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Administrative data

Description of key information

oral: NOAEL = 1000 mg/kg bw/day (BASF SE/Evonik, 30S015/08095, 2014, OECD Guideline 408)

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
Study period:
2013/2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline tudy + GLP
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes (incl. certificate)
Limit test:
yes
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
- Housing: animals were housed together (5 animals per cage) in H-Temp polysulfonate cages type 2000P
- Diet: ground Kliba maintenance diet mouse/rat “GLP”, meal ad libitum
- Water: ad libitum
- Acclimation period: 8 days

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

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

The stability of Oxooel 9 N in corn oil at room temperature for a period of 7 days was proven before the start of the administration period.
The test-substance preparations were produced at least weekly and stored at room temperature. The administration volume was 4 mL/kg body weight.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
once daily
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg/d
Basis:
actual ingested
No. of animals per sex per dose:
10 males and 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: limit test



Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Cage side observations: yes, daily

DETAILED CLINICAL OBSERVATIONS: Yes
prior to the administration period and thereafter at weekly intervals

BODY WEIGHT: Yes
- Time schedule for examinations: weekly intervals

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily visual inspection of the water bottles

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to administration and at day 91
- Dose groups that were examined: 0 and 1000 mg/kg/d

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the administration period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals:all animals

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the administration period
- Animals fasted: Yes
- How many animals: all

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of the administration period
- Metabolism cages used for collection of urine: Yes


FUNCTIONAL OBSERVATION BATTERY Yea
- Time schedule for examinations: at the end of administration period
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity /home cage observation / open field observation

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Other examinations:
Sperm parameters:
Immediately after necropsy and organ weight determination the right testis and cauda epididymis were taken from all male animals. Sperm motility examinations were carried out in a randomized sequence. Sperm head count (testis and cauda epididymis) and morphology were evaluated in all groups.
Estrous cycle:
Estrous cycle length and normality were evaluated daily for all female animals for a minimum of 3 weeks prior to necropsy.
Statistics:
DUNNETT's test (two-sided), one-way analysis using KRUSKALWALLIS test (two-sided)
Details on results:
CLINICAL SIGNS AND MORTALITY
One female animal of test group 1 (100 mg/kg bw/d) was sacrificed in a moribund condition on study day 77. Upon histological analysis, no findings were noted that could explain the cause of this moribund state.

BODY WEIGHT AND WEIGHT GAIN
No significant and test substance-related changes of mean body weights in both sexes were observed. The same was true for mean body weight change values. For male animals of test group 2 (300 mg/kg bw/d) mean body weight change value was significantly higher on study day 14 (+9.4%). This single event was assessed as spontaneous in nature and not to be of toxicological relevance.

CLINICAL EXAMINATION
Salivation after treatment from slight to moderate was observed in all male and female animals of test groups 2 and 3 (300 and 1000 mg/kg bw/d) as well as in 6 of 10 male and 1 of 10 female animals of test group 1 (100 mg/kg bw/d). From the temporary, short appearance immediately after dosing (or shortly before) it was concluded that salivation was induced by a bad taste of the test substance or local affection of the upper digestive tract:
On study days 76 and 77, one female animal of test group 1 (100 mg/kg bw/d) showed poor general condition, paresis of both hindlimbs and apathetic behavior. These findings were not related to the test substance. One female animal of test group 3 (1000 mg/kg bw/d) had a swollen eyelid between study days 87 to 93. The finding was assessed as being incidental and not related to treatment.

WATER AND FOOD CONSUMPTION
No test substance-related findings were observed.

OPHTHALMOSCOPIC EXAMINATION
No treatment-related findings were observed.
One female animal of test group 3 (1000 mg/kg bw/d) had a swollen eyelid between study days 87 to 93. Therefore, on the day of examination the animal was not able to open the eyelid completely. The finding was assessed as being incidental and not related to treatment. In addition, all other apparent findings were assessed as being incidental in nature since they occurred in the control group and did not show a dose-response relationship.

HAEMATOLOGY
No treatment-related changes among hematological parameters were observed.

CLINICAL CHEMISTRY
No treatment-related adverse changes among clinical chemistry parameters were observed.
At the end of the administration period in males of test groups 1 and 3 (100 and 1000 mg/kg bw/d) alanine aminotransferase (ALT) activities were decreased. Creatinine levels were increased in males of test group 2 (300 mg/kg bw/d), but decreased in females of the same test group. Urea values were decreased in females of test groups 1 and 2 (100 and 300 mg/kg bw/d). However, all mentioned parameters were not dose-dependently altered and, therefore, these changes were regarded to be incidental and not treatment-related.
In males of test groups 2 and 3 (300 and 1000 mg/kg bw/d) total bilirubin levels were decreased and potassium levels were increased. The decrease of bilirubin values without any signs of anemia was most probably due to an increased conjugation rate in the liver and an accelerated excretion via the bile. This effect was regarded as adaptive and not adverse. Potassium was the only changed electrolyte in these individuals. Therefore, this alteration was regarded as treatment-related, but not adverse.

URINALYSIS
No treatment-related adverse changes among urinalysis parameters were observed.
In male animals of test groups 2 and 3 (300 and 1000 mg/kg bw/d) the incidences of granulated and epithelial cell casts and transitional epithelial cells and, additionally, in males of test group 1 (100 mg/kg bw/d) the incidence of casts were higher compared to controls. As shown in the histopathological investigation these findings were due to the “inducible α2u globulin nephropathy syndrome” which is described as species- and gender-specific effect in male rats (G. C. Hard et al., 1993). Therefore, the finding is not relevant as adverse effect for humans.
In males of test group 2 (300 mg/kg bw/d) higher incidences of blood were found in the urine. This was due to four individuals with blood values grade 2 and 3. Blood grade 2 was only found in one male of test group 3 (1000 mg/kg bw/d). This effect was regarded as not dose-dependent and, therefore, as being incidental and not treatment-related.


FOB
Deviations from "zero values" were obtained in several animals. However, as most findings were equally distributed between test-substance treated groups and controls, were without a dose-response relationship or occurred in single animals only, these observations were considered to have been incidental.
Regarding the single and overall motor activity, no test substance-related deviations to the control were noted for male and female animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d).

ORGAN WEIGHTS
Statistically significant deviations of the absolute weight in adrenal glands and brain as well as the relative weight decrease of the cuada epipidymis in males occurred without dose dependency. Thus, they were not considered to be treatment-related. The absolute weight increases of the thyroid gland were all below the maximal historical control value (28.8 g) and occurred without concurrent relative weight increased. Therefore, they were regarded as incidental. The statistically significant absolute and relative weight increase in the kidneys of males had a histopathological correlate and was considered to be treatment-related. The statistically significant absolute and relative liver weight increase in males and females of test groups 2 and 3 (300 and 1000 mg/kg bw/d) were both above the maximal historical control range values (absolute/relative, male: 9.261 g / 2.39%, female: 5.771 g / 2.723%) and were regarded as treatment-related.


GROSS PATHOLOGY
All gross findings occurred either individually or were equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment-related findings were observed in the kidneys of males and in the liver and thyroid glands of male and females.
When compared with the control group 0, a dose-dependent increased accumulation and dissemination of eosinophilic droplets was observed in the proximal convoluted tubules of male test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d). Eosinophilic droplets were positive in the Mallory-Heidenhain stain and in the immunhistochemical stain with an antibody to α2uglobulin. In test group 3, only one male showed minimal granular casts. Multifocal basophilic tubules increased rather in incidence than grading when compared with the control group, but without a clear dose-dependent relationship.
A dose dependent diffuse hepatocytic hypertrophy was seen in males and females starting from test group 2 (300 mg/kg bw/d).
Hypertrophy/hyperplasia in males and females of test groups 2 and 3 (300 and 1000 mg/kg bw/d) was regarded as treatment-related. Altered colloid was considered to be treatmentrelated in males of test group 2 and 3 (300 and 1000 mg/kg bw/d) and in females of the high dose (1000 mg/kg bw/d).
All other findings occurred either individually or were equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.


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

SPERM PARAMETERS
Concerning the motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as the sperm head counts in the testis and in the cauda epididymidis no treatment-related effects were observed.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related, adverse effects were observed up to limit dose
Critical effects observed:
not specified
Endpoint conclusion
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Additional information

Sub-acute study (OECD 407)

In a subacute toxicity study (OECD 407, BASF AG, 30S015/08095, 2004) the test substance was administered to 5 Wistar rats/sex/dose by gavage at dose levels of 0, 100, 300, and 1000 mg/kg bw/day for 28 days. There were no compound related adverse effects in mortality, clinical signs, food consumption, hematology, clinical chemistry, urinanalysis, organ weights, or gross and histologic pathology. In the 1000 mg/kg bw/day group the body weight was lower (not significantly) in male animals from study day 14 onwards. Body weight change was significantly lower by -17.47% in males on study day 21.In conclusion, with regard to body weight parameters the oral administration of the test substance by gavage over a period of 4 weeks revealed signs of toxicity in male Wistar rats at dose levels of 1000 mg/kg bw/day but none in female Wistar rats. Salivation was considered to be related to either the bad taste of the test substance or local affection of the upper digestive tract. This finding was not considered to be an adverse and toxicologically relevant effect. Therefore, under the conditions of the present study, the no observed adverse effect level (NOAEL) was 300 mg/kg bw/day for male and 1000 mg/kg bw/day for female Wistar rats. This subacute study in the rat is acceptable and satisfies the guideline requirements for a subacute oral study OECD 407 in rats.

Subchronic Study (OECD 408):

Oxooel 9N was administered by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0 (test group 0), 100 (test group 1), 300 (test group 2) and 1000 mg/kg bw/d (test group 3) over a period of 3 months. With regard to clinical examinations, signs of general systemic toxicity were not observed even at a dose level of 1000 mg/kg bw/d. In addition, no test substance-related effects on estrous cycle length and the number of cycles were obtained. Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose level of 1000 mg/kg bw/d. Furthermore, no test substance-related effects on sperm parameters were obtained.

Regarding pathology, target organs were the kidneys of male animals and the liver and thyroid glands of male and female animals. In the kidneys of males, a dose-dependent increase of α2u-globuline was observed starting from test group 1 (100 mg/kg bw/d), which explains the significant absolute and relative weight increase in the kidneys of males of test groups 2 and 3 (300 and 1000 mg/kg bw/d). α2u-Globulin is a male and rat-specific poor soluble protein synthesized in the male rat liver, filtered by the glomerulus and reabsorbed in the S2 segment of the proximal tubules where it is slowly hydrolyzed by lysosomal digestion. Reversible binding of the test-substance or their metabolites to α2u-globulin decreases the effectiveness of its lysosomal digestion, resulting in strong accumulation of this protein that is visible in form of characteristic eosinophilic droplets. Although the increase of eosinophilic droplets is considered treatment-related, this finding does not represent a risk for humans since they do not synthesize this protein (Durham and Swenberg, 2013). Multifocal basophilic tubules representing areas of regeneration increased when compared with the control group, but without a clear dose-dependent relationship. Basophilic tubules and granular casts resulted from cell injury induced by intracellular overload of eosinophilic droplets and are characteristic of the rat-specific α2u-nephropathy.

In the liver, diffuse hepatocellular hypertrophy was observed in males and females correlating with statistically significant liver weight increase starting from test group 2 (300 mg/kg bw/d). Since no signs of liver hepatotoxicity (necrosis/single cell necrosis, inflammation, fibrosis, fatty change, etc.) were detected, the hepatocellular hypertrophy associated with liver weight increase was considered to be treatment-related and adaptive (Hall et al., 2012).

In the thyroid gland of males of test groups 2 and 3 (300 and 1000 mg/kg bw/d), minimal to slight hypertrophy/hyperplasia of the follicular epithelium and altered colloid occurred without a clear dose-response. In females of the same test groups, hypertrophy/hyperplasia increased dose-dependently with altered colloid in test group 3 only (1000 mg/kg bw/d). Since all of these changes were not accompanied by significantly increased relative organ weights, this strongly suggests an increased thyroxin catabolism occurring in the liver due to hepatic enzyme induction. This well-known phenomenon is characteristic for rodents (Curran et al., 1991) and there is no convincing evidence that humans exposed to chemicals that induce hepatic microsomal enzymes have increased risk for any thyroid gland disorder (Capen, 1997). Thus, the thyroid gland findings were regarded as treatment-related but not adverse.

All other findings occurred either individually or were equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Based on these findings, the NOAEL of the 90 -day oral repeated dose toxicity study according to OECD 408 guideline is 1000 mg/kg/d. Considering that the weight effects observed in animals in the 28 -day repeated dose toxicity study were not confirmed in the study in which animals were exposed three times longer, it can be concluded that these gender-specific weight effects are incidental and do not need to be taken into account.

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for repeated oral dose toxicity under Regulation (EC) No. 1272/2008.