Registration Dossier

Administrative data

Description of key information

Valid repeated dose toxicity studies with the test substance are available. In a 5 day oral range finding study (no guideline followed), the lowest dose level of 200 mg/kg bw/d was established as LOAEL (rats, RCC743523, 1999). In a follow up 28 day subacute oral toxicity study conducted according to OECD guideline 407 the NOAEL was 30 mg/kg bw/d (rats, RCC 743512, 2000). In a 90 day subchronic oral toxicity study conducted according to OECD guideline 408 the NOAEL was 50 mg/kg bw/d (rats, RCC835424, 2002). Based on the results of these studies the NOAEL of 50 mg/kg bw/d was used as key value for chemical assessment.

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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted according to GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
(1998)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain as stated in the report: HanIbm:WIST (SPF)
- Source: RCC Ltd. Biotechnology & Animal Breeding Division, CH-4414 Fullinsdorf /Switzerland
- Age at delivery: 6 weeks
- Age at study initiation: 7 weeks
- Weight at study initiation: 130-162g (males), 111-134g (females)
- Housing: in groups of 5 in Makrolon type-4 cages
- Diet ad libitum: pelleted standard Provimi Kliba 3433 rat maintenance diet
- Water ad libitum: tap water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3
- Humidity (%): 30-70%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations of test article in polyethylene glycol were prepared daily by homogenisation with a magnetic stirrer and were used immediately at room temperature.

VEHICLE:
- Concentration in vehicle: 0, 2.5, 5, 10 and 30 mg/ml
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration, homogeneity and stability (after 3 hours) of the dose formulations were determined in samples taken after experimental start. Concentration and homogeneity of the dose formulations were further determined in samples taken at monthly intervals. The individual concentrations varied in the range from -9% to +5% of the mean concentrations. In addition, nominal concentrations were achieved in the samples and the substance was stable for at least 3 h. The analyses were performed by HPLC.
Duration of treatment / exposure:
91/92 days
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
12.5, 25, 50 and 150 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
Groups 1 and 5 (0 and 150 mg/kg bw/d): 15 males; 15 females (10 animals of each sex were sacrificed after the last treatment. Remaining animals were sacrificed after 4 week recovery period). Groups 2, 3 and 4 (12.5, 25 and 50 mg/kg bw/d): 10 males; 10 females
Control animals:
yes, concurrent no treatment
Details on study design:
The dose selection was based on a 28 day subacute oral toxicity study with 30, 150 and 750 mg/kg bw/d (RCC 743512).
The test article was administered daily by oral gavage to rats of both sexes at dose levels of 12.5, 25, 50 and 150 mg/kg bw/d for a period of 91/92 days. A control group was treated with the vehicle only. The groups comprised 10 animals per sex which were sacrificed after 90 days of treatment. Additional 5 rats per sex and group were used at 0 and 150 mg/kg body weight/day. These animals were treated for 91 days and then allowed a 28-day treatment-free recovery period after which they were sacrificed.
Observations and examinations performed and frequency:
CLINICAL SIGNS AND MORTALITY
The animals were observed for clinical signs once daily. Detailed clinical observations were performed once before study begin and weekly thereafter. Mortality of animals was checked twice daily.
BODY WEIGHT
The weight of each animal was recorded on the day of commencement of treatment, weekly during treatment and recovery and before necropsy.
FOOD CONSUMPTION
The food consumption was recorded once during the pretest period and weekly thereafter.
OPHTHALMOSCOPIC EXAMINATION
Ophthalmoscopic examinations were performed during acclimatization on all animals, and during week 13 on animals of the control and high dose groups and during week 17 on the remaining animals of the control and high dose groups.
The ophthalmoscopic examinations of both eyes of all animals were performed after the application of a mydriatic solution using an ophthalmoscope. A description of any abnormality was recorded. For unilateral findings the contralateral eye was without abnormalities.
HEMATOLOGY AND CLINICAL CHEMISTRY
After 13 weeks (end of treatment) and 17 weeks (end of recovery) animals were fasted in metabolism cages for 18 h before blood sampling. Blood was collected under light isoflurane anesthesia from the orbital sinus for hematology and clinical chemistry. Following parameters were examined at hematology: Erythrocyte count, Reticulocyte maturity index, Hemoglobin, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Hemoglobin concentration distribution width, Hematocrit, Met-hemoglobin, Mean corpuscular volume, Total leukocyte count, Red cell volume distribution width, Platelet count, Reticulocyte count, Differential leukocyte count (neutrophils, eosinophils, basophils, lymphcytes, monocytes, large unstained cells), Coagulation (thromboplastin time, activated partial thromboplastin time).
Following parameters were examined at clinical chemistry:
Glucose, Urea, Creatinine, total Bilirubin, total Cholesterol, Triglycerides, Phospholipids, Aspartate aminotransferase, Alanine aminotransferase, Lactate dehydrogenase, Creatine kinase, Alkaline phosphatase, Gamma-glutamyl-transferase, Sodium, Potassium, Chloride, Calcium, Phosphorus (inorganic), total Protein, Protein, electrophoresis (pre-albumin, albumin, alpha-1 globulin, alpha-2 globulin, beta globulins, gamma globulins) Albumin/Globulin ratio.
URINALYSIS
After 13 weeks (end of treatment) and 17 weeks (and of recovery) urine was collected for urinalysis during the 18 h fasting period (see hematology and clinical chemistry). Following parameters were examined at urinalysis: Volume, Specific gravity, Osmolality, Color, Appearance, pH, Proteins, Glucose, Ketones, Urobilinogen, Bilirubin, Blood, Nitrites, Sediment.
FUNCTIONAL OBSERVATIONAL BATTERY
During week 13, forelimb and hind limb grip strength measurements were performed using a push-pull strain gauge. Locomotor activity was measured quantitatively for a 60-minute period and the total activity of this time period was recorded. Low beams count was reported in 15-minute intervals as well as the total activity of the measuring period.

Sacrifice and pathology:
GROSS PATHOLOGY
All surviving animals were weighed and necropsied. Macroscopic examinations were performed and samples of tissues and organs were collected and kept in fixative. Following tissues were preserved at necropsy:
Adrenals, Aorta, Bone (sternum), Bone marrow (femur), Brain (telencephalon, cerebellum, pons), Caecum, Colon, Duodenum, Esophagus, Eyes with optic nerve, Harderian gland, Heart, Ileum, Jejunum, Kidneys, Larynx, Lacrimal gland, Liver, Lungs, Lymph nodes (mesenteric, thoracic), Mammary gland, Nasal cavity, Ovaries, Pancreas, Pituitary, Prostate gland, Rectum, Salivary glands (parotid and mandibular), Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord, Spleen, Stomach, Testes with epididymides, Thymus, Thyroids / Parathyroids, Tongue, Trachea, Urinary bladder, Uterus (corpus, cervix), Vagina, All gross lesions.
ORGAN WEIGHTS
Following organs from all animals sacrificed at termination were weighed: Adrenals, brain, epididymides, heart kidneys, liver, ovaries, spleen, testes, thymus, thyroids/parathyroids and uterus.
HISTOPATHOLOGY
Histopathological examinations were performed in all animals at 0 and 150 mg/kg bw/day as well as in animals found dead or sacrificed moribund. Following tissues were examined histopathologically:
Adrenals, Aorta, Bone marrow (femur), Brain (telencephalon, cerebellum, pons), Caecum, Colon, Duodenum, Esophagus, Eyes with optic nerve, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs, Lymph nodes (mesenteric, thoracic), Ovaries, Pancreas, Pituitary, Prostate gland, Rectum, Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord, Spleen, Stomach, Testes with epididymides, Thymus, Thyroids / Parathyroids, Trachea, Urinary bladder, Uterus (corpus, cervix), Vagina, All gross lesions.
Samples were embedded, cut at an approximate thickness of 2 to 4 micrometers and stained with hematoxylin and eosin. All tissues not required for microscopic examinations were kept in fixative. From the animals of the low and middle dose groups, adrenal glands, liver and spleen of both sexes were examined.

Statistics:
The following statistical methods were used to analyze the grip strength, locomotor activity, food consumption, body weight, organ weights and ratios:
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables were 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.
- Fisher's exact-test was applied to the macroscopic findings and ophthalmoscopy.
For clinical laboratory data, quantitative data was analyzed by a one-way analysis of variance (ANOVA) when the variances were considered homogeneous according to Bartlett. Alternatively, if the variances were considered to be heterogenous (p<0.05), a nonparametric Kruskal-Wallis test was used. Treated groups were then compared to the control groups using Dunnett's test if the ANOVA was significant at the 5% level and by Dunn's test in the case of a significant Kruskal-Wallis test (p<0.05).
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
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
see below
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
see below
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
see below
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see below
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see below
Details on results:
HEMATOLOGY AND CLINICAL CHEMISTRY
Hematology
After 13 week treatment with 150 mg/kg bw/d test substance, significantly lower red blood cell count, lower hemoglobin levels, reduced hematocrit and increased red cell distribution width were noted in males and females. Although most values remained within the historical control, these differences were considered to be treatment related and indicative of slight anemia.
In addition, significant increases in the absolute and relative reticulocyte counts, as well as concomitant fluorescence shift towards high fluorescence in the reticulocyte fluorescence ratios were noted in both sexes at 150 mg/kg bw/day. These differences were considered to be compensatory changes to the anemia.
The number of large unstained cells was significantly higher in females at 150 mg/kg bw/day and also exceeded the historical control data. A treatment relationship could not be excluded. However, the toxicological significance is unclear. At 12.5, 25 and 50 mg/kg bw/day the number of large unstained cells was not increased.
No further treatment related findings were noted in the hematology parameters after 13 weeks of treatment. Parameters reaching significance in either sex and/or either dose are given in table 3.
At 150 mg/kg bw/d the white blood cell count of females was significantly increased compared to control. After recovery period the white blood cell count of females was decreased (not significantly). In addition, this finding was not seen in males and was therefore considered to be incidental.
Further differences, which were considered to be incidental, consisted of: reduced relative basophil count in males treated with 50 mg/kg/day or 150 mg/kg bw/day and increased lymphocyte count in females treated with 150 mg/kg bw/day. Both findings were considered to be due to a deviant control value and therefore not treatment related. The platelet count was significantly increased in females at 150 mg/kg bw/day, but remained within the limits of the historical control data and did not show a clear dose-response relationship.
In females treated with 150 mg/kg/day, the prothrombin and partial thromboplastin times were significantly increased when compared with control. The increased prothrombin time was also noted in females treated with 12.5 mg/kg bw/day.
After recovery (4 weeks), the elevated hematocrit persisted in males of the high dose group. All other findings were considered to be reversible.
Further findings after recovery consisted of lower mean corpuscular hemoglobin concentrations, increased platelet count and number of large unstained cells in males as well as decreased met-hemoglobin in females. These parameters were unaffected after treatment and are considered to be incidental.
Clinical Chemistry:
Treatment related changes in the clinical chemistry were seen at 150 mg/kg bw/day only.
At 150 mg/kg bw/d inorganic phosphorus levels were significantly increased in males after treatment and after recovery (2.16 vs. 1.99 mmol/l at control and 1.99 vs. 1.78 mmol/l at control). Although this effect was not seen in females, a treatment relationship could not be excluded. In addition, at 150 mg/kg bw/d significantly increased total bilirubin levels were seen in females (3.12 vs. 1.85 µmol/l at control).
Further treatment related differences were seen in the absolute protein fractions or in the relative fractions of protein electrophoresis. The findings consisted of:
-increased relative alpha globulins in females (0.181 vs. 0.171 at control for alpha1 globulin and 0.701 vs. 0.062 at control for alpha2 globulin).
-increased absolute alpha globulins in females (13.05 vs. 12.03 g/l at control for alpha1 globulin and 5.09 vs. 4.35 g/l at control for alpha2 globulin, respectively)
-increased relative beta globulin in males treated with 150mg/kg bw/day after treatment and after recovery (0.193 vs. 0.179 at control and 0.183 vs. 0.165 at control, respectively).
-increased absolute beta globulin level in males after treatment and recovery (13.18 vs. 12.05 g/l at control and 12.22 vs. 10.74 g/l at control, respectively).
-the relative albumin levels were reduced in females treated with 150 mg/kg bw/day, and subsequently the albumin/globulin ratio was slightly reduced in females at 150 mg/kg bw/day (1.295 vs. 1.427).
In addition, in females significantly increased alpha2 globulins were noted at 12.5 and 50 mg/kg bw/d after treatment. Since these effects were not dose dependent they were considered to be incidental.
Further significant effects, such as increased creatinine level, cholesterol levels, alanine aminotransferase, alkaline phosphatase activity and decreased chloride levels were seen. These effects were however considered to be of no toxicological relevance, since they were either within the historical control data, not dose dependent, resulted from an unusually high or low control value or were only seen after recovery but not after treatment.
All other clinical biochemistry parameters were considered to be unaffected.

Significant findings seen after the recovery period were considered to be incidental rather than treatment related. Effects were either within the range of the historical control data or were caused by a high control value. Findings consisted of:
-elevated sodium level in males at 150 mg/kg bw/day
-decreased potassium level in males at 150 mg/kg bw/day
-marginally reduced urea in males at 150 mg/kg bw/day. This decrease was not within the range of the historical control. However, no differences were noted after 13 weeks and the possibility of a late treatment related effect was considered unlikely.
URINALYSIS
The urine output of males was increased at 150 mg/kg bw /day 9.57 vs. 7.69 ml after 13 weeks. The difference exceeded the limits of the historical control data. Thus, a treatment relationship could not be excluded. The increased urine output was fully reversible after recovery.
Other differences such as increased urinary pH values in males at 50 and 150 mg/kg bw/d), reduced erythrocyte count in females at 150 mg/kg bw/d and increased leukocyte count in males at 150 mg/kg bw/d, were considered to be incidental. Differences were either within the range of the historical control data, showed no clear dose response relationship or were only seen after recovery but not after treatment.
All other urinalysis parameters compared favorably with the controls.
ORGAN WEIGHTS
After 13 weeks increased relative and absolute liver weights (both sexes) and increased kidney weights (females) were seen at 150 mg/kg bw/d. The liver-to-brain weight ratios were also increased in these rats but these changes did not attain statistical significance. Details are given in table 4.
The absolute and relative liver weights of females dosed with 12.5 and 50 mg/kg bw/day were significantly increased. Since absolute and relative organ weights of females at 25 mg/kg bw/day were only marginally higher than those of the controls and the liver weights of females treated with 12.5 or 50 mg/kg/day were comparable (i.e. no clear dose-response relationship), these differences were considered to be not treatment related.
A significantly increased relative kidney weight was noted in females treated at 150
mg/kg bw/day in comparison to control. In the absence of morphologic findings, this finding was considered to be incidental. The kidney weights of the females treated with 12.5, 25 and 50 mg/kg/day were unaffected.
After 17 weeks the increased absolute and relative liver weights persisted in females at 150 mg/kg bw/d in comparison to control. The liver-to-brain weight ratio of these females was significantly higher than the control females. These changes were considered to be indicative of partial reversibility of findings noted after 13 weeks. Significantly elevated absolute kidney weights were noted in males at150 mg/kg bw/day compared to controls. Since the relative kidney weight was comparable to control, this difference was considered to be a body weight effect. In addition the kidney-to-brain weight ratio was significantly increased in these males, when compared with the controls.
No further changes in organ weights were seen after 17 weeks.
HISTOPATHOLOGY
After 13 weeks changes considered to be treatment related were present in the adrenal glands, liver and spleen of animals of both sexes given 150 mg/kg/day and in the liver of female rats given 50 mg/kg/day.
At 150 mg/kg bw/d a greater incidence and severity of minimal or slight increased cortical coarse vacuolation of adrenal glands was seen in 9/10 males and 10/10 females.
A minimal or slight hypertrophy of centrilobular hepatocytes in 9/10 males, 10/10 females given 150 mg/kg bw/day and 3/10 females given 50 mg/kg bw/day was seen. The finding correlated with the increased liver weight seen in animals of both sexes given at 150 mg/kg bw/d and females given 50 mg/kg bw/day.
At 150 mg/kg bw/d the level of extramedullary hematopoesis of the spleen was increased compared to control in 7/10 males and 7/10 females.
No treatment related histopathological changes were seen in animals given 12.5 or 25 mg/kg bw/day.
After recovery the hypertrophy of centrilobular hepatocytes and the level of extramedullary hematopoiesis of the spleen were fully reversible. The incidence of increased cortical coarse vacuolation was, however, still increased in males (2/5and females (2/5) compared to controls (0/5 both sexes).
Dose descriptor:
NOEL
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects were seen
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Hypertrophy of centrilobular hepatocytes was noted in females only.
Dose descriptor:
LOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified

Table 3: Changes in selected hematological parameters after 13 weeks and after recovery (17 weeks).

Parameter

MALES

FEMALES

13 weeks

17 weeks

13 weeks

17 weeks

Dose (mg/kg bw/d)

Dose (mg/kg bw/d)

0

12.5

25

50

150

0

150

0

12.5

25

50

150

0

150

RBC (T/l)

8.72

8.67

8.50

8.58

8.08**

8.58

9.92

7.64

7.67

7.49

7.51

7.08**

7.71

7.83

HB (mmol/l)

9.46

9.40

9.31

9.29

8.93**

9.60

9.76

8.87

8.93

8.75

8.80

8.28**

9.30

9.54

HCT (rel)

0.47

0.47

0.46

0.47

0.45**

0.48

0.50**

0.44

0.44

0.44

0.44

0.42**

0.46

0.47

RDW (rel)

0.13

0.13

0.14*

0.13

0.14**

0.12

0.12

0.12

0.12

0.12

0.12

0.13**

0.11

0.13

MCHC (mmol/l)

19.97

20.01

20.18

19.94

19.99

19.94

19.51*

20.11

20.09

21.00

19.92

19.81

20.20

20.47

LUC (rel)

0.005

0.006

0.004

0.007

0.006

0.005

0.001

0.005

0.006

0.005

0.005

0.006

0.006

0.005

LUC (G/l)

0.027

0.031

0.024

0.036

0.037

0.020

0.032*

0.0147

0.016

0.012

0.014

0.023*

0.02

0.014

Platelets (G/l)

870

943

908

939

986

755

891*

905

1011

848

906

1039*

778

804

PT (rel)

0.880

0.870

0.895

0.850

0.892

0.01

0.009

0.953

0.986

0.967

0.950

1.018**

0.902

0.958

APTT (sec)

19.15

18.79

19.06

19.27

18.64

18.44

18.75

19.12

20.24*

19.89

19.88

20.49**

19.22

18.58

Retic (%)

0.023

0.021

0.024

0.023

0.030**

0.020

0.017

0.030

0.030

0.029

0.028

0.036**

0.018

0.017

Retic (G/l)

198

181

202

193

238**

167

155

226

226

214

208

257*

142

129

HFR (%)

0.44

0.39*

0.45

0.45

0.56**

0.37

0.34

0.49

0.46

0.48

0.51

0.60**

0.35

0.34

MFR (%)

0.237

0.262

0.247

0.241

0.199**

0.259

0.281

0.225

0.238

0.230

0.216

0.195

0.266

0.269

LFR (%)

0.321

0.350

0.304

0.307

0.245**

0.369

0.376

0.283

0.301

0.291

0.277

0.206**

0.385

0.390

MET-HB (%)

0.009

0.009

0.009

0.009

0.009

0.010

0.009

0.010

0.010

0.010

0.010

0.010

0.010

0.008*

WBC (G/l)

5.25

5.44

5.06

5.08

6.13

4.53

5.77

2.95

2.84

2.68

2.93

3.70**

0.349

0.341

Eo (rel)

0.019

0.018

0.021

0.016

0.018

0.023

0.016

0.027

0.023

0.021

0.023

0.018

0.025

0.024

Eo (G/l)

0.102

0.096

0.105

0.080

0.100

0.094

0.090

0.078

0.061

0.057

0.065

0.070

0.078

0.064

Baso (rel)

0.008

0.006

0.007

0.005*

0.005**

0.008

0.007

0.005

0.005

0.005

0.005

0.003

0.009

0.008

Baso (G/l)

0.042

0.031

0.035

0.027

0.031

0.036

0.042

0.013

0.015

0.013

0.015

0.014

0.028

0.022

Lymph (rel)

0.744

0.767

0.728

0.766

0.745

0.689

0.734

0.727

0.700

0.687

0.730

0.749

0.719

0.701

Lymph (G/I)

3.935

4.198

3.680

3.900

4.633

3.172

4.260

2.17

2.03

1.84

2.13

2.91**

2.35

1.89

Mono (G/l)

0.107

0.102

0.094

0.103

0.110

0.100

0.132

0.058

0.057

0.059

0.056

0.084*

0.072

0.056

*/** Dunnet-test p<0.05 or p<0.01

Abbreviations: RBC = Erythrocyte count, HB = Hemoglobin, HCT = Hematocrit, RDW = red cell distribution width, MCHC = Mean corpuscular hemoglobin concentration, LUC = Large Unstained Cells, PLATELETS = Platelet count, RETIC = Reticulocyte count, HFR = high, MFR = middle, LFR = low Reticulocyte fluorescence ratios, MHT-HB = Methemoglobin, WBC = Total leukocyte count, PT = Thromboplastin time(=prothrombin time), APTT = Activated partial thrombopastin time, Eo = Eosinophile count, Baso = Basophile count, Lymph = Lymphocyte count

Table 4: Absolute and relative organ weights (g and g/100g bw, respectively) after treatment (week 13) and recovery (week 17).

Organ

 

MALES

FEMALES

13 weeks

17 weeks

13 weeks

17 weeks

Dose (mg/kg bw/d)

Dose (mg/kg bw/d)

0

12.5

25

50

150

0

150

0

12.5

25

50

150

0

150

Body weight

Abs.

377

368

384

368

374

379

420

220

225

218

224

212

230

227

 

Thymus

Abs.

0.32

0.30

0.29

0.28

0.28

0.21

0.27

0.27

0.27

0.27

0.27

0.24

0.263

0.21

Rel.

0.09

0.08

0.08

0.08

0.07

0.06

0.06

0.12

0.12

0.12

0.12

0.12

0.10

0.09

Thyroids

Abs.

0.02

0.02

0.03

0.02

0.02

0.03

0.03

0.02

0.02

0.02

0.02

0.02

0.02

0.02

Rel.

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

Heart

Abs.

1.04

1.04

1.05

0.95

1.00

1.03

1.11

0.73

0.73

0.73

0.75

0.74

0.79

0.77

Rel.

0.28

0.28

0.28

0.26

0.27

0.28

0.27

0.33

0.33

0.34

0.34

0.35

0.34

0.34

Brain

Abs.

2.02

2.03

2.04

2.05

2.06

2.06

2.12

1.91

1.94

1.92

1.91

1.90

1.96

1.87

Rel.

0.54

0.56

0.54

0.56

0.55

0.55

0.51

0.87

0.87

0.88

0.86

0.90

0.86

0.83

Adrenal glands

Abs.

0.06

0.06

0.06

0.06

0.05

0.06

0.06

0.07

0.08

0.07

0.07

0.07

0.07

0.07

Rel.

0.016

0.016

0.016

0.016

0.014

0.017

0.015

0.03

0.04

0.03

0.03

0.03

0.03

0.03

Liver

Abs.

9.26

9.33

9.78

9.37

10.43

8.65

10.04

5.54

6.43*

5.99

6.46*

7.03**

5.73

6.10

Rel.

2.45

2.53

2.54

2.55

2.79**

2.29

2.40

2.51

2.88**

2.75

2.88**

3.31**

2.49

2.70

Spleen

Abs.

0.72

0.71

0.73

0.70

0.73

0.71

0.81

0.54

0.51

0.55

0.55

0.55

0.60

0.53

Rel.

0.192

0.193

0.191

0.188

0.196

0.186

0.192

0.24

0.23

0.25

0.25

0.26

0.26

0.23

Kidneys

Abs.

2.24

2.28

2.27

2.18

2.18

2.13

2.42*

1.44

1.53

1.48

1.53

1.56

1.46

1.52

Rel.

0.60

0.62

0.60

0.59

0.58

0.57

0.58

0.65

0.69

0.68

0.68

0.74**

0.63

0.67

Testes

Abs.

3.59

3.71

3.83

3.66

3.73

3.81

3.66

 

Rel.

0.96

1.01

1.00

1.00

1.00

1.01

0.88

Epididymides

Abs.

1.45

1.45

1.48

1.40

1.42

1.51

1.48

Rel.

0.385

0.394

0.389

0.381

0.383

0.403

0.357

Ovaries

Abs.

 

0.102

0.118

0.106

0.113

0.096

0.098

0.124

Rel.

0.05

0.05

0.05

0.05

0.05

0.04

0.06

Uterus

Abs.

0.99

0.98

1.12

1.01

1.20

0.87

0.89

Rel.

0.45

0.44

0.51

0.45

0.57

0.38

0.39

*/** Dunnett-test *, p<0.05; **,p<0.01;

Conclusions:
Based on the results of this study, 25 mg/kg body weight/day was established as the NOEL and 50 mg/kg body weight/day as the NOAEL.
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

In a 5 days dose range-finding toxicity study, the test item was administered daily by oral gavage to SPF-bred Wistar rats of 2 rats/sex/dose at dose levels of 0 (vehicle control, PEG 300), 200, 600 or 1000 mg/kg body weight for a period of five days. All animals survived until scheduled necropsy. No clinical signs of toxicity were evident.

Body weight and food intake were affected in all treated groups. Organ weighing revealed increased absolute and relative liver weights in all treated animals and lower absolute and relative mean thymus weights in males treated at 200 mg/kg/day and in both sexes treated at 600 mg/kg/day and 1000 mg/kg/day.

 

In the oral subacute 28 day toxicity study (OECD guideline 407) doses of 30, 150 and 750 mg/kg bw/d were applied to rats via gavage. Satellite groups of control and high dosed animals were allowed a 14 -day treatment free recovery period.

One male treated with 750 mg/kg/day was found dead on the day of necropsy. Four females of the high dose group were found dead between days 3-6, three females died during the first blood sampling intervall and one female of this group was killed in extremis. These were considered to be test article-related deaths. All other animals survived until scheduled necropsy.

Test article-related clinical signs were observed in the high dose group and one animals of the mid dose group only. The symptoms included salivation in animals of both sexes of the 750 mg/kg bw /d group (days 5, 25-28) and in one female of the mid dose group (days 25-28). All other observations were restricted to the high dose group. Piloerection was noted during days 3-28 and persisted in the last surviving female until day 41. Hypothermia (days 6-30), hardened abdomen (days 20-30) and sedation (days 19-28) were observed in females only. Locomotor activity of the high dose group animals was decreased.

Treatment with 750 mg/kg bw/d resulted in decreased body weight, whereas food consumption was unaffected.

Application of 750 mg/kg bw/d caused an increase in cell volume, in high fluorescence reticulocytes, normoblast counts, in met-hemoglobin level and polychromatophilia, as well as increased numbers of segmented leukocytes. Changes in the mid dose group were confined to red blood cell parameters whereas the observed effects were more pronounced in the high dose group. Hematological parameter of the low dose group were unaffected. After recovery period, the red blood cell parameter changes were still present.

Clinical biochemistry revealed increased activity of several enzymes and changes in electrolyte, glucose and protein levels which was much more evident in the 750 mg/kg group than in the 150 mg/kg group. The effects on biochemistry parameters suggest differences in lipid metabolism and liver enzyme activation.

A higher urine production (females) and lower pH were noted in the high dose group only.

After 4 weeks' treatment, higher liver weights were noted in males at all dose levels and in females of the mid- and high dose group. Higher kidney weights were observed in females at 150 mg/kg/day and 750 mg/kg/day, whereas adrenal weights were higher in both sexes at 750 mg/kg/day. Thymus weights were lower in the high dose group and remained lower also after recover period.

Treatment relatedgross macroscopic changes in the high dose group included stomach foci, thickening of the thyroid gland, size reduction of the thymus in males as well as stomach foci and size reduction in spleen and thymus in females.

Microscopic changes were observed in the mid and high dose groups. At 150 mg/kg bw/d, animals showed slight liver hypertrophy and increased extramedullary hematopoiesis (females). Animals of the high dose group showed hypertrophy of the zona fasciculata, single cell death in the kidneys, slight hypertrophy and periportal vacuolation of the liver, foci of alveolar macrophages, increased extramedullary hematopoiesis and lymphocyte depletion in the spleen, stomach focal erosions, slight degeneration of testes germinal epithelium and moderate lymphocyte depletion in the thymus.

In males at the end of the dosing period there was full reversibility of the changes seen in the adrenal glands, liver, spleen and stomach and evidence of partial recovery from the lymphocyte depletion seen in the thymus. Degeneration of the germinal epithelium in the testes was still present in all the males of the recovery study.

 

 

90 day oral toxicity study in rats                                                            

 In the oral subchronic 90 day toxicity study (OECD guideline 408) doses of 12.5, 25, 50 and 150 mg/kg bw/d were applied by gavage to groups of male and female Wistar rats. Satellite groups of control and high dosed animals were allowed to recover for 4 weeks after last treatment.

 All animals survived until scheduled necropsies. Clinical signs of toxicity were not observed in any treatment group. Also grip strength and locomotor activity as well as food consumption and body weight gain were unaffected by the treatment.

Effects in hematology were noted only in the high dose group.A reductionin red blood cell counts and hematocrit, as well as increased red cell distribution width and reticulocytosis were observed in both sexes. The number of large unstained cells was elevated in females only. After 4 weeks' recovery, the elevated hematocrit persisted in male animals.

Changes in biochemical parameters were seen only in the high dose group. Inorganic phosphorus levels were increased in males after 13 weeks' treatment and 4 weeks' recovery. After 13 weeks, elevated total bilirubin levels were noted in females. Additionally, diverse differences in the protein fractions of electrophoresis of both sexes and albumin of females were determined.

Urinanalysis revealed an increased output in male animals of the 150 mg/kg bw group.

After 13 weeks, higher liver weights were noted in both sexes treated with 150 mg/kg/day. The liver weight in females was still elevated after the post-observation period but showed tendency to be reversible.

Microscopical findings in the high dose group included increased cortical coarse vacuolation of the adrenal glands, hypertrophy of centrilobular hepatocytes and an increase in the magnitude of extramedullary hematopoiesis in the spleen. In the 50 mg/kg bw group, hypertrophy of centrilobular hepatocytes was noted in females only. After the recovery period, increased cortical coarse vacuolation of the high dose group was still present.

Based on the results of this study, 25 mg/kg body weight/day was established as the NOEL and 50 mg/kg body weight/day as the NOAEL.

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is notconsidered to be classified for repeated dose toxicity under Directive 67/548/EEC.

 

 

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. As a result the substance is not considered to be classified for repeated dose toxicity under Regulation (EC) No. 1272/2008.