Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study, for read-across justification refer to IUCLID section 13.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid
Details on test material:
refer to confidential details on test material

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: 42 ± 1 days
- Weight at study initiation:
- Housing: 5 animals per cage, polysulfonate cages, floor area about 2065 cm2, Dust-free wooden bedding
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours light from 06.00-18.00 h, 12 hours dark from 18.00-06.00 h

IN-LIFE DATES: From: 31 Oct 2014 To: 03 Feb 2015

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food): ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland
- Storage temperature of food: room temperature

Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
1000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
3000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
12000 ppm (males); 15000 ppm (females)
Basis:
nominal in diet
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: 4 week dose finding study
- Rationale for animal assignment (if not random): random
- Section schedule rationale (if not random): random
Positive control:
not required

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once a day


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the start of the administration period and weekly thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations:Prior to the start of the administration period on day -1 and on study day 91
- Dose groups that were examined: all animals (day -1), control and high dose animals (day 91)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: towards the end of the administration period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: towards the end of the administration period
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table [No.?] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: towards the end of the administration period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the administration period
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity / Home cage observations / Open field observations

Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)

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

1. Anesthetized animals
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

Organ/tissue fixation: The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution or in modified Davidson’s solution:

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. Epididymides (modified Davidson’s solution)
12. Esophagus
13. Extraorbital lacrimal glands
14. Eyes with optic nerve (modified Davidson’s solution)
15. Femur with knee joint
16. Harderian glands
17. Heart
18. Ileum
19. Jejunum (with Peyer’s patches)
20. Kidneys
21. Larynx
22. Liver
23. Lungs
24. Lymph nodes (mesenteric and axillary lymph nodes)
25. Mammary gland (male and female)
26. Nose (nasal cavity)
27. Ovaries
28. Oviducts
29. Pancreas
30. Parathyroid glands
31. Pharynx
32. Pituitary gland
33. Prostate
34. Rectum
35. Salivary glands (mandibular and sublingual glands)
36. Sciatic nerve
37. Seminal vesicles
38. Skeletal muscle
39. Skin
40. Spinal cord (cervical, thoracic and lumbar cord)
41. Spleen
42. Sternum with marrow
43. Stomach (forestomach and glandular stomach)
44. Testes (modified Davidson’s solution)
45. Thymus
46. Thyroid glands
47. Trachea
48. Urinary bladder
49. Uterus
50. Vagina

All gross lesions, liver and Thyroid glands were microscopically examined for all animals, all the other organs and tissues that have been fixed were examined for the control and the high dose group.
Statistics:
Body weight, body weight change: A comparison of each group with the control group was performed using DUNNETT's test (two-sided) for the hypothesis of equal means

Feces, rearing, grip strength forelimbs, grip strength hindlimbs, 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 WILCOXON test (two-sided) for the equal medians

Blood parameters: For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. 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 WILCOXON-test (two-sided) for the hypothesis of equal medians; For parameters with unidirectional changes: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) with Bonferroni-Holm adjustment for the hypothesis of equal medians

Urinalysis parameters (apart from pH, urine volume, specific gravity, color and turbidity: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians

Urine pH, volume, specific gravity, color and turbidity: Non-parametric one-way analysis using KRUSKAL-WALLIS test. 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 WILCOXON-test (two-sided) for the hypothesis of equal medians. Urine color and turbidity are not evaluated statistically.

Pathology (Weight parameters): 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 WILCOXON-test (two-sided) for the equal medians

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
Test group 3: 12000 ppm in males and 15000 ppm in females
(875 mg/kg bw/d in males and 1361 mg/kg bw/d in females)

Clinical Examinations

• No treatment-related, adverse findings were observed.

Clinical Pathology

• Increased alkaline phosphatase (ALP) activities, urea and potassium levels were observed in male animals.
• Increased γ-glutamyltransferase (GGT) activities were observed in females.
• Decreased total protein and albumin levels were observed in females.
• Increased incidences of granulated and epithelial casts were observed in males.

Pathology

• Increased mean absolute and relative liver weights were observed in males and females (in combination with clinical pathology).
• Minimal diffuse hepatocellular hypertrophy was observed in 6 (out of 10) male and in 7 (out of 10) female animals (in combination with clinical pathology parameters).


Test group 2: 3000 ppm
(209 mg/kg bw/d in males and 244 mg/kg bw/d in females)

Clinical Examinations, Clinical Pathology and Pathology

• No treatment-related, adverse findings were observed.


Test group 1: 1000 ppm
(71 mg/kg bw/d in males and 85 mg/kg bw/d in females)

Clinical Examinations, Clinical Pathology and Pathology

• No treatment-related, adverse findings were observed.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
3 000 ppm
Based on:
other: test item in the diet
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Effect level:
209 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Dose descriptor:
NOAEL
Effect level:
244 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Regarding clinical pathology, slightly increased alkaline phosphatase (ALP) activities in males of test group 3 (12000 ppm) and slightly increased γ-glutamyl transferase (GGT) activities in females of the same test group (15000 ppm) indicated a liver cell swelling because of liver enzyme induction. Higher urea levels in males of test group 3 (12000 ppm) and decreased total protein and albumin levels in females of the same test group (15000 ppm) were due to an increased protein metabolism in the liver.

 

Regarding pathology, target organs were the liver and the thyroid glands.

The absolute (♂ +16% / ♀ +26%) and relative (♂ +25% / ♀ +28%) liver weights were significantly increased in males and females of test group 3 (12000 [males] and 15000 [females] ppm).The increased liver weights correlated with a minimal diffuse hepatocellular hypertrophy that was observed in 6 (out of 10) male and in 7 (out of 10) female animals in this treatment group. These findings were considered to bea correlate for a microsomal enzyme induction in the liver. They were regarded to betreatment-related and adverse taking the changes in clinical pathology parameters into account.

The incidence of follicular hypertrophy/hyperplasia and of altered colloid in the thyroid glands was minimally increased in male animals of test group 3 (12000 ppm). These findings wereregarded to be secondary to an induced UDP-glucuronyl transferase activity in hepatocellular hypertrophy.The well-known phenomenon is characteristic for rodents (Curran et al., 1991) and leads to an accelerated degradation of T4 with a compensatory increase of TSH causing thyroid gland hypertrophy/hyperplasia of follicular cells. These findings were regarded to be treatment-related but not relevant for humans.

Applicant's summary and conclusion