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Description of key information

OECD 408 study (Repeated Dose Oral Toxicity 90 days study) conducted to recognised training guidelines with CLP certification.

Repeated dose toxicity study (28 days) conducted to generally recognised scientific standards.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

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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
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
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
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
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
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
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.
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
Critical effects observed:
not specified
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.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Principles of method if other than guideline:
Dose-range finding study with low number of animals per group, limited set of investigations
GLP compliance:
no
Remarks:
performed with laboratory standard but no formal GLP, since the study was designed as a dose-range finder for a longer-term study
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
initial age: approx. 5 weeks; initial weight (average): 125 g (males), 114 g (females)
single housing
Route of administration:
oral: gavage
Vehicle:
other: Methocel, 0.25% aqueous solution
Details on oral exposure:
The test substance was suspended in methocel, dosing volume: 10 ml/kg bw
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily
Remarks:
Doses / Concentrations:
0, 30, 100, 300, 1000 mg/kg/day
Basis:
other: nominal in vehicle
No. of animals per sex per dose:
3/sex/group
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
Clinical observations: daily
Body weight: twice weekly
Food consumption: once weekly
Sacrifice and pathology:
organ weights, macroscopic and microscopic examinations on limited set of organs (12 organs)
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
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
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:
not specified
Details on results:
Liver weight increase at doses ≥300 mg/kg/day in both sexes
Kidney weight increase in females at 1000 mg/kg/day
hepatocellular hypertrophy and peripheral liver cell proliferation at 1000 mg/kg/day, especially in females
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
Critical effects observed:
not specified
Conclusions:
The liver is considered as the target organ for the test substance. A NOAEL was established at 300 mg/kg/day.
Executive summary:

In this study conducted generally accepted scientific standards, the repeat dose toxicity NOAEL of the test material (EC 231-272-0) was determined to be 300 mg/kg bw/day. The test was conducted in rats, with dose levels of 30, 100, 300, and 1000 mg/kg bw/day. Increased liver weight was observed in the ≥ 300 mg/kg bw/day groups, as well as more significant liver changes at 1000 mg/kg bw/day, especially in females. Thus the liver is considered a target organ for the test substance.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Procedure and observations

Toxicity after repeated dose administration was examined in a subacute 28d study and in a subchronic 90d study.

First, the test item was administered orally by gavage to groups of 3 male and 3 female Wistar rats at dose levels of 0 mg/kg body weight/day, 30, 100 mg/kg bw/d, 300 mg/kg bw/d and 1000 mg/kg bw/d for 28 consecuitive days. A clinical observation (daily) was performed in all animals. Body weights and food consumption were determined once or twice a week, respectively. A limited set of selected organs was examined macros- and microscopically. Increase in liver weight at doses ≥300 mg/kg/day in both sexes were observed as well as kidney weight increases in females at 1000 mg/kg/day and hepatocellular hypertrophy and peripheral liver cell proliferation at 1000 mg/kg/day, especially in females.

In a second study, rats (10/sex/dose) were administered to the substance for 90d at concentrations of 50, 150 and 400 mg/kg bw (BASF 2014). All animals were periodically checked for mortality, clinical signs, body weight gain and food consumption. An FOB, analysis of blood and urine were also performed. Organ weighing and gross necropsy was done in all treatment groups, histopathological examination for control and high dose group only. Transient salivation and slight transient gait ataxia (males) were observed in high dose animals. 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, calcium oxalate and crystals of unknown origin were found in the urine sediment. An increase in relative and absolute kidney (high dose) and liver weight (mid and high dose) in males and females was recorded. Centrilobular hypertrophy correlating to the observed weight increase was seen in all male and female animals of the high dose group with grading. In the kidney of top dose males an increased severity of basophilic tubules was observed, accompanied by granular casts. These findings were also present in test group 2 (granular cast and slight basophilic tubules).

In preparation for a teratogenicity study in rabbits, a dose range finding studies with non-pregnant rabbits was performed (BASF 2016). In this study, the same clinical symptoms as in rats were observed. Rabbits were sacrificed in extremis after a single dose of 500 mg/kg bw. The lower dose group of 150 mg/kg bw was tolerated with clinical symptoms. No effects were observed at 50 mg/kg bw.

Discussion

The slight transient gait ataxia in high dose males immediately after the administration was seen as treatment-related and adverse. Furthermore, basophilic tubules in conjunction with granular casts in the mid and high dose group (150 and 400 mg/kg bw) 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 (400 mg/kg bw) was regarded to be treatment - related and non-adverse.

The NOAEL is considered to be 50 mg/kg bw.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
most sensitive study for this endpoint

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. Effects observed are not indicative of serious effects at a dose level of 100 mg/kg for subchronic exposure. As a result the substance is not considered to be classified for repeated dose toxicity under Regulation (EC) No. 1272/2008.