantitative parameters: No test substance-related effects were observed.
- 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.
- Motor activity measurement:
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 (20, 100 and 500 mg/kg bw/d).
In male animals of test group 1 (20 mg/kg bw/d) interval No. 8 as well as the overall motor activity were significantly higher. In male animals of test group 3 (500 mg/kg bw/d) interval No. 2 was significantly lower when compared to the mean control value. Since no dose-response relationship occurred, a relation to treatment was excluded.

Ophthalmological examinations
No treatment-related findings were observed.

Estrous cycle
No test substance-related effects on estrous cycle length and the number of cycles were obtained.

Hematology
Test Group 3 (500 mg/kg bw/d):
In males and females: At the end of the administration period, absolute and relative eosinophil counts were decreased (absolute eosinophil counts in females not statistically significantly). This change was regarded as adverse. Deviation of mean absolute/ relative eosinophil counts vs ctrl was -80.9%/-78.7% in males and -39.76%/ -41.6% in females.
In females: absolute reticulocyte counts were significantly increased (192.3 Giga/L), but the values were within the historical control range (females, absolute reticulocytes 124.2-199.7 Giga/L). Therefore, the mentioned changes in this paragraph were regarded as incidental and not treatment-related.
Test Group 2 (100 mg/kg bw/d):
In males: absolute eosinophil counts (Deviation vs ctrl. -35.96%) were already significantly lower compared to controls. However, in this test group this was the only changed clinical pathology parameter among these individuals. Therefore, this alteration was regarded as treatment-related, but non-adverse (ECETOC Technical Report No. 85, 2002).
In females: relative neutrophil counts were significantly higher (Deviation vs ctrl. +31.5%) and relative lymphocyte counts (Deviation vs ctrl. -5.7%) were significantly lower compared to controls. Both parameters were not dose-dependently changed. Absolute reticulocyte counts were significantly increased, but the values were within the historical control range (females, absolute reticulocytes 124.2-199.7 Giga/L). Therefore, the mentioned changes in this paragraph were regarded as incidental and not treatment-related.

No adverse and treatment related effects on these hematological parameters were observed in the low dose group. Other hematological parameters investigated in treated animals did not show adverse and treatment related changes.

Clinical chemistry
Test Group 3 (500 mg/kg bw/d):
In males and females: At the end of the administration period, total protein values were significantly decreased (Deviation vs ctrl.-2.99% and - 2.61% in males and females respectively). These alterations were regarded as adverse.
In males: glucose (Deviation vs ctrl. +15.21%) and inorganic phosphate (Deviation vs ctrl. +17.98%) levels were increased. These alterations were regarded as adverse. Higher chloride levels (mean 102.6 mmol/L) were regarded as incidental and not treatment-related, because the values were within historical control ranges (males, chloride 98.8-105.9 mmol/L).
In females: lower calcium levels (mean 2.52 mmol/L) were regarded as incidental and not treatment-related, because the values were within historical control ranges (females, calcium 2.50-2.69 mmol/L).

No adverse and treatment related effects on these clinical chemistry parameters were observed in the mid and low dose group. Other clinical chemistry parameters investigated in treated animals did not show adverse and treatment related changes.

Urinalyses
Test Group 3 (500 mg/kg bw/d):
At the end of the administration period, in male and female rats urine pH values were decreased. Additionally, in females of this test group specific gravity of the urine was increased without a significant lower urine volume, and significantly higher incidences of crystals of unknown origin were found. These changes were regarded as adverse.


No adverse and treatment related effects on these urinalysis parameters were observed in the mid and low dose group. Other urinalysis parameters investigated in treated animals did not show adverse and treatment related changes.

Sperm parameters
Test Group 3 (500 mg/kg bw/d):
At the end of the administration period, in males motility of the sperms (mean 0.8 vs 85.7% in controls) and the sperm head counts in the cauda epididymidis (mean 317 vs 731.9 Mio/g in controls) were significantly decreased, whereas the incidence of abnormal sperms in the cauda epididymidis was significantly increased (mean 94.4 vs 5.0 % in controls). Among the abnormal sperms mostly those with bent heads or missing heads or broken tails or combined abnormalities were found. Spermatid cell counts in the testis were not affected in this test group (109.8 vs 98.6 Mio/g in controls). The mentioned changes were regarded as adverse.
Test Group 1 (100 mg/kg bw/d):
Motility of the sperms in males was significantly lower compared to controls (mean 78.0 vs 85.7% in controls) and the mean was marginally below the historical control range (motility of sperms 79-93%). However, no other sperm analysis parameter in this test group was significantly changed and the values of the sperm head counts in the cauda epididymidis (627.5 Mio/g) and the incidences of abnormal sperms (5.35%) were within historical control ranges (sperm head counts in the cauda epididymidis 485-890 Mio/g; abnormal sperms 5.0-6.8%). In addition, no histopathologic findings were observed in the male sex organs of this test group (see Histopathology). Therefore, the slightly lower motility of the sperms in males was regarded as treatment-related but non-adverse (ECETOC Technical Report No. 85, 2002).

No adverse and treatment related effects on sperm parameters were observed in the low dose group.

Organ weights
Absolute organ weights (significant changes):
Test Group 3 (500 mg/kg bw/d):
Males: Cauda epididymis (71% of control); Epididymides: (83% of control)
Females: Liver (114% of control); Ovaries (81% of control)

Relative organ weights (significant changes):
Test Group 3 (500 mg/kg bw/d):
Males: Cauda epididymis (77% of control); Heart (108% of control); Kidneys (110% of control); Liver (117% of control)
Females: Kidneys (111% of control); Liver (118% of control)

The significantly decreased weights of the cauda epididymis (absolute and relative) and epididymides (absolute) showed a histopathological correlate and were considered treatment-related.
The significantly increased liver weights in male (relative) and female animals (absolute and relative) were slightly above historical control values and showed a histopathological correlate which was regarded as treatment-related.
The significant weight decrease of the ovaries (91.6 mg), as well as the significant relative weight increase of the heart in males (0.288%) and kidneys in females (0.73%) were all within the historical control values (ovaries: 82.5 – 114.2 mg; heart: 0.248 – 0.31%; kidneys: 0.652 – 0.746%). None of these organs showed histopathological changes and therefore the weight changes were not considered to be treatment-related. The significant relative weight increase of the kidneys in males (0.672%) was minimally above the historical control range (0.544 – 0.652%) but showed no histopathological correlate. This weight increase was considered secondary to the decreased terminal body weight (-8%) and was not attributed to the treatment. All other mean relative weight parameters did not show significant differences when compared to the control group.

Gross lesions
All findings observed occurred individually and were considered incidental or spontaneous in origin and without any relation to treatment.


Histopathology
Treatment-related findings were observed in the left epididymis of males, and in the liver of male and female animals in test groups 3 (500 mg/kg bw/d) with incidences and gradings according to the tables below:

Epididymides: Treatment-related findings were observed in the left epididymis of males in test groups 3 (500 mg/kg bw/d)
Ductal Atrophy in 1/10 (grade 1), 5/10 (grade 2) and 4/10 (grade 3) animals.
Oligospermia in 5/10 (grade 1) and 5/10 (grade 2) animals
The ductal atrophy was observed at the distal corpus and proximal cauda, and was characterized by narrowing of the ductal diameter and lumina, associated with increased epithelial height. In some animals, an intraductal folding of the epididymal epithelium giving rise to a pseudoglandular epithelial pattern (cribiform change) was also noted. The ductal atrophy was associated with oligospermia.

Liver: Treatment-related findings were observed in the liver of male and female animals in test groups 3 (500 mg/kg bw/d)
Centrilobular hypertrophy (grade 1) in 5/10 males (vs. 1/10 controls) and in 6/10 females (vs. 0/10 controls).

All other findings observed 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.

Effect levels

Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
clinical biochemistry
urinalysis
organ weights and organ / body weight ratios
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (nominal)
System:
male reproductive system
Organ:
cauda epididymis
Treatment related:
yes

Applicant's summary and conclusion