Registration Dossier

Administrative data

Description of key information

- oral: NOAEL = 50 mg/kg/day (4 weeks, gavage)
- oral: NOEL = 15 mg/kg/day (4 weeks, gavage)

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:
2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
including 14-day recovery period
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: HanBrl:WIST (SPF)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd, Biotechnology & Animal Breeding Division, CH-4414 Fullinsdorf / Switzerland
- Age at study initiation: 7 weeks
- Weight at study initiation: Males: 139-161 grams (mean 149 grams), Females: 108-134 grams (mean 120 grams)
- Housing: In groups of five in Makrolon type-4 cages with wire mesh tops and standardized softwood bedding
- Diet: Pelleted standard Provimi Kliba 3433 (batch nos. 77/01, 119/01) rat maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst/ Switzerland) was available ad libitum.
- Water: Community tap-water from Itingen was available ad libitum in water bottles
- Acclimation period: Under test conditions after health examination

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30-70 %.
- Air changes (per hr): 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light/12 hours dark, music during the light period.

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
(PEG 300)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was weighed into a glass beaker on a tared Mettler balance and the vehicle added. The mixtures were prepared using a magnetic stirrer and stored at room temperature (17-23°C). Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration, homogeneity and stability (after 2 hours) of the dose formulations were determined in samples taken after experimental start. Concentration and homogeneity of the dose formulations were determined in samples taken during week 3 of the treatment according to a HPLC method supplied by the Sponsor.
Duration of treatment / exposure:
Test duration: 28 days
Frequency of treatment:
Dosing regime: 7 days/week
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
450 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
0 and 450 mg/kg bw/day: 10 males and 10 females
15, 50, 150 mg/kg bw/day: 5 males and 5 females
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for dose selection : a dose range-finding study in rats was conducted HF 380/542 - CG 88 4339). Groups of 5 male and 5 female Sprague Dawley rats were treated at dose
levels of 100, 300, and 3000 mg/kg/day for 14 days. At 100 mg/kg, no clinical signs or clinical pathology changes were found. At the higher doses, treatment-related effects were observed with a dose effect relationship : reduction in body weight gain and food consumption during the first week, slight decrease in haemoglobin, erythrocyte count and packed cell volume, increase in cholesterol and alkaline phosphatases, increase in liver and adrenals weight and decrease in testes weight (3000 mg/kg). Based on these results, the following doses were chosen : 0 - 10 - 50 - 500 mg/kg.
Further information on the dose-range finding study other than given above are not available.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Observations for mortality/viability were recorded twice daily. The animals were observed for clinical signs once before commencement of administration; twice daily on days 1-3; as well as once daily on days 4-28 and once daily during days 29-42 (recovery).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were observed in their home cages, outside their home cages in a standard arena and in the hand. These observations were performed in random sequence once before commencement of administration and once weekly (weeks 1 -3) thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded weekly during pretest, treatment and recovery and before necropsy,

FOOD CONSUMPTION:
The food consumption was recorded once during the pretest period and weekly thereafter.


OPHTHALMOSCOPIC EXAMINATION: No data


HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 4 and 6 weeks
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes approximately 18 hours before blood sampling
- How many animals: all animals
- Parameters checked: Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume,Mean corpuscular hemoglobin, Mean corpuscular hemoglobinconcentration, Platelet count, Reticulocyte count, Reticulocyte fluorescence ratios, Nucleated erythrocytes (normoblasts), Heinz bodies, Methemoglobin, Total leukocyte count, Differential leukocyte count, Red blood cell morphology, Thromboplastin time (=prothrombin time), Activated partial thromboplastin time,


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 4 and 6 weeks
- Animals fasted: Yes approximately 18 hours before blood sampling
- How many animals: all
- Parameters checked: Glucose, Urea, Creatinine, Uric Acid, Bilirubin, total, Cholesterol, total, Triglycerides, Phospholipids, Aspartate aminotransferase, Alanine aminotransferase, Lactate dehydrogenase, Creatine kinase, Alkaline phosphatase, Gamma-glutamyl transferase, Calcium, Phosphorus, Sodium, Potassium, Chloride, Albumin, Protein, total, Globulin, Albumin/Globulin ratio,


URINALYSIS: Yes
- Time schedule for collection of urine: after 4 weeks
- Metabolism cages used for collection of urine: Yes (into a specimen vial.)
- Animals fasted: Yes (Urine was collected during the 18-hour fasting period)
- Parameters checked: Volume (18-hour), Specific gravity, Osmolality, Color, Appearance, pH, Protein, Glucose, Ketone, Bilirubin, Blood, Nitrite, Urobilinogen, Urine sediment, Red blood ceils, Crystals (Calcium oxalate), Crystals (Triple phosphate)


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During week 4
- Dose groups that were examined: all animals
- Battery of functions tested: grip strength / motor activity

Sacrifice and pathology:
GROSS PATHOLOGY: Yes, Adrenal glands, Aorta, Bone (sternum, femur including joint), Bone marrow (femur), Brain (cerebrum, cerebellum, pons), Cecum, Colon, Duodenum, Epididymides (fixed in Bouin's solution), Esophagus, Eyes with optic nerve (fixed in Davidson's solution), Harderian gland (fixed in Davidson's solution), Heart, Ileum, with Peyer's patches, Jejunum with Peyer's patches, Kidneys, Larynx, Lacrimal gland (exorbital), Liver, Lungs (infused with formalin at necropsy), Lymph nodes (mesenteric, mandibular), Mammary gland area, Nasal cavity, Ovaries, Pancreas, Pituitary gland, Prostate gland, Rectum, Salivary glands (mandibular, sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord (cervical, midthoracic, lumbar), Spleen, Stomach, Testes (fixed in Bouin's solution), Thymus, Thyroid (incl. parathyroid gland), Tongue, Trachea, Urinary bladder (infused with formalin at, necropsy), Uterus, Vagina, Gross lesions,

HISTOPATHOLOGY: Yes, from the animals of control and high-dose groups; Adrenal glands, Bone marrow (femur), Brain (cerebrum, cerebellum, pons), Cecum, Colon, Duodenum, Epididymides (fixed in Bouin's solution), Heart, Ileum, with Peyer's patches, Jejunum with Peyer's patches, Kidneys, Liver, Lungs (infused with formalin at necropsy), Lymph nodes (mesenteric, mandibular), Ovaries, Prostate gland, Rectum, Sciatic nerve, Seminal vesicles, Spinal cord (cervical, midthoracic, lumbar), Spleen, Stomach, Testes (fixed in Bouin's solution), Thymus, Thyroid (incl. parathyroid gland), Trachea, Urinary bladder (infused with formalin at, necropsy), Uterus, Vagina, Gross lesions,
As test item-related morphologic changes were detected in the organs of high-dose animals,the same organs (males: the adrenal glands, epididymides, femur with bone marrow, kidneys, spleen and testes; females: femur including bone marrow, liver and lung) from animals of the mid- and low-dose groups were examined.
Other examinations:
The following organ weights were recorded on the scheduled dates of necropsy: Brain, Thymus, Spleen, Ovaries, Heart, Liver, Kidneys, Adrenals, Testes, Epididymides
Statistics:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Student's t-test was applied to grip strength and locomotor activity.
• Fisher's exact-test was applied to the macroscopic findings.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
During daily observations, a number of test item-related clinical signs were noted in males and females treated with 450 mg/kg/day. Piloerection was noted from days 8-14 in several rats, whereas hunched posture was noted during days 9-12 in females only. In both cases, the severity was generally slight and the finding receded after a few days. Emaciation (days 8-10) and "waggling gait" (days 9-12), noted in several females, showed a similar pattern of reversibility. Dark feces and pale feces were noted in rats treated with 450 mg/kg/day from days 12-22 and from days 24 to 28 of treatment, respectively. The latter finding persisted for two days of the recovery period. These test item-related findings were considered likely to be related to the compensated anemia seen in the hematology parameters. During weekly observations, piloerection was noted during weeks 2 and 3 in a small number of rats treated with 450 mg/kg/day. During the functional observational battery at week 4, pale feces was noted in two males treated with 150 mg/kg/day and two males treated with 450 mg/kg/day.
No test item-related clinical signs of toxicity were seen during daily or weekly observations (weeks 1 -3) in rats treated with 15, 50 or 150 mg/kg/day.
Mortality:
no mortality observed
Description (incidence):
All animals survived until scheduled necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weights were reduced in males treated with 150 mg/kg/day from treatment day 22 (-7.8% on treatment day 28) and in both sexes treated with 450 mg/kg/day (-11.3% and -13.8% on treatment day 28 in males and females, respectively) from treatment day 8 onwards when compared with the control rats. These differences were considered to be test item-related. The body weights of the remaining groups compared favorably with those of the controls. During the recovery period, the mean body weights of rats previously treated with 450 mg/kg/day remained lower than those of the controls, although the mean body weight gain improved.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
The mean daily food consumption was reduced during the first measurement interval in both sexes at 450 mg/kg/day when compared with that of the controls, but improved thereafter to levels comparable to that of the controls. The mean daily food consumption of the remaining groups was unaffected. No differences persisted during the recovery period.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Test item-related changes in the hematology parameters noted after four weeks' treatment in males and females at 150 mg/kg/day and 450 mg/kg/day were considered to represent different compensated stages of anemia with reticulocytosis. Although reduced erythrocytes were noted in females treated with 150 and 450 mg/kg/day, compensatory reticulocytosis was more clearly expressed in the males treated with 150 mg/kg/day and 450 mg/kg/day. Hemoglobin and hematocrit were reduced in both sexes at 450 mg/kg/day and hematocrit was reduced in females treated with 150 mg/kg/day. Commensurate changes in the mean cell hemoglobin, mean cell hemoglobin concentration and mean corpuscular volume were noted in the males treated with 450 mg/kg/day. These differences were considered to be test item-related. Increased leukocyte counts in males at 150 mg/kg/day (p<0.05) and both sexes at 450 mg/kg/day (p<0.01) reflected higher absolute segmented neutrophil counts in males at 150 mg/kg/day and 450 mg/kg/day and higher absolute lymphocyte counts in both sexes at 450 mg/kg/day. All other differences in the hematology parameters noted after four weeks' treatment were considered to be incidental.

After the two week recovery period, the red blood cell count of the females previously treated with 450 mg/kg/day remained lower than that of the controls. In males previously treated with 450 mg/kg/day, the mean cell hemoglobin concentration remained lower, the absolute and relative reticulocyte counts higher and the left shift in the reticulocyte fluorescence ratios persisted. All other differences noted after the two-week recovery period when compared to the control values were considered to be incidental.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Test item-related changes noted after four weeks' treatment in rats treated at 450 mg/kg/day included increased creatinine (males), total bilirubin (males), total cholesterol (both sexes), triglycerides (both sexes), phospholipids (both sexes), albumin (males), total protein (males) and calcium (both sexes) levels were increased when compared to the controls. Gamma glutamyl transferase activity was higher in females at 450 mg/kg/day. These findings were considered to be related to the test item and probably indicative of minor changes in liver metabolism. All remaining changes noted after four weeks' treatment were considered to be incidental. With the exception of the elevated gamma glutamyl transferase activity noted after two weeks' recovery in females previously treated with 450 mg/kg/day, all findings noted after four weeks' treatment were reversible.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No test item-related changes in the urinalysis parameters were noted after four weeks' treatment.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No clinical signs of neurological impairment were noted during the functional observational battery at week 4.
All other findings were considered to be unrelated to the treatment with the test item.
Grip Strength: The fore- and hindlimb grip strength of the test item-treted rats compared favorably with those of the control rats.
Locomotor Activity: No test item-related differences in the locomotor activity was noted at any dose level.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
After four weeks' treatment, increased liver weights were noted in females at 50 mg/kg/day, whereas increased liver and kidney weights were noted in both sexes at 150 mg/kg/day and 450 mg/kg/day. Reduced testes and epididymide weights were noted in the males at 450 mg/kg/day. These changes were considered to be test item-related. All remaining changes noted after treatment were considered to be incidental
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Small testes ("diminished in size") were observed in males at 450 mg/kg/day. This test item related finding was noted at the end of treatment and recovery period. The remaining macroscopic findings were considered to be typical background findings unrelated to the treatment with the test item.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic examination demonstrated that under the conditions of this experiment, the test item produced morphological alterations in the male genital system, male kidneys, hematopoietic tissues spleen and bone marrow, especially in males, and female liver and lung. The testes of males at 450 mg/kg/day exhibited reduced spermatogenesis, in some cases associated with the occurrence of spermatic giant cells and tubular atrophy. The epididymides contained cellular debris and reduced amount of spermatozoa. These changes were not reversible during the recovery period, but they are considered as potentially reversible after a prolonged period of time. The kidneys of males at 150 and 450 mg/kg/day exhibited increased occurrence of tubular hyaline change, probably representing the microglobulins. Following the recovery period, this change was still slightly more prominent at 450 mg/kg/day compared to the controls. The bone marrow had fatty atrophy increased in males and females at 450 mg/kg/day. Splenic extramedullary hematopoietic activity was increased in males at 50, 150 and 450 mg/kg/day. These changes in hematopoietic tissues were accompanied by decreased hemoglobin and hematocrit values and increased proportion of reticulocytes in the peripheral blood, especially in males, and they probably reflect mild effects on the erythropoietic activity. Slight centrilobular hepatocellular hypertrophy occurred in several females at 450 mg/kg/day, and the presence of pulmonary alveolar foam cells was slightly increased in females at 450 mg/kg/day.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: haematology
Dose descriptor:
NOEL
Effect level:
15 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects at this dose level
Critical effects observed:
yes
Lowest effective dose / conc.:
150 mg/kg bw/day (actual dose received)
System:
haematopoietic
Organ:
spleen
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Under the conditions of this experiment, the test item produced morphological alterations in the male genital system, hematopoietic tissues spleen and bone marrow, especially in males, and female liver and lung.

 

The effects on testes occurred only at 450 mg/kg/day and consisted mainly in reduced spermatogenesis. They were correlated with macroscopic observation of testes diminished in size and reduced organ weights of testes and epididymides. Microscopic examination of the affected testes demonstrated the presence of spermatogonia, spermatocytes and to some degree of round spermatids. Some round spermatids showed abnormal central clearing of their nuclei. The elongated spermatids were mostly missing. The changes were not reversible during the recovery period, but their reversibility after a prolonged period of time is probable, as the spermatogenic epithelium may readily regenerate. Findings in the epididymides were obviously secondary to testicular lesions.

 

The increased presence of hyaline change in renal tubules of males at 150 and 450 mg/kg/day probably represented accumulation of microglobulin (also known as uroglobulin), known to occur in male rats and to be species-specific. Fatty atrophy of the bone marrow was minimally increased in males and females at 450 mg/kg/day. Splenic extramedullary hematopoietic activity was increased in males at 50, 150 and 450 mg/kg/day. These changes in hematopoietic tissues were acompanied by decreased hemoglobin and hematocrit values and increased proportion of reticulocytes in the peripheral blood, especially in males, and they probably reflect mild effects on the erythropoietic activity.

 

Slight centrilobular hepatocellular hypertrophy occurred in several females at 450 mg/kg/day, but it was not accompanied by changes in activity of liver enzymes. The presence of pulmonary alveolar foam cells was slightly increased in females at 450 mg/kg/day. This was probably unrelated to hyperlipidemia occurring at this dose level, because hyperlipidemia also occurred in males, where no increase in pulmonary alveolar foam cells was observed. The increase in average organ weight of the liver and kidneys relative to body weight was partly due to the decreased body weight, as there were no morphological changes accounting for these weight differences.

 

According to the results of pathology the NOEL is 15 mg/kg/day. Also at 50 mg/kg/day the effects were few, marginal in extent, and not adverse in character.

Table: Findings in male rats

 

 

28 days treatment

 

14 days recovery

Dose Level[mg/kg bw/day]

Control

50

150

450

Control

450

Clinical Signs

 

 

 

 

Pale feces

Piloerection

Dark feces

Pale feces

 

 

Body Weight Gain1)[g]

68.2

64.1

 

60.2 *

 (-12%)

48.1**

(-30%)

87.6

69.9

Hematology

 

 

 

 

 

 

 Hb[mmol/L]

9.95

10.23

9.82

9.51**

9.81

9.48

 HCT[L/L]

0.47

0.48

0.46

0.45**

0.47

0.47

 MCV[fL]

56

55.2

53.0*

52.0**

56.4

57.1

 MCHC[mmol/L]

20.9

21.4*

21.3

21.3*

20.8

20.3**

 Platelets[G/L]

1016

1143

1220*

1275**

1008

1122

 RETIC[T/L]

0.2487

0.2621

0.2903*

0.3152**

0.2734

0.3646**

 HFR[%]

8.6

8.1

10.7

13.6**

6.7

11.1*

 LFR[%]

50.3

51.2

45.9

44.9*

56.1

51.4

 WBC[G/L]

6.5

6.7

8.5*

10.0**

7.1

5.9

 SEG[G/L]

0.83

0.97

1.39

1.55**

0.85

0.95

 LYMPH[G/L]

5.60

5.67

7.11

8.28**

6.21

4.82

 PT[sec]

11.6

12.1

11.1**

11.3

11.8

11.4

Clinic. Biochem.

 

 

 

 

 

 

 Creatinine[mmol/L]

44.4

45.0

44.0

49.2**

44.6

45.0

 Bilirubin[mmol/L]

1.27

1.53

1.36

2.56**

1.82

1.84

 Cholest.[mmol/L]

1.66

1.50

1.64

2.20**

1.83

1.97

 Trigl.[mmol/L]

0.43

0.43

0.48

0.78**

0.44

0.48

 Phos. lip.[mmol/L]

1.47

1.38

1.48

2.00**

1.59

1.75

 Albumin[g/L]

39.1

39.5

40.0

41.5**

38.3

39.8**

 ALAT(GPT)[mkat/L]

0.42

0.40

0.35**

0.32**

0.46

0.42

 Protein[g/L]

62.2

62.3

63.7

65.0**

63.4

65.7**

 Calcium[mmol/L]

2.78

2.80

2.80

2.89**

2.73

2.79

 

 

Organ /Body Weight Ratio1)

 

 

 

 

 

 

 Liver

2.89

2.92

3.29** (+14%)

3.73** (+29%)

2.67

3.03** (+13%)

 Kidney

0.68

0.70

0.79** (+16%)

0.87** (+27%)

0.69

0.71

 Testes

1.23

1.12

1.25

0.70** (-43%)

1.07

0.68* (-36%)

 Epididymides

0.358

0.338

0.344

0.267** (-25%)

0.361

0.228** (-37%)

Pathology2)

 

 

 

 

 

 

 Bone marrow:

  Fatty atrophy

 

1.0 (4)

 

1.5 (4)

 

1.3 (3)

 

1.6 (5)

 

1.3 (4)

 

1.0 (3)

 Kidney:

 Tubular hyaline change

 

1.0 (4)

 

2.0 (4)

 

2.8 (5)

 

3.4 (5)

 

2.0 (2)

 

2.4 (1)

 Spleen:

 Extramed. hematopoiesis

 

2.2 (5)

 

2.6 (5)

 

3.0 (5)

 

3.4 (5)

 

2.6 (5)

 

3.8 (5)

 Testes:

 Reduced Spermatogenesis

 Spermatic giant cells

 Tubular atrophy

 

-

-

-

 

-

-

-

 

-

-

-

 

3.8 (5)

3.0 (3)

4.0 (1)

 

-

-

-

 

4.0 (4)

1.0 (1)

2.8 (4)

 Epididymides:

 Cellular debris

 Reduced Spermatozoa

 

-

-

 

-

-

 

-

-

 

2.8 (4)

5.0 (4)

 

-

-

 

1.6 (5)

5.0 (4)

1)The numbers in the parenthesis refers to the percentage increase over the control. Values are given only with statistical significance.

2)The numbers refers to the mean severity and the number in the parenthesis to the numbers of animals affected.

*/** Statistically significant at 5% or 1% level (Dennett-test).

Table: Findings in female rats

 

28 days treatment

14 days recovery

after 28 days treatment

Dose Level[mg/kg bw/day]

Control

50

150

450

Control

450

Clinical Signs

 

 

 

Piloerection

Hunched post.

Emaciation

‘Waggling gait’

Dark feces

Pale feces

 

 

Body Weight Gain1)[g]

41.7

44.7

37.8

 

23.8 **

 (-43%)

46.8

38.8

Hematology

 

 

 

 

 

 

 RBC[T/L]

8.07

7.68*

7.67*

7.32**

7.89

7.48*

 Hb[mmol/L]

9.38

9.15

9.15

8.57**

9.78

9.45

 HCT[L/L]

0.45

0.43

0.4*

0.41**

0.47

0.46

 Met-Hb[%]

0.5

0.6

0.6

0.7*

0.3

0.4

 WBC[G/L]

4.2

4.1

5.5

6.2**

4.8

5.0

 LYMPH[G/L]

3.47

3.39

4.69

5.51**

4.10

4.39

 PT[sec]

11.6

11.5

10.8**

11.0**

12.6

12.2

Clinic. Biochem.

 

 

 

 

 

 

 Cholest.[mmol/L]

1.57

1.77

2.05

3.09**

1.83

1.97

 Trigl.[mmol/L]

0.29

0.30

0.34

1.05**

0.33

0.31

 Phos. lip.[mmol/L]

1.56

1.69

1.91

2.68**

1.68

1.50

 G-GT[nkat/L]

4.67

4.57

5.67

22.42**

7.70

12.80**

 Calcium[mmol/L]

2.73

2.76

2.78

2.81*

2.76

2.75

 

Organ /Body Weight Ratio1)

 

 

 

 

 

 

 Liver

2.98

3.33*

(+12%)

3.42**

(+15%)

4.81**

 (+61%)

2.98

3.28

 Kidney

0.70

0.79

0.81*

 (+16%)

0.88**

 (+26%)

0.71

0.72

Pathology2)

 

 

 

 

 

 

 Bone marrow:

  Fatty atrophy

 

1.0 (5)

 

1.3 (3)

 

1.5 (4)

 

1.6 (5)

 

1.5 (2)

 

1.5 (2)

 Liver:

 Hypertrophy

 

-

 

-

 

-

 

2.0 (3)

 

-

 

-

 Lung:

 Foam cells

 

-

 

1.0 (3)

 

1.0 (1)

 

2.3 (3)

 

1.0 (1)

 

1.8 (4)

1)The numbers in the parenthesis refers to the percentage increase over the control. Values are given only with statistical

significance.

2)The numbers refers to the mean severity and the numbers in the parenthesis to the numbers of animals affected

*/** Statistically significant at 5% or 1% level (Dennett-test).

Table: Body weights (g) of males (PEG300 = vehicle)

PEG300

15 mg/kg bw

50 mg/kg bw

150 mg/kg bw

450 mg/kg bw

Day 1

199

189

199

193

201

Day 8

240

230

239

230

219**

Day 15

278

269

275

265

245**

Day 22

308

301

305

289

274**

Day 28

335

326

327

309*

297**

Table: Body weights (g) of females (PEG300 = vehicle)

PEG300

15 mg/kg bw

50 mg/kg bw

150 mg/kg bw

450 mg/kg bw

Day 1

0

0

0

0

0

Day 8

20

21

20

19

9**

Day 15

39

42

38

37

24**

Day 22

55

59

53

50

36**

Day 28

68

72

64

60*

48**

Conclusions:
The test material was determined to have a 28-day repeat dose (oral) NOEL of 15.0 mg/kg bw/day, and a NOAEL of 50.0 mg/kg bw/day (haemotological findings) in the rat under the conditions of the test.
Executive summary:

In this guideline (OECD 407) study conducted with GLP certification, the 28-day (oral) repeat dose toxicity NOAEL of the test material (EC 438-340-0) was determined to be 50 mg/kg bw/day. The repeat dose toxicity NOEL of the test material was determined to be 15.0 mg/kg bw/day. The test was conducted in the rat, with dose levels of 15, 50, 150 and 450 mg/kg bw/day. A target system critical effect (haematopoietic spleen) occurred at 150 mg/kg bw/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subacute
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

In a subacute toxicity study, doses of 0, 15, 50, 150 and 450 mg/kg bw/day of test article were administered daily via gavage to rats of both sexes for 28 days (OECD 407; RCC, 2002). Each five males and five females were treated and sacrificed at the end of the 28 day treatment period. In addition, from the control and high dose group each 5 animals of either sex were sacrificed after a recovery phase of 14 days. At the dose of 450 mg/kg body weight, test-item related findings were reduced food consumption, reduced body weight gain, clinical signs of intoxication (piloerection, hunched posture, dark feces), increased liver and kidney weights, reduced testes and epididymides weights, and changes in clinical biochemical and hematological parameters. In addition, morphological alterations were noted in testes, kidney, spleen, bone marrow, liver and lung.

At the dose of 150 mg/kg body weight, the findings were reduced body weight gain, increased organ/body weight ratios in liver and kidney, changes in hematological parameters and morphological alterations in kidney and spleen. The body weight changes seen at 150 mg/kg/day were minor and less than 10% altered in ration to controls. The alterations described in hematology at 150 mg/kg/day confined mainly of changes in red blood cell parameters and were minimal and confidently within the historical range of normal variations.

Furthermore, increased liver and kidney weights were reported for the 150 mg/kg/day dose group. The deviation from control was mostly small and without statistical significance. The relative weights were without statistical flag. No histopathological correlate was noted and reversibility of these findings could be shown for the high-dose group (450 mg/kg/day). Hyaline changes in renal tubules were reported in male rats at doses of 150 mg/kg/day and above. It is a well known male rat nephropathy without any relevance for human beings. Consequently, the NOAEL is 50 mg/kg/day. Findings at 150 mg/kg bw do not fulfill criteria as adverse as specified 1272/2008/EEC. In principle the above findings were reproduced in a 14 -day test study in 2013. This study was performed as a dose-range finding study for the screening study for reproductive performance with a different rat strain. No mortality occurred during the study period. At 300 mg/kg bw/day, clinical signs of toxicity were noted during the observation period. These consisted of hunched posture (noted for all animals on several days), piloerection (noted for all animals on two days), and rales (noted for one animal on one day). Salivation seen after dosing among all animals at 150 and 300 mg/kg bw/day was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). At both dose levels, a dose related effect on body weights and body weight gain was noted. Two females at 150 mg/kg bw/day showed a body weight loss of -3 or -4% on Day 14, and two females at 300 mg/kg bw/day showed a body weight loss of -10% or -2% on Day 14 (of which the latter one showed -11% on Day 10). Food consumption before or after allowance for body weight was lower at both dose levels in a dose related manner. At 150 mg/kg bw/day, slightly lower food consumption values were noted on Days 5-10 of study. At 300 mg/kg bw/day, food consumption was reduced, mainly on Days 1-10 of treatment. At 150 mg/kg bw/day, glucose concentration was increased for one female (no. 6) and cholesterol was increased for all animals. At 300 mg/kg bw/day, total protein and albumin levels were decreased, increased concentrations were noted for glucose, cholesterol and potassium. At 150 mg/kg bw/day, one female (no. 6) showed an enlarged liver and adrenal glands. At 300 mg/kg bw/day, all females showed an enlarged liver and two females showed enlarged adrenal glands. At both dose levels, increased weights of the liver (all animals) and adrenal glands (one female at 150 mg/kg bw/day and two females at 300 mg/kg bw/day) were noted. The kidneys and spleen weights among the dose groups were similar to control levels.

Justification for classification or non-classification

Classification according to GHS (1272/2008/EEC, as amended for the fifth time in Directive EC 944/2013

Based on the relevant findings, the substance does not have to be classified for repeated dose toxicity.