Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 Feb 2015 - 30 Mar 2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
Presentation of data in written and graphical format is limited.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
(1998)
Deviations:
yes
Remarks:
no urinalysis was performed; no details on analytical verification of the doses are reported
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

Species:
mouse
Strain:
Balb/c
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH, Sulzfeld, Germany
- Age at study initiation: young healthy adult mice
- Weight at study initiation: 17.8 - 24.9 g (males), 16.2 - 19.6 g (females)
- Housing: animals were kept in groups of 5 in cages
- Diet: complete feed for mice breeding, ground (REF V1120-100, ssniff Spezialdiäten GmbH, Soest, Germany) and gelatine, gold, extra pure, 180 Bloom (REF 4274.3, Carl Roth GmbH & Co. KG, Karlsruhe, Germany) formulated with the appropriate amount of test material, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Mixing appropriate amounts with (complete feed for mice + gelatine): The diet was prepared as follows (calculation for a final mass of 1 kg feed): 0, 2.33, 4.67 and 6.67 g of the test material were added to 970, 967.67, 965.33 and 963.33 g complete feed for mice, 30 g gelatine and 1000 g tap water to achieve dietary doses of 0, 350, 700 and 1000 mg/kg bw
- Storage temperature of food: at room temperature (15 - 25 °C)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The diet was monthly subjected to quality control, but no further information on test material content is reported.
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily, 7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
2 330 ppm
Dose / conc.:
4 670 ppm
Dose / conc.:
6 670 ppm
Dose / conc.:
350 mg/kg bw/day (nominal)
Remarks:
315 mg/kg bw/day (females) and 326 mg/kg bw/day (males) acutally ingested
Dose / conc.:
700 mg/kg bw/day (nominal)
Remarks:
654 mg/kg bw/day (females) and 654 mg/kg bw/day (males) acutally ingested
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
967 mg/kg bw/day (females) and 934 mg/kg bw/day (males) actually ingested
No. of animals per sex per dose:
20
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Test doses are chosen on the basis of results from a previous acute toxicity test and provided by sponsor. In addition a preliminary test for dose finding was carried out.
Positive control:
Not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The animals were observed daily for symptoms of systemic toxicity and twice daily for signs of morbidity and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made in all animals once prior to first exposure and once a week thereafter. These observations include respiration (dyspnoe, respiration frequenz), skin/membranes (cyanosis, paleness, oedema), fur (piloerection), eyes (exophtalmus, conjunctivitis, ptosis), motion (cataleptic posture, abnormal posture, abnormal gait, stereotyped movements, unusual movements, myoclonic muscle twiches, tremor), secretions (nose secretions, increased/coloured lacrimation, salivation) and excretions (diarrhea), muscle tone (body tension, tonic/clonic convulsions) and aggressiveness/irritability.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were determined prior to first exposure of the test material and once a week thereafter during the observation period.

FOOD CONSUMPTION AND COMPOUND INTAKE: YES
- Food consumption for each animal determined and mean weekly diet consumption calculated as g food/week: Yes

FOOD EFFICIENCY: YES

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmoscopical examinations (corneal opacity, retina degeneration) were carried out prior to first exposure of test material and towards the end of exposure period (not earlier than week 11).
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were taken 5 days before the end of exposure period of all mice to obtain citrate blood for testing the clotting ability and at the end of exposure period of all mice finishing the study by puncture of the retro-orbital plexus prior to necropsy.
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: all animals
- Parameters examined: erythrocyte count (Ery), hematocrit (PCV), haemoglobin (Hb), total leucocyte count (Leuc), differential leucocyte count (segmented neutrophil granulocytes (Seg), lymphocytes (Lymph), monocytes (Mono), eosinophil granulocytes (Eosin), basophil granulocytes (Baso), thrombocytes (Throm))

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were taken 5 days before the end of exposure period of all mice to obtain citrate blood for testing the clotting ability and at the end of exposure period of all mice finishing the study by puncture of the retro-orbital plexus prior to necropsy.
- Animals fasted: Yes, overnight fasting
- How many animals: all animals
- Parameters examined: total protein (TP), albumin (ALB), triglyceride (TG), cholesterol (CHOL), glucose (GLU), total bilirubin (BILI), blood urea nitrogen (BUN), creatinine (CREA), alkaline phosphatase (AP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), inorganic phosphorus (P), calcium (Ca), potassium (K), sodium (Na)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Functional tests were carried out prior to first exposure of test material and towards the end of exposure period (not earlier than week 11).
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity, grip strength, motor activity
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- A full necropsy was performed with all animals after termination of the study.

HISTOPATHOLOGY: Yes
- For histological examination samples of the following organs were fixed: salivary glands, esophagus, trachea, thyroid glands, parathyroid glands, stomach, duodenum, jejunum, ileum, caecum, colon, liver, spleen, lung, pancreas, thymus, brachial lymph nodes, skin, mammary gland, urinary bladder, kidneys, heart, skeletal muscle, peripheral nerve, aorta, mesenteric lymph nodes, adrenal glands, brain, vertebral column with spinal cord, sternum, skull including pituitary gland, ovaries, oviducts, uterus, vagina, epididymis, prostate, seminal vesicle, coagulating gland, testes and eyes.
Other examinations:
ORGAN WEIGHTS
- The following organs were weighed upon necropsy: brain, heart, liver, spleen, kidneys, adrenal glands, thymus, testes, epididymis, ovaries, uterus.
Statistics:
For all animals in the high dose and control groups the body weight, the feed consumption, the feed efficiency and the motor activity at each day / week were analyzed descriptively and displayed in a line graph. The comparison of the high dose group with the control group was performed applying a mixed linear model for repeated measurements. Modelling was performed involving all groups and the least-squares means (LS mean values) were calculated for each group. The high dose group was compared to the control group using the t-test for LS mean differences. The relative organ weight and the parameters of haematology and clinical biochemistry were analyzed descriptively. The comparison of the high dose group with the control group was performed using the Kruskal-Wallis test. All statistical tests were performed two-sided. Significance levels calculated to be less than 0.05 were marked with an asterisk.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
1/20 females in the high dose group died 5 days before the end of exposure period after blood sampling (non-adverse)
Mortality:
mortality observed, treatment-related
Description (incidence):
1/20 females in the high dose group died 5 days before the end of exposure period after blood sampling (non-adverse)
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Low/mid dose group: significant decrease of food consumption in females (non-adverse)
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
All dose groups (females) and mid dose group (males): significant increase of erythrocytes, hematocrit, haemoglobin (non-adverse)
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Low/mid dose groups (females): significant increase of CHOL, TP, UREA, BUN, CREA, TG; high dose group (females): significant increase of TP, CREA; All dose group (males): significant increase/decrease of various clinical chemistry parameters (non-adverse)
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
All dose groups (females): significant decrease of heart, spleen weight (relative); low/high dose groups (females): significant decrease of kidney weight (relative); all dose groups (males): significant decrease of adrenal weight (relative) (non-adverse)
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Reddish discoloration of the liver in 1/20 females in the high dose group; the animal died due to a blood sampling error (non-adverse)
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Reddish discoloration of the liver in 1/20 females in the high dose group; the animal died due to a blood sampling error. Lesions were observed in several tissues; however, none of the lesions noted had a special character (non-adverse)
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
1/20 females in the high dose group died due to a blood sampling error. No mouse exhibited adverse physical symptoms within the observation period (non-adverse).

BODY WEIGHT AND WEIGHT GAIN
There were no significant differences between the animals in the test and control groups regarding the body weight development. The body weight of all mice was gaining during the study period.

FOOD CONSUMPTION AND COMPOUND INTAKE
In the female low dose group and mid dose group the feed consumption of the animals was significantly lower than in the control group. There were no significant differences in all male test groups as well as in the female high dose group compared to the control group regarding feed consumption. Since there is no dose-response-relationship in this respect this finding is considered to be incidental.

FOOD EFFICIENCY
No significant differences between the animals in the test and control groups were recorded with respect to the feed consumption efficiency.

OPHTHALMOSCOPIC EXAMINATION
Ophthalmoscopic examination of the animals did not reveal any abnormal findings.

HAEMATOLOGY
In the female low dose group the parameters erythrocytes, hematocrit, haemoglobin, leucocytes, thrombocytes, lymphocytes and eosinophils are significantly higher than in the control group and the parameter thrombocytes is significantly lower. In the female mid dose group the parameters erythrocytes, hematocrit, haemoglobin, leucocytes, monocytes are significantly higher than in the control group. In the female high dose group the parameters erythrocytes, hematocrit and hemoglobin are significantly higher than in the control group. Furthermore, the parameter thrombocytes is significantly lower in the female high dose group than in the control group. In the male low dose group the parameter thrombocytes is significantly higher than in the control group and the parameter leucocytes is significantly lower. In the male control group for the parameters segs, lymphocytes, monocytes, eosinophils, basophils and bands the sample size is equal to one. In the male mid dose group the parameters erythrocytes, hematocrit and hemoglobin are significantly higher than in the control group. The reported significant increase of the parameters erythrocytes, hematocrit and hemoglobin indicate hemoconcentration. These findings might be seen in conjunction with the significant decrease of relative organ weights of spleen and/or kidney. Since the values of the parameters with statistical significance are in the reported reference range (reference range of literature and/or in-house reference range it is considered, that these findings are only of minor importance. For all other parameters no significant differences between the high dose and control group were detected refer to table 1).

CLINICAL CHEMISTRY
In the female low dose group the parameters CHOL, TP, UREA, BUN, CREA, TG, K and P-inorganic are significantly higher than in the control group, whereas the parameters Ca and Na are significantly lower. In the female mid dose group the parameters CHOL, TP, UREA, BUN, CREA, TG, Ca, P-inorganic and ALB are significantly higher than in the control group. In the female high dose group the parameters TP and CREA are significantly higher than in the control group. These findings might be seen (as well as the appropriate hematological findings indicating hemoconcentration) in conjunction with the significant decrease of relative organ weights of spleen and kidney. In the male low dose group the parameters TP, UREA, BUN and K are significantly higher than in the control group, whereas the parameters AP, AST and Na are significantly lower. In the male control group for the parameter ALB the sample size is equal to two. In the male mid dose group the parameters TP, Ca and P-inorganic are significantly higher than in the control group, whereas the parameter Na is significantly lower. In the male high dose group the parameters Na and Ca are significantly lower than in the control group. In the male high dose group the parameters Na and Ca are significantly lower than in the control group. Since the values of the parameters with statistical significance are in the reported reference range (reference range of literature and/or in-house reference range) it is considered, that these findings are only of minor importance. For all other parameter no significant differences between the high dose and control group were detected (refer to table 2).

NEUROBEHAVIOUR
Motor activity did not show significant differences between the high dose and control group.

ORGAN WEIGHTS
Significant decreases of relative organ weights were observed in the female low dose group (heart, spleen, adrenal gland and kidney), female mid dose group (heart, spleen and adrenal gland), female high dose group (heart, spleen, kidney) as well as in the male low dose group (adrenal gland), male mid dose group (adrenal gland, kidney) and male high dose group (heart, adrenal gland). For all other relative organ weights no significant differences between animals of the and control groups were detected. The pathologist points out that the significant decrease of the female spleen and kidney weights might be seen in conjunction with different blood parameters indicating hemoconcentration (increased erythrocyte count, hematocrit and hemoglobin, total protein, creatinine). The origin of significantly decreased heart weights in male and female animals is unclear. A morphological correlate of this change was not observed during histopathology (high dose group). The significant decrease in adrenal weight in male animals is interpreted as measurement inaccuracy. Due to the earlier loss of x-zonal adrenal tissue in males, adrenal glands in early adult male animals have a generally lower adrenal weight than early adult female animals (refer to table 3). The observed effects were not considered to be treatment-related as no macroscopic or microscopic abnormalities were found in these organs.

GROSS PATHOLOGY
The reddish discoloration of the liver in one female (1/20) of the high dose group was caused by a blood sampling error. Apart from the liver lesion gross findings are interpreted as incidental (scoliosis, peritoneal mass of stomach, urinary calculus), hormonal cycle dependent (fluid filled uteri) and traumatic or sequels of trauma (dull/ shrunken eye globes, hemorrhages, skin wounds, lymphadenopathy of inguinal lymph nodes, alopecia).

HISTOPATHOLOGY: NON-NEOPLASTIC
The observed liver lesions of 5/10 female in the high dose, which were detected to a slightly higher number (to a lesser degree in severity) comprised focal/multifocal necrosis, single cell necrosis, hepatocyte karyocytomegaly and inflammation focal/multifocal. Hepatocyte necrosis can occur spontaneously or can be induced or exacerbated by treatment. Focal/multifocal necrosis can occasionally be seen in untreated rodents and may also be a terminal event due to impaired blood flow. Karyomegaly is also regarded as an incidental finding, especially in older mice. Variations in cell size may be attributed to increase of nuclear size and ploidy. Inflammation, focal/multifocal was diagnosed only if the leukocyte infiltration was associated with degenerative changes, edema and vascular injury since mononuclear infiltrates are frequent incidental background findings in rodents Hepatocyte necrosis as well as inflammatory lesions were examined to a slightly higher number and degree in the livers of animals of the high dose group. Hepatocyte karyocytomegaly was observed to a comparable extent in control male and male mice of the high dose group. This change was not seen in female control mice whereas female mice of the high dose group exhibited a comparable degree of hepatocyte karyocytomegaly as control male mice and male mice of the high dose group. Lesions that were present to a comparable extent in control and high dose animals were considered as background findings, due to ambient conditions or terminal asphyxia. Lesions such as glandular stomach cysts, adrenocortical nodules and mesonephric duct remnants are congenital lesions and are thus interpreted as irrelevant for the assessment of subchronic toxicity.

HISTORICAL CONTROL DATA
The observed haematology and clinical chemistry parameters with statistical significance were in the reported reference range (reference range of literature and/or in-house reference range).

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
967 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: The NOAEL is corresponding to the highest dose level tested.
Dose descriptor:
NOAEL
Effect level:
934 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: The NOAEL is corresponding to the highest dose level tested.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Haematology data (statistically significant parameters)

Parameter

Dose Group

N

Mean

STD

Median

Min

Max

p-Value

Females

Erythrocytes [T/l]

control

low dose

medium dose

high dose

10

9

10

9

10.39

11.18

11.93

11.22

0.40

0.48

0.89

0.77

10.37

11.37

12.04

11.47

9.81

10.34

10.45

9.79

11.14

10.34

13.21

12.00

 

0.0029*

0.0007*

0.0222*

Haematocrit [i/L]

control

low dose

medium dose

high dose

10

9

10

9

0.49

0.53

0.58

0.52

0.02

0.03

0.04

0.04

0.49

0.53

0.57

0.54

0.46

0.48

0.52

0.44

0.51

0.56

0.65

0.56

 

0.0040*

0.0002*

0.0330*

Haemoglobin [g/L]

control

low dose

medium dose

high dose

10

9

10

9

157.20

168.78

181.70

171.00

6.34

7.85

14.53

12.85

157.00

172.00

181.00

176.00

146.00

154.00

161.00

147.00

169.00

176.00

203.00

185.00

 

0.0068*

0.0004*

0.0271*

Leucocytes [G/L]

control

low dose

medium dose

high dose

10

10

10

9

2.81

4.10

4.89

3.23

0.87

1.62

1.29

0.92

2.90

3.75

4.50

3.10

1.60

2.00

3.10

2.20

4.30

7.70

7.80

4.70

 

0.0447*

0.0013*

0.3259

Basophils [%]

control

low dose

medium dose

high dose

7

9

8

5

0.57

0.00

0.13

0.40

0.79

0.00

0.35

0.55

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

2.00

0.00

1.00

1.00

 

0.0358*

0.1787

0.7812

Thrombocytes [G/L]

control

low dose

medium dose

high dose

10

9

10

9

211.90

283.78

237.30

159.11

48.32

72.75

49.09

37.95

210.00

283.00

212.00

160.00

129.00

190.00

175.00

102.00

298.00

376.00

321.00

210.00

 

0.0498*

0.4053

0.0179*

Lymphocytes [G/L]

control

low dose

medium dose

high dose

7

9

8

5

1.74

2.98

3.03

2.00

0.57

1.23

0.63

0.55

1.90

2.70

3.05

1.90

0.70

1.50

2.20

1.30

2.50

5.60

4.10

2.50

 

0.0337*

0.0026*

0.5122

Eosinophils [G/L]

control

low dose

medium dose

high dose

7

9

8

5

0.09

0.27

0.28

0.08

0.09

0.15

0.29

0.04

0.10

0.20

0.20

0.10

0.00

0.10

0.00

0.00

0.20

0.60

0.90

0.10

 

0.0090*

0.1076

1.0000

Monocytes [G/L]

control

low dose

medium dose

high dose

7

9

8

5

1.43

1.33

0.08

0.02

0.53

0.81

0.05

0.04

1.00

1.00

0.10

0.00

1.00

1.00

0.00

0.00

2.00

3.00

0.10

0.10

 

0.5153

0.0231*

0.8020

Segs [G/L]

control

low dose

medium dose

high dose

7

9

8

5

0.53

0.84

1.16

0.62

0.28

0.42

0.40

0.22

0.50

1.00

1.10

0.50

0.30

0.30

0.70

0.40

1.00

1.40

2.00

0.90

 

0.1081

0.0051*

0.4042

Males

Erythrocytes [T/l]

control

low dose

medium dose

high dose

10

10

10

10

10.62

11.30

11.95

10.98

0.85

1.20

0.93

0.69

11.06

11.62

12.21

11.04

9.14

8.62

10.49

9.87

11.50

12.78

13.29

12.21

 

0.0588

0.0065*

0.5957

Haematocrit [i/L]

control

low dose

medium dose

high dose

10

10

10

10

0.52

0.57

0.60

0.52

0.04

0.05

0.05

0.03

0.53

0.58

0.61

0.52

0.46

0.46

0.53

0.45

0.59

0.65

0.67

0.57

 

0.0527

0.0031*

0.7036

Haemoglobin [g/L]

control

low dose

medium dose

high dose

10

10

10

10

162.80

170.70

181.60

168.10

12.81

18.86

14.26

11.50

167.50

177.00

185.50

168.50

141.00

128.00

159.00

151.00

176.00

194.00

202.00

189.00

 

0.0638

0.0139*

0.5201

Leucocytes [G/L]

control

low dose

medium dose

high dose

10

10

10

10

6.18

3.71

6.25

7.72

2.07

1.36

2.17

2.38

5.75

3.50

6.60

7.35

3.80

2.50

2.30

5.10

10.30

7.20

9.30

11.60

 

0.0022*

0.6772

0.1037

Thrombocytes [G/L]

control

low dose

medium dose

high dose

10

10

10

10

210.50

290.90

257.40

194.90

47.38

87.84

79.46

36.80

195.00

280.00

242.00

186.50

146.00

168.00

160.00

153.00

292.00

464.00

422.00

266.00

 

0.0343*

0.1402

0.3845

*: p<0.05

N: number of animals

STD: standard deviation

Table 2: Clinical chemistry data (statistically significant parameters)

Parameter

Dose Group

N

Mean

STD

Median

Min

Max

p-Value

Females

CHOL [mmol/L]

control

low dose

medium dose

high dose

9

10

8

6

3.57

4.23

4.65

4.00

0.35

0.53

0.73

0.42

3.40

4.20

4.70

4.05

3.10

3.60

3.70

3.20

4.20

5.00

5.80

4.40

 

0.0077*

0.0032*

0.0750

TP [g/L]

control

low dose

medium dose

high dose

9

10

7

5

49.73

60.83

60.53

56.08

3.57

5.16

3.86

3.53

50.10

59.80

60.30

56.20

42.60

53.50

56.20

51.00

54.80

68.20

66.90

59.40

 

0.0004*

0.0009*

0.0196*

Urea [mmol/L]

control

low dose

medium dose

high dose

9

10

9

7

10.02

12.18

11.20

10.73

0.73

1.64

0.98

0.92

9.80

12.45

11.10

10.90

9.00

9.60

9.20

9.20

11.00

14.80

12.40

12.00

 

0.0079*

0.0151*

0.1118

BUN [mmol/L]

control

low dose

medium dose

high dose

9

10

9

7

4.68

5.69

5.23

5.01

0.33

0.75

0.46

0.43

4.60

5.80

5.20

5.10

4.20

4.50

4.30

4.30

5.10

6.90

5.80

5.60

 

0.0069*

0.0115*

0.0892

CREA [µmol/L]

control

low dose

medium dose

high dose

9

10

7

6

23.89

38.70

31.86

30.50

5.73

7.10

7.15

5.21

21.00

38.00

31.00

31.00

18.00

27.00

23.00

22.00

33.00

50.00

45.00

37.00

 

0.0009*

0.0383*

0.0381*

TG [mmol/L]

control

low dose

medium dose

high dose

9

9

5

3

1.61

2.41

2.25

1.66

0.20

0.54

0.56

0.23

1.67

2.33

2.06

1.61

1.24

1.73

1.79

1.46

1.85

3.37

3.13

1.92

 

0.0009*

0.0062*

0.7811

Ca [mmol/L]

control

low dose

medium dose

high dose

9

10

9

5

2.10

1.91

2.34

2.08

0.13

0.12

0.10

0.19

2.10

1.90

2.40

2.10

1.90

1.80

2.20

1.80

2.30

2.10

2.50

2.30

 

0.0071*

0.0014*

0.8908

K [mmol/L]

control

low dose

medium dose

high dose

10

9

7

5

26.33

52.97

26.61

25.70

3.23

4.52

3.95

4.89

26.05

54.30

26.70

24.80

21.40

44.20

21.00

21.20

32.00

58.70

33.50

34.00

 

0.0002*

0.9223

0.5403

Na [mmol/L]

control

low dose

medium dose

high dose

10

9

9

6

150.30

126.22

147.22

151.33

3.33

4.18

5.02

3.98

150.50

126.00

148.00

151.50

144.00

118.00

136.00

146.00

155.00

135.00

152.00

156.00

 

0.0002*

0.2019

0.6234

P-inorganic [mmol/L]

control

low dose

medium dose

high dose

9

10

9

6

9.86

11.86

12.00

10.57

0.92

1.94

1.18

0.99

10.30

12.85

12.60

10.75

8.60

7.40

9.30

9.20

11.10

13.40

13.10

11.60

 

0.0159*

0.0023*

0.1400

ALB [g/L]

control

low dose

medium dose

high dose

5

9

8

2

6.56

12.74

24.36

6.30

8.18

7.35

6.33

1.84

3.50

13.20

24.90

6.30

1.40

0.90

15.10

5.00

20.90

22.00

33.10

7.60

 

0.2053

0.0128*

0.4386

Males

AP [µkat/L]

control

low dose

medium dose

high dose

10

7

5

8

1.43

0.95

1.39

1.86

0.23

0.30

0.34

0.33

1.45

1.00

1.28

1.85

0.97

0.55

1.02

1.42

1.75

1.33

1.88

2.30

 

0.0046*

0.8058

0.0098*

AST [µkat/L]

control

low dose

medium dose

high dose

8

10

4

9

2.93

1.64

2.59

3.03

0.88

0.84

0.94

1.10

2.70

1.58

2.59

3.06

1.71

0.36

1.49

0.82

4.01

2.90

3.69

4.58

 

0.0129*

0.4969

0.7728

BILI-total [µmol/L]

control

low dose

medium dose

high dose

6

10

4

9

11.28

6.32

2.65

16.81

6.06

6.17

2.82

9.00

11.35

3.50

1.60

18.40

3.40

1.60

0.60

0.10

18.60

20.20

6.80

28.80

 

0.1035

0.0247*

0.1749

TP [g/L]

control

low dose

medium dose

high dose

9

8

7

8

52.27

66.95

61.07

55.34

6.56

4.88

4.15

6.87

50.30

68.45

58.20

56.60

44.30

57.80

57.40

45.50

67.40

72.70

67.10

63.00

 

0.0015*

0.0095*

0.2682

Urea [mmol/L]

control

low dose

medium dose

high dose

8

9

7

10

9.11

11.46

9.41

8.59

1.35

1.45

0.85

0.98

9.30

11.00

9.50

8.50

6.20

9.90

8.30

7.10

10.90

13.70

10.60

10.20

 

0.0024*

0.8162

0.1965

BUN [mmol/L]

control

low dose

medium dose

high dose

8

9

7

10

4.24

5.36

4.39

3.99

0.62

0.67

0.37

0.46

4.30

5.10

4.40

3.95

2.90

4.00

3.90

3.30

5.10

6.40

4.90

4.80

 

0.0028*

0.7266

0.1962

TG [mmol/L]

control

low dose

medium dose

high dose

5

7

6

8

2.00

2.60

2.82

3.42

0.21

0.95

1.42

1.12

1.96

2.45

3.38

3.32

1.75

1.51

0.10

1.66

2.31

4.02

4.01

5.55

 

0.3718

0.0679

0.0281*

K [mmol/L]

control

low dose

medium dose

high dose

10

9

5

8

23.42

47.58

23.58

26.94

4.37

9.30

4.73

2.69

21.70

47.10

22.60

27.35

20.10

32.20

18.70

22.30

32.90

62.70

30.60

31.00

 

0.0003*

1.0000

0.0558

Na [mmol/L]

control

low dose

medium dose

high dose

10

9

9

8

157.00

130.00

152.00

151.63

2.62

9.54

4.27

2.88

157.00

132.00

152.00

151.00

153.00

115.00

145.00

147.00

162.00

147.00

157.00

157.00

 

0.0002*

0.0119*

0.0026*

Ca [mmol/L]

control

low dose

medium dose

high dose

9

7

8

7

2.29

2.21

2.56

2.07

0.14

0.13

0.11

0.11

2.30

2.20

2.55

2.10

2.00

2.00

2.40

1.90

2.40

2.40

2.70

2.20

 

0.2297

0.0009*

0.0082*

P-inorganic [mmol/L]

control

low dose

medium dose

high dose

9

7

8

7

10.92

11.56

12.18

11.16

0.60

1.15

0.63

0.68

10.90

11.10

12.50

11.00

9.90

10.00

11.40

10.00

11.70

13.00

12.80

12.00

 

0.3665

0.0038*

0.3661

*: p<0.05

N: number of animals

STD: standard deviation

Applicant's summary and conclusion

Conclusions:
The test material was examined for toxicity after repeated exposure in a subchronic study with mice according to OECD 408 and in compliance with GLP. Based on no treatment-related findings of toxicological relevance, the NOAEL is therefore considered to be 934 and 967 mg/kg bw/day for males and females, respectively.