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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:
2013-2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
CAS: 7473-98-5
Batch identification: .0007989197
Purity: 99.6%
Physical state/appearance: liquid/ colourless to yellowish, clear
Storage conditions: ambient (room temperature), under light exclusion
Stability: 07 Nov 2014

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, Sulzfeld, Germany
- Age when supplied: 30 ± 1 day(s)
- Age at the start of administration period: 42 ± 1 day(s)
- Housing: 5 animals per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: one week

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

IN-LIFE DATES: From: 15.01.2013 To: 30.04.2013

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
drinking water containing 1% carboxymethylcellulose
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: All test item formulations were prepared once weekly.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis was carried out at in-house as a part of this study. All studies were carried out in compliance with the Principles of Good Laboratory Practice
Duration of treatment / exposure:
The test item was administered daily for 92 days to male and for 93 days to female animals
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
400 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on 28d study
- Rationale for selecting satellite groups: no satellite groups, no post-observation period
Positive control:
no

Examinations

Observations and examinations performed and frequency:
Mortality: A check for moribund and dead animals was made twice daily on working day and once daily on Saturday, Sunday and public holidays. If animals were in a moribund state, they were sacrificed and dissected.

Clinical observation: All rats were checked daily for any abnormal clinical signs prior administration, as well as, within 2 hours and 5 hours post administration.

Detailed Clinical Observation: Detailed clinical observation (DCO) was 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 during “handling”
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. exophthalmos
15. feces (appearance/ consistency)
16. urine
17. pupil size

Food consumption: was determined weekly over a period of 1 day and calculated as mean food consumption grams per animal and day.

Water consumption: was monitored daily by visual inspection of water bottles for any abnormal changes in volume.

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 body weight on the respective day of weighing and body weight on day 0 was calculated as body weight change.

Functional Observational Battery: FOB was performed in all animals per sex and group towards the end of the administration period, started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests, as well as, reflex tests

Ophthalmoscopy: Prior to the start of administration period, eyes of all animals were examined with an ophthalmoscope (HEINE OPTOTECHNIK, Herrsching, Germany) after treatment with a mydriatic agent. At the end of the administration period, eyes of the control and highest dose group animals were re-examined on study day 85.

Hematology: In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence (table 1 and 2).

Urinalysis: For urinalysis the individual animals were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight. Urine samples were evaluated in a randomized sequence (table 5).

Clinical chemistry: In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence (table 3 and 4)
Sacrifice and pathology:
HISTOPATHOLOGY: Yes (see table 6)

GROSS PATHOLOGY:
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

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
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
45. Thymus
46. Thyroid glands
47. Trachea
48. Urinary bladder
49. Uterus
50. Vagina
Statistics:
Please refer to tables 7 and 8

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
transient salivation and slight transient gait ataxia
Mortality:
mortality observed, treatment-related
Description (incidence):
transient salivation and slight transient gait ataxia
Body weight and weight changes:
no effects observed
Food efficiency:
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the study in male and female rats of test group 3 (400 mg/kg bw/d) cholesterol and calcium levels were increased
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
calcium oxalate and crystals of unknown origin were found in the urine sediment
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
increase in liver and kidney weights
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
liver and kidney
Details on results:
Clinical observations and Detailed Clinical Observation (DCO)
Salivation in different grades was observed in one of 10 males of test group 2 (150 mg/kg bw/d) and in 8 of 10 male animals of test group 3 (400 mg/kg bw/d). The transient salivation showed a dose-dependency and was therefore seen as treatment-related but not adverse.
Additionally, four male animals of the high dose group, male animal no. 33 showed a slight transient gait ataxia immediately after the administration on study day 66. The slight transient gait ataxia immediately after the administration was seen as treatment-related and adverse.

Clinical chemistry
At the end of the study in male and female rats of test group 3 (400 mg/kg bw/d) cholesterol and calcium levels were increased. Additionally, in females of the same test group creatinine levels were decreased.

Urinalysis
In females of test group 3 (400 mg/kg bw/d) calcium oxalate and crystals of unknown origin were found in the urine sediment. In males of test group 3 (400 mg/kg bw/d) pH values were lower and specific gravity in the urine was higher compared to controls. These findings were regarded as treatment-related, but per se not as adverse effects.

Organ weights
Increase in relative and absolute kidney and liver weight in males and females. The increases in liver weights of test group 3 (400 mg/kg bw/day) animals of both sexes and of test group 2 (150 mg/kg bw/day) males were regarded to be treatment related. As there was an apparent dose - response, the increased weight of kidneys of test group 3 (400 mg/kg bw/day) females, a treatment related effect cannot be ruled out even in the absence of a histopathological correlate.

Histopathology
Centrilobular hypertrophy correlating to the observed weight increase was seen in all male and female animals of test group 3 (400 mg/kg bw/day) with grading.
In the kidney of test group 3 male animals, there was an increased severity of basophilic tubules. This was accompanied by granular casts in 6/10 test group 3 animals as compared to none in the controls. These findings were also present in test group 2: 1/10 animals showing a granular cast and 2/10 with slight basophilic tubules. Basophilic tubules in conjunction with granular casts were considered to be adverse.
An accumulation of alpha 2u protein was excluded by special staining and therefore does not explain the observed findings.
Although there was no histopathological correlate, the weight increase of kidneys of female animals of test group 3 was regarded to be treatment - related and non-adverse.

Effect levels

Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Executive summary:

In this guideline (OECD 408) study conducted with GLP certification, the repeat dose toxicity NOAEL of the test material (EC 231-272-0) was determined to be 50 mg/kg bw/day. The test was conducted in rats, with dose levels of 50, 150 and 400 mg/kg bw/day. Non-adverse changes (clinical observations, clinical chemistry, urinalysis, organ weights and histopathology) were observed, primarily in the highest dosage group.