Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Oral (OECD 407), rat: NOAEL 1000 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 Feb - 9 May 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
(1995)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
Commission directive 96/54/EEC of 30 Sept 1996
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
MINISTERIUM FÜR RAUMORDNUNG UND UMWELT DES LANDES SACHSEN-ANHALT, Germany
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 42 - 51 days
- Weight at study initiation: 190.0 g ± 5.7 g (males), 162.6 g ± 6.6 g (females)
- Housing: while acclimatisation in groups of up to 3 in Makrolon cages Type 3 separately according to sex, afterwards individually in Makrolon Type 3 cages, granulated soft wood bedding
- Diet: pelleted standard diet ALTROMIN 1324 (ALTROMIN, Lage/Lippe, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 7 days (males), 9 days (females)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.0 - 22.5
- Humidity (%): 30 - 55, with a short falling below to 25% on 4 days
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Separate formulations were prepared for each dose level by mixing a weighed quantity of the test item with an appropriate volume of vehicle, warming up to approximately 70 °C and cooling down to 30 - 40 °C each day shortly before administration.

VEHICLE
- Concentration in vehicle: 30, 60, 100 mg/mL
- Amount of vehicle: 10 mL/kg bw
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
28 days and 14 days post-exposure recovery period (5 animals of vehicle control and high dose group)
Frequency of treatment:
once daily, 7 days/week
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 (dose groups and negative control), 10 (high dose group and vehicle control)
Control animals:
yes, concurrent vehicle
other: negative control
Details on study design:
- Dose selection rationale:
The dose levels were selected after a pre-experiment using 1000 mg/kg bw/day in 4 male and 4 female rats each over a period of 11 days. No signs of mortality, clinical signs or pathological findings were noted. Body weight and food consumption was normal over the period of pre-experiment.

- Post-exposure recovery period in satellite groups: 14 days
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
off-cage examinations: changes in skin, fur, eyes, mucous membranes, gait, posture and response to handling, occurrence of secretions and excretions, autonomic activity, presence of clonic or tonic movements and stereotypies or bizarre behaviour

BODY WEIGHT: Yes
- Time schedule for examinations: at the start of the study and then weekly thereafter and at the end of the study before food withdrawal

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as mg food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to killing at the termination of the study one day after the last administration of the test item or one day after the 14 day recovery period
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes (overnight)
- How many animals: 5 (dose groups and negative control), 10 (high dose group and vehicle control)
- Parameters examined: Erythrocyte count (RBC), Haemoglobin concentration (HB), Packed cell volume (HCT), Platelet count (PLT), Total leucocyte count (WBC), Leucocyte differential count, Prothrombin time (PT), Fibrinogen concentration (FIB)
These parameters (except FIB) were also determined in the animals of the satellite groups at the end of the recovery period.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to killing at the termination of the study one day after the last administration of the test item or one day after the 14 day recovery period
- Animals fasted: Yes (overnight)
- How many animals: 5 (dose groups and negative control), 10 (high dose group and vehicle control)
- Parameters examined: Alanine aminotransferase (ALT), Alkaline phosphatase (AP), Aspartate aminotransferase (AST), Albumin (Alb), Creatinine (Crea), Glucose (Gluc), Total cholesterol (Chol), Total protein (Prot), Urea, Potassium (Pot), Sodium (Sod)
The parameters ALT, AP, Chol and Sod were also determined in all animals of the satellite groups at the end of the recovery period; Prot and Alb alone in the female animals.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: prior to administration, in the last week of dosing and in the last week of the recovery period
- Dose groups that were examined: all
- Battery of functions tested:
sensory reactivity: Reactivity to noises: hit on the cage with a metal plate; reactivity to light: test of the pupillary reflex with an electric torch; reactivity to touch: the animal was slightly touched on the back with a spatula; grip strength: animal sitting and holding on to the cage grid with all four extremities was pulled on the tail horizontal to the back for approximately 1 to 2 sec; motor activity: observation of locomotion and kind of movement (running, jumping and so on) and of coordination of movement (anomalies of posture or gait)

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals were examined externally. The cranial, thoracic and abdominal cavities were opened and examined macroscopically. The following organs of all animals were weighed after trimming off fat and other contiguous tissue: adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes and thymus. From bilateral organs both were weighed and reported separately.
HISTOPATHOLOGY: Yes
Whole organs or samples of the tissues of all animals listed below were fixed in 10 percent formaldehyde and preserved in 4 percent formaldehyde:
Adrenals, Sciatic nerve, Bone marrow (from sternum), Small and large intestine (including Peyer' s patches), Brain (3 sections), Spinal cord (from second trachelo-vertebra [Axis]), Epididymides, Spleen, Heart, Stomach, Kidneys, Testes, Liver, Thymus, Lungs, Thyroid, Lymph nodes (mesenteric and submandibular), Trachea, Urinary bladder, Ovaries, Uterus, Prostate
All the listed organs from animals of water control, vehicle control and high dose group were examined microscopically after staining with Hemalum-Eosin. Paired organs were both examined.
Statistics:
Group means and standard deviations were calculated for all numerical data. Sexes were analysed separately. The statistical significance compared to the vehicle control was proved by means of the following different statistical methods. The statistical significance was declared at the two-sided 5% level.
• Welch t-test:
- for all data of body weights, body weight gain and food consumption
- for the comparison of only two mean values at the satellite groups and at the comparison of water control to vehicle control for the parameters of haematology (excluding differential leucocyte count), coagulation, clinical biochemistry and absolute and relative organ weights
• Dunnett test:
- for the comparison of the mean values of the three dose groups to vehicle control for the parameters of haematology (excluding differential leucocyte count), coagulation, clinical biochemistry and absolute and relative organ weights
• Calculation of mean value, standard deviation and range:
- for the parameters of differential leucocyte count
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Only some slight clinical symptoms (slight rough hair coat, slight squatting position, faeces soft) were shortly observed in single animals. These symptoms are not caused by the administration of the test item. None of the animals showed further alterations of its general state of well-being and behaviour.
Mortality:
no mortality observed
Description (incidence):
None of the animals died during the course of investigation.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The body weights or the body weight gain of all animals of all dose groups were not influenced by the administration of the test item. The few statistically significantly calculated differences to the vehicle control group in the body weight gain were incidental.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Calculation of food concumption showed statistically significant increases in the male animals (and partly also in der female animals) of the high dose group (1000 mg/kg bw) compared to the vehicle control group. However these statistically significant values of the dose groups were in the range of the historical control data in the testing facility and also in the range of the negative control. The values of the vehicle control were decreased compared to the water control group and also to the historical control data in the testing facility. Therefore, it was deemed these changes were caused by the administration of the vehicle and not by the test item administration.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
None of the haematological parameters was statistically significantly changed by the test item administration. The parameters of the leucocyte differential count were all in the normal range for the strain used.
The prothrombin time was statistically significantly decreased in the female animals in all three dose groups without dose dependence. This was the reason for the determination of this parameter also in the satellite groups. A statistically significant decrease was observed no more after the recovery period, i.e. the effect was reversible.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The serum cholesterol level was increased in all animals of all dose groups, statistically significantly in all dose groups of females, at 1000 mg/kg bw/day in males and in females of satellite groups. This effect was irreversible in the female animals in the course of the recovery period.
Serum total protein and albumin level were statistically significantly increased in the female animals at 600 and 1000 mg/kg bw/day. Values for high dose group were in the limit range of historical control data of the testing facility. Therefore, this effect could have been caused by the test item administration. This effect was reversible in the course of the recovery period.
Alanine aminotransferase (ALT) activity was statistically significantly decreased in males in all dose groups and at 600 mg/kg bw/day in females. Alkaline phosphatase (AP) was decreased in females of all dose groups, statistically significantly at 300 and 1000 mg/kg bw/day, the same tendency was also observed in males of all groups. There was no dose-dependence and all values were in the range of the historical control data of the testing facility.
Serum sodium level was statistically significantly decreased in males at 600 and 1000 mg/kg bw/day and statistically significantly increased in females at 600 and 1000 mg/kg bw/day. All values were in the range of the historical control data and therefore it was deemed this change was incidental and not treatment related.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The absolute and the relative weights of the liver were increased in all treated animals, statistically significant for the absolute weight in the male animals at 600 and 1000 mg/kg bw/day and for the relative weight in all dose groups of male animals and at 600 and 1000 mg/kg bw/day of female animals. This effect was irreversible in the female animals in the course of the recovery period. A change was observed no more in male animals at the end of the recovery period. The liver weight was also increased in the animals of the vehicle control group compared to the negative control group, statistically significantly in female animals. Therefore, the liver is reported as a possible target organ. Nevertheless, all increased weights of liver are in the range of the historical control data in the testing facility for the strain used.
Further isolated statistically significant differences in weights of heart, kidneys, spleen, left testis, right ovary and thymus were deemed to be incidental since the values were in the range of the historical control data in the testing facility for the strain used or the differences were not confirmed by the other counterpart of bilateral organs or by comparing the relative weight.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No abnormalities were found in any animal.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Intracellular vacuoles (with an extend of 5 to 50 % of the scrutinised area) were observed in the hepatocytes of all animals of the vehicle control group and at 1000 mg/kg bw/day without difference between both groups. These vacuoles are an indication of fat deposits into the cells, which were dissolved in the course of preparation of microscopic slides. An appearance of leucocytes (especially basophil and eosinophil granulocytes), lymphocytes and a formation of cell detritus or microhaemorrhages as indications of cell damage were not observed. These vacuoles were not observed in the hepatocytes of the animals of the negative control group. Therefore, it was deemed this change was caused by the administration of the vehicle and not caused by the test item administration.
The other occasional histological findings were incidental, they were in the physiological range of the animals used and the effects in the lungs (emphysemic areas with / without haemorrages, atelectatic area) could be caused probably by the euthanasia or by the ether anaesthesia for blood sampling.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects up to the highest dose tested
Critical effects observed:
no

Table 1. Results of Prothrombin time

 

 

 

Vehicle control

300

mg/kg bw/day

600  

mg/kg bw/day

1000  

mg/kg bw/day

Control water

Female animals:

 

Prothrombin time [sec]

 

Difference to vehicle control

mean

37.7

31.4

31.9

31.3

36.9

± SD

2.31

1.51

1.30

4.65

4.34

n

5

5

5

5

5

t-cal

-

- 3.548

- 3.309

- 3.639

- 0.364

t-tab

-

2.590

2.590

2.590

2.439

significant

-

yes

yes

yes

no

 

Table 2. Results of Serum Cholesterol

 

 

 

Vehicle control

300  

mg/kg bw/day

600  

mg/kg bw/day

1000  

mg/kg bw/day

Control water

Male animals:

 

Cholesterol [mg/dL]

 

Difference to vehicle control

mean

48.2

63.8

61.6

79.8

43.8

± SD

6.46

6.57

8.41

33.06

5.54

n

5

5

5

5

5

t-cal

-

1.396

1.199

2.828

- 1.156

t-tab

-

2.590

2.590

2.590

2.317

significant

 

no

no

yes

no

Satellite groups:

 

males

females

Vehicle control

1000 mg/kg bw

Vehicle control

1000 mg/kg bw

 

Cholesterol [mg/dL]

 

Difference to vehicle control

mean

61.2

58.4

62.8

82.6

± SD

22.53

13.74

11.58

14.35

n

5

5

5

5

t-cal

-

- 0.237

-

2.401

t-tab

-

2.396

-

2.326

significant

-

no

-

yes

 

Table 3. Results of absolute liver weights

Test-parameters

 

Vehicle control

300  

mg/kg bw/day

600  

mg/kg bw/day

1000  

mg/kg bw/day

Control water

Male animals:

Liver [mg]

 

 

 

Difference to vehicle control

mean

9909.4

12856.2

12317.0

13341.0

9241.0

± SD

1628.96

890.33

2448.67

1603.60

1377.78

n

5

5

5

5

5

t-cal

-

2.689

2.197

3.131

- 0.701

t-tab

-

2.590

2.590

2.590

2.319

significant

-

yes

no

yes

no

Female animals:

Liver [mg]

 

 

 

Difference to vehicle control

mean

8688.6

9118.2

9988.8

9942.6

6512.4

± SD

1238.63

1543.91

1462.45

1172.21

793.90

n

5

5

5

5

5

t-cal

-

0.498

1.508

1.455

- 3.308

t-tab

-

2.590

2.590

2.590

2.380

significant

-

no

no

no

yes

Satellite groups:

 

males

females

Vehicle control

1000 mg/kg bw

Vehicle control

1000 mg/kg bw

Liver [mg]

 

 

 

Difference to vehicle control

mean

11219.6

11060.2

7375.6

8527.6

± SD

777.312

998.888

807.567

178.430

n

5

5

5

5

t-cal

-

- 0.282

-

3.115

t-tab

-

2.333

-

2.696

significant

-

no

-

yes

 

Table 4. Results of relative liver weights

Test-parameters

 

Vehicle control

300  

mg/kg bw/day

600  

mg/kg bw/day

1000  

mg/kg bw/day

Control water

Male animals:

Liver [%]

 

 

 

Difference to vehicle control

mean

3.2707

4.2672

4.1937

4.4646

3.1964

± SD

0.48901

0.21619

0.49273

0.36217

0.34443

n

5

5

5

5

5

t-cal

-

3.880

3.593

4.648

- 0.278

t-tab

-

2.590

2.590

2.590

2.354

significant

-

yes

yes

yes

no

Female animals:

Liver [%]

 

 

 

Difference to vehicle control

mean

3.9401

4.1289

4.6978

4.7363

3.0386

± SD

0.31497

0.54358

0.51594

0.42324

0.29933

n

5

5

5

5

5

t-cal

-

0.651

2.614

2.747

- 4.639

t-tab

-

2.590

2.590

2.590

2.307

significant

-

no

yes

yes

yes

Satellite groups:

 

males

females

Vehicle control

1000 mg/kg bw

Vehicle control

1000 mg/kg bw

Liver [%]

 

 

 

Difference to vehicle control

mean

3.2628

3.2752

3.2638

3.5592

± SD

0.25795

0.14702

0.31751

0.15132

n

5

5

5

5

t-cal

-

0.093

-

1.878

t-tab

-

2.418

-

2.481

significant

-

no

-

no

 

Conclusions:
Based on the results of this study, the NOAEL for systemic toxicity was set at 1000 mg/kg bw/day since no adverse effects related to the test substance administration were observed up to concentrations of 1000 mg/kg bw/day, the highest dose tested.
Executive summary:

The test substance was tested in a repeated dose oral toxicity study according to OECD Guideline 407 and in compliance with GLP (2001). The dose levels for the main study were selected based on a dose range-finding toxicity study in 4 male and 4 female rats using a dose level of 1000 mg/kg bw/day over a period of 11 days. No mortality or clinical signs were observed in these animals. Body weight gain and food consumption was normal and necropsy revealed no pathological findings. Therefore, for the main study, dose levels of 300, 600 and 1000 mg/kg bw/day were chosen. 5 Wistar rats per sex and dose (10 animals per sex for vehicle control and high dose group) were treated daily via gavage with the test substance for 28 days. The vehicle control group received corn oil and the negative control received water. 5 animals per sex from the vehicle and high dose group, respectively, were maintained untreated for additional 14 days to investigate reversibility of possible effects. In the main study, no mortality occurred and there were no clinical symptoms caused by the test substance. Body weights, body weight gain and food consumption were not influenced by the test substance. None of the haematological parameters was changed, but the prothrombin time was reversibly decreased in the female animals without dose dependence. Serum cholesterol was increased in all animals, irreversible in female animals. Serum total protein and albumin level were increased in female animals at 600 and 1000 mg/kg bw/day. The absolute and relative weights of liver were increased in all treated animals, irreversible in female animals. An increase of liver weight was also observed in the animals of the vehicle control group compared to the negative control group, statistically significant in female animals. No abnormalities were found in any animal in the course of the macroscopic pathological examination. Intracellular vacuoles as an indication of fat deposits were observed in the hepatocytes of all histologically examined liver tissues of the animals of the vehicle control group and the 1000 mg/kg bw/day dose group. This effect seems to be mainly caused by the administration of the vehicle (corn oil), because these vacuoles were not observed in animals of the negative control group. This is additionally confirmed by the increased weights of the liver in the animals of the vehicle control group compared to the negative control group. The increased weights of the liver in the animals of all dose groups compared to the vehicle control group and also the increased serum cholesterol level show that the test substance contributes to these effects on the liver. Therefore, the liver is reported as a possible target organ. Nevertheless, all increased liver weights were in the range of historical control data, no macroscopic or histologic pathological effects related to the test substance were observed and liver enzymes were not increased.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises an adequate and reliable study (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.6, of Regulation (EC) No 1907/2006.

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

The test substance was tested in a repeated dose oral toxicity study according to OECD Guideline 407 and in compliance with GLP (2001). The dose levels for the main study were selected based on a dose range-finding toxicity study in 4 male and 4 female rats using a dose level of 1000 mg/kg bw/day over a period of 11 days. No mortality or clinical signs were observed in these animals. Body weight gain and food consumption was normal and necropsy revealed no pathological findings. Therefore, for the main study, dose levels of 300, 600 and 1000 mg/kg bw/day were chosen. 5 Wistar rats per sex and dose (10 animals per sex for vehicle control and high dose group) were treated daily via gavage with the test substance for 28 days. The vehicle control group received corn oil and the negative control received water. 5 animals per sex from the vehicle and high dose group, respectively, were maintained untreated for additional 14 days to investigate reversibility of possible effects. In the main study, no mortality occurred and there were no clinical symptoms caused by the test substance. Body weights, body weight gain and food consumption were not influenced by the test substance. None of the haematological parameters was changed, but the prothrombin time was reversibly decreased in the female animals without dose dependence. Serum cholesterol was increased in all animals, irreversible in female animals. Serum total protein and albumin level were increased in female animals at 600 and 1000 mg/kg bw/day. The absolute and relative weights of liver were increased in all treated animals, irreversible in female animals. An increase of liver weight was also observed in the animals of the vehicle control group compared to the negative control group, statistically significant in female animals. No abnormalities were found in any animal in the course of the macroscopic pathological examination. Intracellular vacuoles as an indication of fat deposits were observed in the hepatocytes of all histologically examined liver tissues of the animals of the vehicle control group and the 1000 mg/kg bw/day dose group. This effect seems to be mainly caused by the administration of the vehicle (corn oil), because these vacuoles were not observed in animals of the negative control group. This is additionally confirmed by the increased weights of the liver in the animals of the vehicle control group compared to the negative control group. The increased weights of the liver in the animals of all dose groups compared to the vehicle control group and also the increased serum cholesterol level show that the test substance contributes to these effects on the liver. Therefore, the liver is reported as a possible target organ. Nevertheless, all increased liver weights were in the range of historical control data, no macroscopic or histologic pathological effects related to the test substance were observed and liver enzymes were not increased.

In conclusion, no adverse effects on systemic toxicity after repeated exposure were observed up to maximal tested doses. Therefore, a NOAEL of 1000 mg/kg bw/day was derived for systemic toxicity in the OECD 407 study in male and female rats.

Justification for classification or non-classification

The available data on repeated oral dose toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.