Registration Dossier

Administrative data

Description of key information

Oral: NOAEL; OECD 408; 90-day rat; gavage; increase in liver nuclear pyknosis, increase in absolute and relative liver weight, increase in kidney weight (females only) were observed in the highest dose groups (18 mg/kg bw/day); reliability = 2.
NOT CLASSIFIED

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
not specified
Species:
rat
Strain:
other: OFA(SD)SPD
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Research Institute for the Breeding of Test Animals (A-2325 Himberg, Austria.)
- Age at study initiation: No details
- Average weight at study initiation: 157 g for males and 136 g for females
- Housing: Kept in pairs in Type III Makrolon cages with sterilized softwood chips for bedding
- Diet: ad libitum - Altromin Maintaining Diet No. 1324, pelletized
- Water: ad libitum -Tap in bottles acidified with sulfuric acid to a pH of 5
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 60
- Photoperiod (hrs dark / hrs light): 10/14
Route of administration:
oral: gavage
Vehicle:
other: deionized water
Details on oral exposure:
The test substance was administered once daily in the mornings Monday- Saturday (inclusive) using a metal esophageal probe. The test substance was freshly dissolved in deionized water.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
13 weeks (90 days)
Frequency of treatment:
Once daily in the mornings Monday-Saturday (inclusive) given a volume of 10 ml/kg at various doses.
Dose / conc.:
2 other: mg/kg/day
Dose / conc.:
6 other: mg/kg/day
Dose / conc.:
18 other: mg/kg/day
No. of animals per sex per dose:
20 (and control group)
160 animals total (80 male, 80 female)
Control animals:
yes
Details on study design:
Male and female animals were separated and subdivided into groups based on increasing body weight using a random number series. The animals were put pairwise into cages according to consecutive numbers. The arrangement of the cages in the room was not randomized. The animals were individually marked in the right ear with brass ear markers. The animals with odd numbers received an additional notch in the left ear muscle.

- Dose selection rationale: The doses were selected based on previously obtained results from a dose identification study (4-week oral gavage).
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes.
- Time schedule: Daily Mon-Sat by caretakers and once per week by the veterinarian.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily Mon-Sat by caretakers and once/week by the veterinarian

BODY WEIGHT: Yes
- Time schedule for examinations: Once/week

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule for examinations: Once/week

WATER CONSUMPTION: Yes
- Time schedule for examinations: Once/week

OPHTHALMOSCOPIC EXAMINATION: Yes
-Performed during last test week

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before substance was administered 6 days prior to study start and at test days 30 and 86
- Anaesthetic used for blood collection: Yes (light ether narcosis)
- Animals fasted: No
- How many animals: 10 males and 10 females from all groups

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before substance was administered 6 days prior to study start and at test days 30 and 86
- Animals fasted: No
- How many animals: 5 males and 5 females from all groups (at first two collection time points); 10 males and 10 females from all groups (at last collection time point)

URINALYSIS: Yes
- Time schedule for collection of urine: 6th, 7th, 28th, 29th, 84th, 85th days.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- How many animals: 10 males and 10 females from all groups
Sacrifice and pathology:
All animals that were still living at the end of the test and any that died prematurely during the study were sacrificed, dissected and examined on the 91st-93rd test days. Organs were weighed, fixed, and stained with hematoxillin and eosin. Eyes were fixed according to Susa method. The histopathology evaluation took place on a Zeiss photo microscope III.
Gross and relative organ weight and histopathology was performed on selected organs.

Organs weighed and prepared for histopathology: kidneys, suprarenal bodies, spleen, testicles, heart, liver, brain, and hypophysis (pituitary gland). Organs in pairs were weighed together if obvious size differences were not present. All organs except for the liver were analyzed histopathologically from the control and high dose groups. The liver was analyzed histopathologically from the control and all test groups.
Statistics:
The mean, standard deviation and sample size of the median together with the smallest and largest values are presented in this study. Frequencies above approximately 10 (e.g., number of neutrophils) and measured values were examined with simple analysis of variance followed by the Scheffe test for uniformity of the group means. Frequencies below approximately 10 (e.g., number of monocytes) and ordinal data (e.g., urine evaluation) were compared using the H-test. The Chi-square test and/or Fisher's Exact Test was used for events that were counted (numerated events). A value of p <=0.05 was established as the significance limit.

All testing methods were taken from the book "Applied Statistics" by Sachs L (Springer Publishing House, 4th edition, 1974)
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:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
In addition to spontaneous alterations and probably random differences between the groups, the following substance-related effects were observed or measured:
-An abundance of incriminating, degenerative lesions of the liver in the group receiving the highest dose (statistically significant only in one case: pycnosis).
-A definite and dose-related increase in the absolute and relative liver weights in the highest dose group.
-An increase in kidney weight, although statistically significant only with females in the group receiving the highest dose.

All toxic effects mentioned above were only observed in the highest dose group (18 mg/kg of body weight).
Dose descriptor:
NOAEL
Effect level:
6 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: 1) an increase in the amount of serious degenerative liver damage (nuclear pyknosis), 2) a clear and dose-dependent increase in the absolute and relative liver weight, and 3) an increase in the kidney weight (females only).
Critical effects observed:
yes
Lowest effective dose / conc.:
18 mg/kg bw/day (nominal)
System:
haematopoietic
Organ:
liver
Treatment related:
yes
Critical effects observed:
yes
Lowest effective dose / conc.:
18 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes
Conclusions:
The rat oral 90 day gavage NOAEL identified in this study for the test substance was 6 mg/kg bw.
Executive summary:

Male and female rats were orally gavaged with the test substance for 90 days at levels of 2, 6, and 18 mg/kg bw (6 days per week). The following statistically significant test-substance related effects were observed in the 18 mg/kg bw: 1) an increase in the amount of serious degenerative liver damage (nuclear pyknosis), 2) a clear and dose-dependent increase in the absolute and relative liver weight, and 3) an increase in the kidney weight (females only). Based on this information, the rat oral 90 day gavage NOAEL identified in this study for the test substance was 6 mg/kg bw.

Endpoint conclusion
Dose descriptor:
NOAEL
6 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Organ:
kidney
liver

Additional information

Male and female rats were orally gavaged with the test substance for 90 days at levels of 2, 6, and 18 mg/kg bw (6 days per week). The following statistically significant test-substance related effects were observed in the 18 mg/kg bw: 1) an increase in the amount of serious degenerative liver damage (nuclear pyknosis), 2) a clear and dose-dependent increase in the absolute and relative liver weight, and 3) an increase in the kidney weight (females only). Based on this information, the rat oral 90-day gavage NOAEL identified in this study for the test substance was 6 mg/kg bw.

Justification for classification or non-classification

Based on results of repeated oral studies, the substance does not need to be classified for repeated dose toxicity according the EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.