Registration Dossier

Administrative data

Description of key information

The NOAEL value for repeated dose oral toxicity was 0.2 mg/kg bw/day in rats.

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:
03 Aug 2018 to 15 Oct 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
other: Wistar Han IGS rats (Crl:WI(Han))
Details on species / strain selection:
This rat strain was used because it is routinely used at the test facility for this type of studies.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 11 to 12 weeks
- Weight at study initiation: Males: 308.1 to 370.5 g. Females: 207.4 to 246.3 g
- Housing: The rats were housed in Makrolon cages with a bedding of wood shavings and strips of paper and a wooden block as environmental enrichment. During the pre-treatment and pre-mating period the females were housed 3-4 to a cage and the males 4-5 to a cage. After allocation the animals were housed four rats to a cage (separated by sex). For mating, one male and one female were housed together. Males were transferred to their home cages after mating. Mated females were housed individually in Makrolon cages, which were placed in another cage rack.
- Diet: The rats received a cereal-based (closed formula) rodent diet (VRF1 (FG)) from a commercial supplier (SDS Special Diets Services, Witham, England) and were fed ad libitum.
- Water: Water was provided ad libitum
- Acclimation period: 9 days

DETAILS OF FOOD AND WATER QUALITY: Food and water was checked for contaminants.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 45-65
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 03 Aug 2018 to 15 Oct 2018
Route of administration:
oral: gavage
Details on route of administration:
The oral route of administration was used because this is an anticipated route of human exposure.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Dilutions of the test substance in the vehicle were prepared daily. During the daily administration all dilutions were continuously stirred on a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle: Corn oil was selected as a vehicle for the preparation of test item formulations as the test item was not soluble in water.
- Concentration in vehicle: 0.04, 0.4 and 2.0 mg/mL corresponding to a dose of 0.2, 2.0 and 10.0 mg/kg bw, respectively.
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): A1800663
- Purity: 100%
- The dosing volume was adjusted based on the latest recorded body weight for each individual animal to maintain a constant dose level in terms of the animal’s body weight. During the gestation period, dose volumes were not adjusted after GD 14. A fixed volume based on the body weight on gestation day 14 was used for females between gestation day 14 up to the end of pregnancy.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses to determine the homogeneity and content of the test substance in dosing dilutions were conducted using an ICP-MS method.
• The homogeneity (and content) of the test substance in the test dilutions was assessed in the batch prepared on 20 August 2018, by analysing three samples of each test dilution (taken at top-, mid- and bottom- of the vial).
• The content of the test substance in each test dilution was determined by analysing one sample of each test dilution prepared on 3, 10 and 17 September 2018.
• Because the test substance was determined by analysing the Nickel content, it was not possible to determine the stability of the test substance. Instead, fresh test dilutions were prepared daily.
Duration of treatment / exposure:
Males: Male animals were dosed during a 2-week premating period, during mating and after mating up to and including the day prior to sacrifice (after 31 days of treatment).
Females: The female animals were dosed during a 2-week premating period, and during mating, gestation and lactation up to and including the day prior to sacrifice on day 14 of lactation, or soon thereafter (one female killed on day 15 of lactation).
Frequency of treatment:
once daily
Dose / conc.:
0.2 mg/kg bw/day (actual dose received)
Remarks:
Group 2: Low dose
Dose / conc.:
2 mg/kg bw/day (actual dose received)
Remarks:
Group 3: Mid dose
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Remarks:
Group 4: High dose
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected in consultation with the sponsor based on the results of an abandoned study (Triskelion Report V21099/02, September 2018) and a 2-week dose-range finding study in rats administered 0.2, 1.0 and 5.0 mg test substance/kg body weight/day (Triskelion Report V21099/03, September 2018).
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
- Time schedule: All the animals were observed once daily for clinical signs of toxicity and twice daily for mortality and morbidity.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: prior to the first exposure and then once weekly throughout the study. The latter observations were conducted a few hours after dosing. Arena testing was not performed during the last days of pregnancy and during the first days of lactation.
- Parameters checked: Signs noted included but were not limited to changes in skin and fur, piloerection, changes in the eyes, gait (including posture), and presence of clonic or tonic movements, stereotypies and bizarre behaviour.

BODY WEIGHT:
- Time schedule for examinations: The body weight of each adult animal was recorded once during the acclimatization period (on day -4) and at initiation of treatment (day 0). Females were weighed once per week during the premating and mating period. Mated females were weighed on days 0, 7, 14 and 20 during presumed gestation and on day 0, 4, 7 and 13 of lactation. Non-mated females were weighed once per week after the mating period. The adult animals were weighed on their scheduled necropsy date in order to calculate the correct organ to body weight ratios.

FOOD CONSUMPTION:
- The food consumption was measured per cage over the same periods as the body weight were measured. The results were expressed in g per animal per day. Food intake was not recorded during the mating period. Food intake of non-mated females was not recorded.

HAEMATOLOGY:
- Time schedule for collection of blood: On the day of scheduled sacrifice
- Anaesthetic used for blood collection: Yes (CO2/O2)
- Animals fasted: Yes, overnight (water was available)
- How many animals: The clinical pathology (haematological and clinical biochemistry) examinations were conducted in five males and five females randomly selected from each main group and from all recovery group animals.
- Parameters checked: Haemoglobin (Hb), Packed cell volume (PCV), Red blood cells (RBC), Reticulocytes, Total white blood cells (WBC), Differential white blood cells (neutrophils, lymphocytes, eosinophils, basophils, monocytes), Prothrombin time and Thrombocyte count. The following parameters were calculated: Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH) and Mean corpuscular haemoglobin concentration (MCHC).

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: On the day of scheduled sacrifice
- Anaesthetic used for blood collection: Yes (CO2/O2)
- Animals fasted: Yes, overnight (water was available)
- How many animals: The clinical pathology (haematological and clinical biochemistry) examinations were conducted in five males and five females randomly selected from each main group and from all recovery group animals.
- Parameters checked: alkaline phosphatase activity (ALP), aspartate aminotransferase activity (ASAT), alanine aminotransferase activity (ALAT), gamma glutamyl transferase activity (GGT), total protein, albumin, ratio albumin to globulin (calculated), urea, creatinine, glucose (fasting), bilirubin (total), cholesterol (total), triglycerides, calcium (Ca), sodium (Na), potassium (K), chloride (Cl), inorganic phosphate (PO4) and bile acids.

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule for examinations: Females on PN13 after sacrifice of their pups.
- Dose groups that were examined: All groups
- Battery of functions tested: See Table 2 in ‘Any other information on materials and methods incl. tables’.
Sacrifice and pathology:
SACRIFICE:
The animals were sacrificed by exsanguination from the abdominal aorta whilst under CO2/O2 anaesthesia and then subjected to macroscopic examination for pathological changes. Adult male animals were sacrificed after 31 days of treatment. On the day of necropsy a vaginal smear was taken of all adult females. Dams were sacrificed on day 14 of lactation, except for one female that was killed on LD15.

GROSS NECROPSY/ ORGAN WEIGHTS: Table 3 in ‘Any other information on materials and methods incl. tables’.
At scheduled necropsy, the organs of the adult animals were weighed (paired organs together) as soon as possible after dissection to avoid drying. Samples of the tissues and organs (see Table 1) of the adult animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde. The reproductive organs, the thyroid, the parathymic lymph nodes and all gross lesions of all male and female animals were preserved. The number of implantation sites in the uterus was counted. The other organs/tissues were preserved of five adult animals/sex/group (surviving males with the lowest identification numbers in each cage; females with a litter were selected).

HISTOPATHOLOGY: Table 3 in ‘Any other information on materials and methods incl. tables’.
Tissues for microscopic examination were embedded in paraffin wax, sectioned, and stained with haematoxylin and eosin, except for sections of the testes which were stained with PAS haematoxylin. Microscopic examination (by light microscopy) was performed as follows:
Males:
• Microscopic examination was performed on the preserved organs of males of the control group (1) and the high-dose group (4). Organs marked with an asterisk (the levator ani plus bulbocavenosus muscle complex, Cowper’s glands and glans penis) were preserved after weighing but not further examined.
• Microscopic examination of the preserved adrenals, heart, liver, lungs and skeletal muscle was extended to males of the low-dose (2) and mid-dose (3) groups.
• Gross lesions and the parathymic lymph nodes were examined microscopically in males of all dose groups.
• Reproductive organs (testes, epididymides, seminal vesicles and prostate) of males that did not mate were examined microscopically.

Females:
Because the female high-dose group was terminated intercurrently, microscopic examination of organs and tissues was focussed on the next lower-dose group (3) which completed the entire study duration.
• Microscopic examination was performed on the preserved organs of all females of the control group (1) and mid-dose group (3).
• Microscopic examination of the preserved adrenals, heart, liver, lungs, and skeletal muscle was extended to females of the low-dose (2) group.
• Gross lesions and the parathymic lymph nodes were examined microscopically in females of all dose groups (including the high-dose females).
• Reproductive organs (ovaries and uterus) of females that were non-mated were examined microscopically.
Other examinations:
Blood sampling for hormone determinations
During scheduled necropsy blood was taken from the aorta under CO2/O2 anaesthesia from all adult male and female animals and plasma was stored in a freezer at ≤-18° C.

Hormone determinations (T4)
Plasma samples were analysed for Thyroxin (T4) hormone levels in all adult males.
Statistics:
The statistical procedures for analysis of data are described in Table 1 in ‘Any other information on materials and methods incl. tables’. Where applicable, high-dose females, and non-mated females were excluded from mean data tables presenting data from the gestation and lactation periods.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were no treatment-related clinical signs in males or, until parturition, in females. Signs noted prior to the death of eight females of the high-dose group included respiratory distress, piloerection and soiled fur/perineum. Two other pregnant high-dose rats delivered, but had litters with mainly dead pups and showed paleness and piloerection after delivery. The females in the other dose-groups did not show treatment-related clinical signs.
Mortality:
mortality observed, treatment-related
Description (incidence):
At parturition, one high-dose rat was found dead and eight high-dose rats were humanely killed because of conditional decline.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See Tables 1 to 8 in 'Any other information on results incl. tables'.
Mean body weights were statistically significantly reduced in high-dose males in the last two weeks of their treatment, and in high-dose females on day 7 and 20 of gestation. In addition, body weight gain was statistically significantly reduced in high-dose males at most stages, and in high-dose females during the first week of the premating period and the last week of gestation. There were no treatment-related differences in body weights or body weight gain in males or females of the low- and mid-dose groups.
Statistically significantly lower body weight gains noted in females prior to the start of the treatment, and in low-dose males in the last week of their treatment were not reflected in significant effects on body weights and are considered chance findings.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
See Tables 9 to 12 in 'Any other information on results incl. tables'.
Statistically significantly reductions in food consumption were noted in high-dose males, and in high-dose females during premating, gestation and (in two remaining rats only) lactation. Food consumption was also statistically significantly reduced in males of the mid-dose group in their last week of treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
See Tables 13 to 16 in 'Any other information on results incl. tables'.
In mid- and high-dose males, red blood cell count, haemoglobin concentration and packed cell volume were statistically significantly decreased, and reticulocytes were increased compared to controls. Prothrombin time was statistically significantly increased in high-dose males. An incidental increase in thrombocyte count in mid-dose males was not confirmed in the high-dose group and was therefore considered a chance finding. In females treated up to 2 mg/kg bw/day there were no significant differences in red blood or coagulation parameters, apart from an incidental increase in haemoglobin concentration in mid-dose females. In high-dose males, total white blood cell count, absolute lymphocyte and eosinophil counts were statistically significantly lower, while the percentage of monocytes was statistically significantly higher than in controls. There were no statistically significant differences in white blood cell counts in males or females of the low- and mid-dose groups.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
See Tables 17 to 18 in 'Any other information on results incl. tables'.
In high-dose males, ASAT activity and urea, chloride and sodium concentrations were statistically significantly increased, while total protein, albumin, glucose and calcium concentrations were statistically significantly decreased compared to controls.
There were no treatment-related differences in clinical chemistry data in males or females of the low- and mid-dose groups. Results obtained in het high-dose female group are not discussed because the measurements were performed in one dam only.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
The results of the neurobehavioral observations and motor activity assessment did not indicate any neurotoxic potential of the test substance in rats.
Weak evidence of an effect of treatment on body temperature was, however, observed in the functional observation test: at the end of the treatment period, body temperature was statistically significantly decreased in the female high-dose group which included by then only two dams.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
See Tables 19 to 22 in 'Any other information on results incl. tables'.
The following statistically significant differences with the controls were noted in absolute organ weights and/or in organ to body weight ratio’s:
• The absolute and relative weights of the heart were increased in mid- and high-dose males. The absolute and relative weights of the heart were also considerably increased in the two surviving high-dose females (no statistics applied).
• The absolute and relative weights of the liver were increased in high-dose males.
• The absolute weights of the epididymides, prostate and seminal vesicles, and the relative weights of the prostate and seminal vesicles were decreased in high-dose males.

The following statistically significant differences were not ascribed to the test substance:
• The relative weight of the kidneys was decreased in low-dose males, but this finding was not confirmed at higher dose levels.
• The absolute weight of the kidneys was lower in high-dose males. Because the relative weight of this organ was not significantly affected, this finding is ascribed to the lower terminal body weights in this group.

There were no statistically significant changes in females of the low- and mid-dose groups. Results obtained in high-dose females are, due to the low number of remaining dams in this group, not further discussed.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
MACROSCOPY OF MORIBUND AND DEAD FEMALES:
Macroscopy showed enlarged parathymic lymphnodes in 6/9 females, a small cecum in 2/9 females, stomach ulcers in 4/9 females and pale lungs in 2/9 females. The remaining findings were considered unremarkable and part of the background pathology of rats of this strain and age.

MACROSCOPY AT SCHEDULED NECROPSY:
At necropsy, the parathymic lymphnodes were enlarged in 5/12 high-dose males and in 2/3 surviving high-dose females. Red discoloration was noted in 4/12 high-dose males (including three males also showing enlarged parathymic lympnodes). In the other groups, including the controls, these findings were observed only incidentally.
The heart was enlarged of 1/12 high-dose males. The remaining findings were considered unremarkable and part of the background pathology of rats of this strain and age.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
MICROSCOPY OF MORIBUND AND DEAD ANIMALS:
Mild to moderate histiocytosis was observed in the parathymic lymphnodes of 7/9 high-dose females which, in most cases, corresponded with the macroscopically observed enlarged parathymic lymphnodes (see above).
The macroscopically pale lungs (see above) of the two high-doses females showed minimal perivascular inflammation.
The remaining findings were considered unremarkable and part of the background pathology of rats of this strain and age.

MICROSCOPY AT SCHEDULED NECROPSY:
Parathymic lymphnode
In the control group 3/24 rats (3 males) showed minimal histiocytosis (including the male with the macroscopically observed enlargement of the parathymic lymphnodes). In the low-dose group, 4/24 rats (4 females) showed minimal histiocytosis. In the mid-dose group 5/21 rats (2 males and 3 females) showed minimal histiocytosis. In the high-dose group 14/15 rats (11 males and the three surviving females) showed mild to moderate histiocytosis of the parathymic lymphnodes. Adrenal gland
In 4/5 males of the mid-dose group and 5/5 males of the high-dose group, mild to moderate vacuolation was observed in the zona fasciculata of the adrenal gland. This finding was not observed in any other group.
Heart
Degeneration of heart muscle tissue, characterized by the presence of fibrotic areas, was observed in 4/5 mid-dose males at minimal to mild degree, and in 6/6 high-dose males at mild to moderate degree. This finding was not observed in any other group.
Skeletal muscle
Minimal to mild degeneration of the skeletal muscle was observed in 5/5 high-dose males. In addition 3/5 high-dose males showed minimal to mild necrosis and 4/5 high-dose males showed minimal to mild mononuclear inflammation. In the mid-dose group, one male showed minimal mononuclear inflammation and minimal necrosis. These findings were not observed in any other group.
Liver
Minimal to mild hyperemia was observed in in the liver of 5/6 high-dose males while minimal hyperemia was observed in the liver of 2/5 mid-dose females. This finding was not observed in any other group.
Lung
Minimal to mild perivascular inflammation was noted in 6/6 high-dose males, in 4/5 mid-dose males and in 5/5 mid-dose females. In addition, minimal to mild accumulation of alveolar macrophages was noted in 6/6 high-dose males, in 5/5 mid-dose males and in 4/5 mid-dose females. Further, the incidence of alveolitis was increased in high-dose males (5/6 high-dose males showed minimal to mild alveolitis). Apart from the incidental occurrence of alveolitis and alveolar macrophages, these findings were not observed in the control group or the low-dose group. Occasional occurrence of alveolar macrophages and alveolitis is in line with our historical control data.
The remaining findings were considered unremarkable and part of the background pathology of rats of this strain and age.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
T4 LEVELS: No statistically significant effects in plasma T4 levels were noted between treatment groups and controls.
Key result
Dose descriptor:
NOAEL
Effect level:
0.2 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
haematology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Key result
Dose descriptor:
NOAEL
Effect level:
0.2 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
10 mg/kg bw/day (actual dose received)
System:
musculoskeletal system
Organ:
heart
other: skeletal muscle
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Table 1. Body weight males during the study - Day(s) Relative to Start Date

Sex: Male

 

Bodyweights

Bodywt

day -x

(g)

[G]

Bodywt
(g)

[G]

Bodywt

(g)

[G]

Bodywt

(g)

[G]

Bodywt

(g)

[G]

Bodywt

(g)

[G]

-4

0

7

14

21

28

0.0 mg/kg

Mean

332.25

339.20

349.24

360.44

364.82

377.88

 

SD

14.42

16.02

16.17

17.21

15.37

14.27

 

N

12

12

12

12

12

12

0.2 mg/kg

Mean

330.89

338.01

346.78

357.60

361.08

368.26

 

SD

12.48

12.55

13.83

13.92

15.38

17.69

 

N

12

12

12

12

12

12

2.0 mg/kg

Mean

330.38

339.37

346.56

358.48

362.31

372.29

 

SD

12.58

13.26

13.00

14.23

14.99

16.79

 

N

12

12

12

12

12

12

10.0 mg/kg

Mean

331.36

336.26

337.01

346.16

343.83**

349.34**

 

SD

14.80

14.44

14.55

17.78

15.84

19.15

 

N

12

12

12

12

12

12

[G] - Ancova/Anova & Dunnett: ** = p < 0.01

 

Table 2. Body weight females premating -Day(s) Relative to Start Date

Sex: Female

 

Bodyweights

Bodywt
day -x

(g)

[G]

Bodywt
(g)

[G]

Bodywt
(g)

[G]

Bodywt
(g)

[G]

-4

0

7

14

0.0 mg/kg

Mean

220.88

227.02

227.25

232.16

 

SD

10.23

9.64

8.57

12.38

 

N

12

12

12

12

0.2 mg/kg

Mean

221.58

225.08

225.74

232.08

 

SD

10.67

10.81

10.50

9.43

 

N

12

12

12

12

2.0 mg/kg

Mean

221.50

226.14

225.57

231.78

 

SD

9.72

10.83

12.34

10.36

 

N

12

12

12

12

10.0 mg/kg

Mean

221.63

225.31

218.46

223.95

 

SD

12.02

11.42

11.87

13.84

 

N

12

12

12

12

[G] - Ancova/Anova & Dunnett

 

Table 3. Body weight females gestation - Day(s) Relative to Mating (Litter: A)

Sex: Female

Bodyweights

Bodywt
(g)

[G]

Bodywt
(g)

[G]

Bodywt
(g)

[G]

Bodywt
(g)

[G]

0

7

14

20

0.0 mg/kg

Mean

232.50

253.30

272.04

335.07

 

SD

11.11

9.41

15.61

10.99

 

N

12

12

12

12

0.2 mg/kg

Mean

229.98

251.06

271.20

334.12

 

SD

11.09

10.20

11.56

14.81

 

N

10

10

10

10

2.0 mg/kg

Mean

228.88

253.80

275.38

338.78

 

SD

8.65

12.96

14.20

19.49

 

N

12

12

12

12

10.0 mg/kg

Mean

224.24

240.39*

258.06

301.00**

 

SD

14.04

15.67

18.39

24.59

 

N

11

11

11

11

[G] - Ancova/Anova & Dunnett: * = p < 0.05; ** = p < 0.01
Litter: A = First litter

 

Table 4. Body weight females lactation - Day(s) Relative to Mating (Litter: A)

Sex: Female

Bodyweights

Bodywt
(g)

[G]

Bodywt
(g)

[G1]

Bodywt
(g)

[G]

Bodywt
(g)

[G]

0

4

7

13

0.0 mg/kg

Mean

259.67

266.33

270.13

278.13

 

SD

13.44

13.83

13.53

12.55

 

N

12

12

12

12

0.2 mg/kg

Mean

254.92

262.06

268.41

274.79

 

SD

11.13

5.75

10.47

9.30

 

N

10

10

10

10

2.0 mg/kg

Mean

258.28

267.38

273.63

275.38

 

SD

13.68

14.75

14.28

11.73

 

N

12

12

12

12

10.0 mg/kg

Mean

245.05n

249.20n

253.95n

260.35n

 

SD

3.75

5.37

1.34

14.64

 

N

2

2

2

2

[G] - Ancova/Anova & Dunnett: n - Inappropriate for statistics

[G1] - Kruskal-Wallis & Dunnett on Ranks: n - Inappropriate for statistics

Litter: A = First litter

 

Table 5. Body weight changes males during the study - Day(s) Relative to Start Date

Sex: Male

 

 

Wgt change
last -x to 0

(g)

[G]

Body wt
change

(g)

[G]

Body wt
change

(g)

[G]

Body wt
change

(g)

[G1]

Body wt
change

(g)

[G]

-4 - 0

0 - 7

7 - 14

14 - 21

21 - 28

0.0 mg/kg

Mean

6.95

10.04

11.20

4.38

13.07

 

SD

3.89

4.01

2.39

3.71

5.10

 

N

12

12

12

12

12

0.2 mg/kg

Mean

7.12

8.77

10.83

3.48

7.18*

 

SD

2.44

3.38

4.72

4.01

4.24

 

N

12

12

12

12

12

2.0 mg/kg

Mean

8.99

7.19

11.92

3.83

9.98

 

SD

3.05

2.82

3.36

2.15

4.32

 

N

12

12

12

12

12

10.0 mg/kg

Mean

4.90

0.75**

9.15

-2.33**

5.52**

 

SD

2.96

3.42

5.02

6.35

6.63

 

N

12

12

12

12

12

[G] - Ancova/Anova & Dunnett: * = p < 0.05; ** = p < 0.01
[G1] - Kruskal-Wallis & Dunnett on Ranks: ** = p < 0.01

 

Table 6. Body weight changes females premating - Day(s) Relative to Start Date

Sex: Female

 

 

Wgt change
last -x to 0

(g)

[G]

Body wt
change

(g)

[G]

Body wt
change

(g)

[G]

-4 - 0

0 - 7

7 - 14

0.0 mg/kg

Mean

6.14

0.23

4.91

 

SD

1.29

6.90

8.48

 

N

12

12

12

0.2 mg/kg

Mean

3.50*

0.66

6.34

 

SD

2.44

5.54

5.99

 

N

12

12

12

2.0 mg/kg

Mean

4.64

-0.58

6.21

 

SD

2.77

6.23

3.99

 

N

12

12

12

10.0 mg/kg

Mean

3.68*

-6.85*

5.49

 

SD

2.05

4.63

6.43

 

N

12

12

12

[G] - Ancova/Anova & Dunnett: * = p < 0.05

 

Table 7. Body weight changes females gestation - Day(s) Relative to Mating (Litter: A)

Sex: Female

 

Body wt
change

(g)

[G]

Body wt
change

(g)

[G1]

Body wt
change

(g)

[G1]

0 - 7

7 - 14

14 - 20

0.0 mg/kg

Mean

20.80

18.74

63.03

 

SD

7.47

11.18

9.84

 

N

12

12

12

0.2 mg/kg

Mean

21.08

20.14

62.92

 

SD

6.35

5.57

8.97

 

N

10

10

10

2.0 mg/kg

Mean

24.93

21.58

63.41

 

SD

5.65

3.54

9.02

 

N

12

12

12

10.0 mg/kg

Mean

16.15

17.67

42.94*

 

SD

4.04

5.96

16.33

 

N

11

11

11

[G] - Ancova/Anova & Dunnett

[G1] - Kruskal-Wallis & Dunnett on Ranks: * = p < 0.05

Litter: A = First litter

 

Table 8. Body weight changes females lactation - Day(s) Relative to Littering (Litter: A)

Sex: Female

 

Body wt
change

(g)

[G]

Body wt
change

(g)

[G]

Body wt
change

(g)

[G]

0 - 4

4 - 7

7 - 13

0.0 mg/kg

Mean

6.67

3.79

8.01

 

SD

10.27

8.08

11.00

 

N

12

12

12

0.2 mg/kg

Mean

7.14

6.35

6.38

 

SD

7.93

6.88

11.65

 

N

10

10

10

2.0 mg/kg

Mean

9.11

6.24

1.76

 

SD

10.42

4.65

8.72

 

N

12

12

12

10.0 mg/kg

Mean

4.15n

4.75n

6.40n

 

SD

1.63

4.03

13.29

 

N

2

2

2

[G] - Ancova/Anova & Dunnett: n - Inappropriate for statistics
Litter: A = First litter

 

Table 9. Food consumption males during the study - Daily Food Cons Per Animal (Gram)

Sex: Male

Day(s) Relative to
Animal Start Date

0 - 7

7 - 14

21 - 28

0.0 mg/kg

Mean

16.7

15.0

14.8

 

SD

0.5

0.5

1.1

 

N

3

3

3

0.2 mg/kg

Mean

16.5

14.4

13.2

 

SD

1.1

0.6

0.5

 

N

3

3

3

2.0 mg/kg

Mean

16.7

14.0

12.8*

 

SD

0.8

0.3

0.4

 

N

3

3

3

10.0 mg/kg

Mean

15.2

12.2**

11.3**

 

SD

1.0

0.7

0.6

 

N

3

3

3

Dunnett: * = p < 0.05; ** = p < 0.01

N=Number of cages

Consumption was measured per cage over the periods shown and expressed as g/animal/day

 

Table 10. Food consumption females premating - Daily Food Cons Per Animal (Gram)

Sex: Female

Day(s) Relative to
Animal Start Date

0 - 7

7 -14

0.0 mg/kg

Mean

12.3

12.2

 

SD

0.9

0.5

 

N

3

3

0.2 mg/kg

Mean

12.1

11.5

 

SD

0.7

0.7

 

N

3

3

2.0 mg/kg

Mean

12.6

11.3

 

SD

0.3

0.3

 

N

3

3

10.0 mg/kg

Mean

10.9

9.5**

 

SD

0.3

0.3

 

N

3

3

Dunnett: ** = p < 0.01

N=Number of cages

Consumption was measured per cage over the periods shown and expressed as g/animal/day

 

Table 11. Food consumption females gestation - Daily Food Cons Per Animal

Sex: Female

Day(s) Relative
to Mating (Litter: A)

0 - 7

7 - 14

14 - 20

0.0 mg/kg

Mean

13.66

14.24

16.45

 

SD

1.42

2.35

2.00

 

N

12

12

12

0.2 mg/kg

Mean

13.20

13.95

14.93

 

SD

1.18

1.41

1.13

 

N

10

10

10

2.0 mg/kg

Mean

13.42

14.57

16.04

 

SD

1.61

1.49

1.41

 

N

12

12

12

10.0 mg/kg

Mean

11.25**

12.57

12.90**

 

SD

1.30

1.06

2.23

 

N

11

11

11

Dunnett: ** = p < 0.01

Litter: A = First litter

N=Number of cages

Consumption was measured per cage over the periods shown and expressed as g/animal/day

 

Table 12. Food consumption females lactation - Daily Food Cons Per Animal

Sex: Female

Day(s) Relative
to Littering (Litter: A)

0 - 4

4 - 7

7 - 13

0.0 mg/kg

Mean

24.05

34.72

41.50

 

SD

2.87

4.11

4.46

 

N

12

12

12

0.2 mg/kg

Mean

23.37

33.23

41.86

 

SD

3.17

3.19

5.87

 

N

10

10

10

2.0 mg/kg

Mean

22.59

33.17

39.91

 

SD

4.37

4.41

4.42

 

N

12

12

12

10.0 mg/kg

Mean

10.09**

16.00**

14.23**

 

SD

0.19

1.98

5.63

 

N

2

2

2

Dunnett: ** = p < 0.01

Litter: A = First litter

N=Number of cages

Consumption was measured per cage over the periods shown and expressed as g/animal/day

 

Table 13. Red blood cell and coagulation parameters -Day: 31 Relative to Start Date

Sex: Male

 

 

 

 

 

 

 

 

 

RBC (10E12/L)

[G]

Hb

(mmol/L)

[G]

PCV
(L/L)

[G]

MCV
(fL)

[G]

MCH
(fmol)

[G]

MCHC (mmol/L)

[G]

Reticulo
cytes

(%)

[G]

Thrombo
cytes

(10E9/L)

[G1]

Prothrom Time

(s)

[G1]

0.0 mg/kg

Mean

8.962

9.76

0.4788

53.44

1.089

20.39

2.228

754.2

17.74

 

SD

0.251

0.30

0.0116

1.03

0.031

0.42

0.148

55.6

0.48

 

N

5

5

5

5

5

5

5

5

5

0.2 mg/kg

Mean

8.702

9.52

0.4672

53.70

1.094

20.38

2.086

778.6

17.92

 

SD

0.210

0.24

0.0111

1.15

0.017

0.31

0.199

24.8

0.47

 

N

5

5

5

5

5

5

5

5

5

2.0 mg/kg

Mean

8.480*

9.20**

0.4534*

53.48

1.086

20.30

3.054*

865.8**

18.12

 

SD

0.308

0.14

0.0123

0.80

0.026

0.35

0.559

39.5

0.19

 

N

5

5

5

5

5

5

5

5

5

10.0 mg/kg

Mean

8.270**

8.80**

0.4410**

53.35

1.065

19.96

3.224**

738.8

19.46**

 

SD

0.316

0.32

0.0144

1.43

0.032

0.42

0.550

131.8

0.89

 

N

5

5

5

5

5

5

5

5

5

[G] - Ancova/Anova & Dunnett: * = p < 0.05; ** = p < 0.01
[G1] - Kruskal-Wallis & Dunnett on Ranks: ** = p < 0.01

 

Table 14 Red blood cell and coagulation parameters -Day: 14 Relative to Littering (Litter: A)

Sex: Female

 

 

 

 

 

 

 

 

 

RBC (10E12/L)

[G]

Hb

(mmol/L)

[G]

PCV
(L/L)

[G]

MCV
(fL)

[G]

MCH
(fmol)

[G]

MCHC (mmol/L)

[G]

Reticulo
cytes

(%)

[G]

Thrombo
cytes

(10E9/L)

[G]

Prothrom Time

(s)

[G1]

0.0 mg/kg

Mean

7.872

9.20

0.4544

57.81

1.170

20.25

2.570

961.8

19.44

 

SD

0.454

0.41

0.0238

3.31

0.057

0.21

1.367

73.3

0.71

 

N

5

5

5

5

5

5

5

5

5

0.2 mg/kg

Mean

7.505

9.03

0.4440

59.16

1.203

20.33

3.230

830.5

19.80

 

SD

0.147

0.22

0.0146

1.34

0.021

0.26

0.478

122.9

0.22

 

N

4

4

4

4

4

4

4

4

4

2.0 mg/kg

Mean

8.310

9.80*

0.4804

57.81

1.180

20.41

2.856

856.0

19.30

 

SD

0.267

0.20

0.0191

1.32

0.022

0.40

0.371

90.3

1.13

 

N

5

5

5

5

5

5

5

5

5

10.0 mg/kg

Mean

7.255n

9.45n

0.4840n

66.80n

1.306n

19.55n

3.310n

701.5n

19.65n

 

SD

0.983

0.92

0.0566

1.25

0.050

0.39

0.311

4.9

1.63

 

N

2

2

2

2

2

2

2

2

2

[G] - Ancova/Anova & Dunnett: * = p < 0.05; n - Inappropriate for statistics
[G1] - Kruskal-Wallis & Dunnett on Ranks: n - Inappropriate for statistics

 

Table 15. Total and differential white blood cell counts -Day: 31 Relative to Start Date

Sex: Male

 

 

 

 

 

 

 

 

 

 

 

WBC
(10E9/L)

[G]

Lympho
Absolute

(10E9/L)

[G]

Neutro Absolute

(10E9/L)

[G]

Eosino
Absolute

(10E9/L)

[G]

Baso
Absolute

(10E9/L)

[G1]

Mono Absolute

(10E9/L)

[G2]

Lympho
cytes

(%)

[G]

Neutro phils

(%)

[G]

Eosino phils

(%)

[G]

Baso phils

(%)

[G1]

Mono cytes

(%)

[G]

0.0 mg/kg

Mean

6.54

5.00

1.25

0.115

0.015

0.125

76.36

19.40

1.70

0.22

1.90

 

SD

1.97

1.63

0.44

0.053

0.009

0.047

6.81

6.33

0.52

0.08

0.22

 

N

5

5

5

5

5

5

5

5

5

5

5

0.2 mg/kg

Mean

6.22

4.74

1.22

0.098

0.014

0.111

76.24

19.60

1.62

0.22

1.78

 

SD

1.33

1.00

0.31

0.031

0.004

0.033

2.04

1.50

0.58

0.04

0.40

 

N

5

5

5

5

5

5

5

5

5

5

5

2.0 mg/kg

Mean

6.28

4.79

1.22

0.114

0.013

0.108

76.06

19.84

1.80

0.20

1.66

 

SD

2.11

1.70

0.36

0.043

0.009

0.056

4.05

3.89

0.31

0.07

0.27

 

N

5

5

5

5

5

5

5

5

5

5

5

10.0 mg/kg

Mean

3.42*

2.29*

0.96

0.042*

0.010

0.107

68.12

26.78

1.32

0.24

3.12**

 

SD

1.26

0.72

0.59

0.012

0.012

0.043

8.83

8.71

0.43

0.22

0.19

 

N

5

5

5

5

5

5

5

5

5

5

5

[G] - Ancova/Anova & Dunnett: * = p < 0.05; ** = p < 0.01

[G1 ]- Kruskal-Wallis & Dunnett on Ranks

[G2] -Ancova/Anova & Dunnett(Log)

 

Table 16. Total and differential white blood cell counts - Day: 14 Relative to Littering (Litter: A)

Sex: Female

 

 

 

 

 

 

 

 

 

 

 

WBC
(10E9/L)

[G]

Lympho
Absolute

(10E9/L)

[G]

Neutro Absolute

(10E9/L)

[G]

Eosino
Absolute

(10E9/L)

[G]

Baso
Absolute

(10E9/L)

[G1]

Mono Absolute

(10E9/L)

[G]

Lympho
cytes

(%)

[G]

Neutro phils

(%)

[G]

Eosino phils

(%)

[G2]

Baso phils

(%)

[G1]

Mono cytes

(%)

[G]

0.0 mg/kg

Mean

8.46

3.79

4.22

0.073

0.012

0.325

44.16

50.46

0.86

0.14

3.84

 

SD

0.98

1.01

0.40

0.014

0.008

0.056

7.64

7.50

0.13

0.09

0.53

 

N

5

5

5

5

5

5

5

5

5

5

5

0.2 mg/kg

Mean

6.43

2.81

3.30

0.059

0.009

0.212

44.28

50.65

0.98

0.13

3.38

 

SD

2.31

1.04

1.28

0.044

0.007

0.097

6.77

7.62

0.78

0.05

1.16

 

N

4

4

4

4

4

4

4

4

4

4

4

2.0 mg/kg

Mean

5.76

2.32

3.07

0.071

0.006

0.245

39.84

53.70

1.24

0.10

4.26

 

SD

2.37

1.00

1.24

0.029

0.004

0.125

3.35

4.03

0.34

0.07

1.00

 

N

5

5

5

5

5

5

5

5

5

5

5

10.0 mg/kg

Mean

4.60n

1.89n

2.33n

0.205n

0.006n

0.140n

35.30n

54.85n

5.85n

0.15n

3.30n

 

SD

2.40

1.87

0.59

0.103

0.000

0.034

22.20

15.77

5.30

0.07

0.99

 

N

2

2

2

2

2

2

2

2

2

2

2

[G] - Ancova/Anova & Dunnett: n - Inappropriate for statistics

[G1] - Kruskal-Wallis & Dunnett on Ranks: n - Inappropriate for statistic

[G2] - Ancova/Anova & Dunnett(Log): n - Inappropriate for statistics

 

Table 17. Clinical chemistry -Day: 31 Relative to Start Date

Sex: Male

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

ALP
(U/L)

[G]

ASAT
(U/L)

[G1]

ALAT
(U/L)

[G]

GGT
(U/L)

[G]

Bilirub
Total

(umol/L)

[G]

Creatin
ine

(umol/L)

[G1]

Bile
Acids

(umol/L)

[G]

Total
Protein

(g/L)

[G]

Albumin
(g/L)

[G1]

Albumin/
Globulin

[G]

Glucose
Plasma

(mmol/L)

[G]

Cholest
erol

(mmol/L)

[G]

Triglyc
erides

(mmol/L)

[G2]

Urea (mmol/L)

[G]

PO4 (mmol/L)

[G]

Ca

(mmol/L)

[G]

Cl

(mmol/L)

[G]

K

(mmol/L)

[G]

Na

(mmol/L)

[G]

T4
(ng/ml)

[G]

0.0 mg/kg

Mean

79.2

63.0

51.8

9.20

1.14

42.6

12.28

65.8

12.6

0.237

7.682

1.812

0.600

5.32

2.744

2.850

105.4

5.76

142.8

451.03

 

SD

9.5

7.2

9.6

2.17

0.32

0.9

8.46

1.3

0.9

0.023

1.360

0.287

0.199

0.94

0.358

0.093

1.1

0.84

1.3

147.38

 

N

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

12

0.2 mg/kg

Mean

103.2

68.2

51.8

11.40

1.04

41.2

18.22

64.6

12.4

0.238

7.290

1.600

0.824

5.08

2.384

2.762

105.4

5.54

142.8

451.59

 

SD

23.0

9.7

12.0

1.95

0.40

1.5

24.05

1.5

1.3

0.028

0.904

0.060

0.487

0.43

0.235

0.026

1.1

0.33

1.3

164.97

 

N

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

12

2.0 mg/kg

Mean

88.6

73.8

46.6

9.80

1.34

41.2

30.42

65.4

12.6

0.239

7.346

1.476

0.658

6.00

2.580

2.868

105.6

5.84

143.2

356.25

 

SD

12.7

6.9

12.2

3.27

0.57

4.0

15.78

3.7

0.9

0.010

0.817

0.287

0.098

0.54

0.174

0.069

0.5

0.53

1.6

112.48

 

N

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

12

10.0 mg/kg

Mean

81.0

215.6**

61.0

8.40

0.98

46.8

10.04

60.2**

10.6*

0.214

5.252**

1.482

0.346

8.06**

2.396

2.716*

110.6**

5.44

145.0*

380.35

 

SD

16.2

67.9

25.4

2.07

0.35

6.0

9.49

0.8

0.5

0.013

0.730

0.225

0.096

1.18

0.318

0.062

1.5

0.38

0.7

109.83

 

N

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

10

The bromocresol polychromatic endpoint kit, used by the Siemens Dimension Clinical Chemistry system for the detection of albumin, has a lower response for rat albumin than for human albumin. Calibration curves in the physiological range for rat albumin showed that the signal for rat albumin was approximately one third of the signal obtained with human QC samples (Triskelion validation report V 21229/10, 2018). Hence, the true plasma albumin concentrations in this rat study were about three times higher than the values shown in this table, and the A/G ratio reported is lower than the actual value.

[G] - Ancova/Anova & Dunnett: * = p < 0.05; ** = p < 0.01

[G1] - Kruskal-Wallis & Dunnett on Ranks: * = p < 0.05; ** = p < 0.01

[G2] - Ancova/Anova & Dunnett(Log)

 

Table 18. Clinical chemistry -Day: 14 Relative to Littering (Litter: A)

Sex: Female

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

ALP
(U/L)

[G]

ASAT
(U/L)

[G1]

ALAT
(U/L)

[G]

GGT
(U/L)

[G]

Bilirub
Total

(umol/L)

[G]

Creatin
ine

(umol/L)

[G2]

Bile
Acids

(umol/L)

[G]

Total
Protein

(g/L)

[G]

Albumin
(g/L)

[G]

Albumin/
Globulin

[G]

Glucose
Plasma

(mmol/L)

[G]

Cholest
erol

(mmol/L)

[G]

Triglyc
erides

(mmol/L)

[G2]

Urea (mmol/L)

[G]

PO4 (mmol/L)

[G]

Ca

(mmol/L)

[G]

Cl

(mmol/L)

[G]

K

(mmol/L)

[G]

Na

(mmol/L)

[G]

0.0 mg/kg

Mean

97.2

95.6

52.8

13.60

0.92

45.0

36.03

61.6

10.8

0.213

7.454

2.084

1.766

8.48

3.792

2.754

99.4

4.98

143.8

 

SD

30.6

7.3

11.5

2.51

0.41

3.3

23.07

2.1

1.3

0.025

0.516

0.407

0.925

1.38

0.496

0.101

1.5

0.25

1.3

 

N

5

5

5

5

5

5

4

5

5

5

5

5

5

5

5

5

5

5

5

0.2 mg/kg

Mean

97.2

102.2

57.2

13.20

1.60

47.2

24.18

58.4

11.0

0.231

7.560

1.938

1.266

9.26

3.750

2.676

99.4

4.90

143.8

 

SD

13.4

18.2

15.1

2.17

0.81

1.3

26.71

3.5

1.6

0.024

0.812

0.280

0.277

0.77

0.422

0.059

1.8

0.19

2.5

 

N

5

5

5

5

5

5

4

5

5

5

5

5

5

5

4

5

5

5

5

2.0 mg/kg

Mean

92.0

133.5

67.5

12.50

1.38

52.0

17.58

61.5

11.0

0.218

7.225

1.958

1.648

9.43

3.443

2.740

99.3

5.05

142.0

 

SD

31.7

35.2

12.7

1.29

0.65

7.2

14.66

2.1

0.8

0.016

1.260

0.494

0.523

1.48

0.101

0.056

1.0

0.37

2.7

 

N

4

4

4

4

4

3

4

4

4

4

4

4

4

4

4

3

4

4

4

10.0 mg/kg

Mean

57.0n

239.0n

39.0n

12.00n

1.40n

48.0n

1.80n

62.0n

11.0n

0.216n

5.650n

1.900n

0.630n

13.30n

3.460n

2.870n

107.0n

5.70n

147.0n

 

 

SD-

-

-

-

-

-

-

-

-

-

SD-

-

-

-

-

-

-

-

-

 

N

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

The bromocresol polychromatic endpoint kit, used by the Siemens Dimension Clinical Chemistry system for the detection of albumin, has a lower response for rat albumin than for human albumin. Calibration curves in the physiological range for rat albumin showed that the signal for rat albumin was approximately one third of the signal obtained with human QC samples (Triskelion validation report V 21229/10, 2018). Hence, the true plasma albumin concentrations in this rat study were about three times higher than the values shown in this table, and the A/G ratio reported is lower than the actual value.

[G] - Ancova/Anova & Dunnett: n - Inappropriate for statistics

[G1] - Kruskal-Wallis & Dunnett on Ranks: n - Inappropriate for statistics

[G2] - Ancova/Anova & Dunnett(Log): n - Inappropriate for statistics

 

Table 19. Absolute organ weights -Day(s): 31 Relative to Start Date

Sex: Male

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Terminal bw

(g)

[G]

Brain
(g)

[G]

Heart
(g)

[G1]

Adrenals
(g)

[G]

Kidneys
(g)

[G]

Liver
(g)

[G]

Spleen
(g)

[G]

Thymus
(g)

[G]

Thyroid
(g)

[G1]

Testes
(g)

[G]

Epididy
mides

(g)

[G]

Prostate
(g)

[G]

Seminal
vesicles

(g)

[G]

LABC
Muscle

(g)

[G]

Cowpers
Glands

(g)

[G]

Glans Penis (g)

[G2]

0.0 mg/kg

Mean

365.67

2.080

1.002

0.0586

2.160

8.242

0.5774

0.2568

0.0160

3.648

1.218

1.054

1.283

1.0848

0.1086

0.1268

 

SD

16.21

0.089

0.058

0.0088

0.142

0.619

0.0854

0.0326

0.0051

0.269

0.136

0.147

0.230

0.1339

0.0228

0.0251

 

N

12

5

5

5

5

5

5

5

12

12

12

12

12

12

12

12

0.2 mg/kg

Mean

357.54

2.068

1.014

0.0532

2.012

8.368

0.5824

0.2870

0.0168

3.488

1.170

0.990

1.412

1.0198

0.1128

0.1168

 

SD

16.20

0.061

0.084

0.0052

0.094

0.688

0.0272

0.0753

0.0034

0.282

0.090

0.190

0.225

0.0914

0.0157

0.0167

 

N

12

5

5

5

5

5

5

5

12

12

12

12

12

12

12

12

2.0 mg/kg

Mean

361.52

2.104

1.206**

0.0600

2.204

8.744

0.6282

0.2856

0.0176

3.567

1.203

0.963

1.323

1.0993

0.1161

0.1203

 

SD

15.79

0.080

0.068

0.0115

0.059

0.765

0.0581

0.0477

0.0069

0.242

0.098

0.167

0.211

0.1526

0.0221

0.0290

 

N

12

5

5

5

5

5

5

5

12

12

12

12

12

12

12

12

10.0 mg/kg

Mean

336.98**

2.028

1.410**

0.0560

1.938*

9.626*

0.5778

0.2760

0.0162

3.514

1.073**

0.655**

0.886**

0.9769

0.1039

0.1207

 

SD

19.59

0.044

0.173

0.0070

0.131

0.936

0.0880

0.1033

0.0036

0.251

0.064

0.098

0.187

0.1106

0.0319

0.0287

 

N

12

5

5

5

5

5

5

5

12

12

12

12

12

12

12

12

[G] - Ancova/Anova & Dunnett: * = p < 0.05; ** = p < 0.01
[G1] - Ancova/Anova & Dunnett(Log): ** = p < 0.01
[G2] - Kruskal-Wallis & Dunnett on Ranks

 

Table 20. Absolute organ weights -Day(s): 14 Relative to Littering (Litter: A)

Sex: Female

 

 

 

 

 

 

 

 

 

 

 

Terminal bw

(g)

[G]

Brain
(g)

[G]

Heart
(g)

[G]

Adrenals
(g)

[G1]

Kidneys
(g)

[G1]

Liver
(g)

[G]

Spleen
(g)

[G1]

Thymus
(g)

[G2]

Thyroid
(g)

[G]

Ovaries
(g)

[G]

Uterus
(g)

[G]

0.0 mg/kg

Mean

255.28

1.962

0.882

0.0736

1.588

8.788

0.5362

0.1724

0.0168

0.0806

0.4788

 

SD

12.94

0.087

0.046

0.0073

0.107

0.952

0.0247

0.0262

0.0045

0.0107

0.0564

 

N

12

5

5

5

5

5

5

5

12

12

12

0.2 mg/kg

Mean

252.33

1.952

0.814

0.0726

1.628

8.718

0.5348

0.1606

0.0146

0.0822

0.5472

 

SD

8.49

0.101

0.090

0.0062

0.083

0.255

0.0514

0.0155

0.0021

0.0095

0.0931

 

N

10

5

5

5

5

5

5

5

10

10

10

2.0 mg/kg

Mean

254.19

1.944

0.936

0.0956

1.634

9.294

0.5200

0.2192

0.0159

0.0863

0.5176

 

SD

12.10

0.102

0.112

0.0264

0.231

1.214

0.1234

0.0853

0.0035

0.0147

0.0888

 

N

12

5

5

5

5

5

5

5

12

12

12

10.0 mg/kg

Mean

244.65n

1.905n

1.810n

0.0925n

1.755n

9.845n

0.4515n

0.2920n

0.0150n

0.1225n

0.7050n

 

SD

9.40

0.064

0.099

0.0007

0.049

0.163

0.0375

0.0537

0.0071

0.0290

0.3196

 

N

2

2

2

2

2

2

2

2

2

2

2

[[G] - Ancova/Anova & Dunnett: n - Inappropriate for statistics

[G1] - Kruskal-Wallis & Dunnett on Ranks: n - Inappropriate for statistics

[G2] - Ancova/Anova & Dunnett(Log): n - Inappropriate for statistics
Litter: A = First litter

 

Table 21. Relative organ weights -Day(s): 31 Relative to Start Date

Sex: Male

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Terminal bw

(g)

[G]

Brain
rel.wgt

(g/kg

 bw)
[G]

Heart rel.wgt

(g/kg

 bw) [G]

Adrenals
rel.wgt

(g/kg

 bw) [G]

Kidneys
rel.wgt

(g/kg

 bw) [G]

Liver
rel.wgt

(g/kg

 bw) [G]

Spleen
rel.wgt

(g/kg

 bw) [G]

Thymus
rel.wgt

(g/kg

 bw) [G]

Thyroid
rel.wgt

(g/kg

 bw) [G1]

Testes
rel.wgt

(g/kg

 bw)
[G1]

Epididy rel.wgt

(g/kg

 bw) [G]

Prostate
rel.wgt

(g/kg

 bw) [G]

Sem ves
rel.wgt

(g/kg

 bw) [G]

LABC Muscle rel.wgt

(g/kg bw) [G]

Cowpers Gl. rel.wgt

(g/kg

 bw) [G1]

Glans Penis rel.wgt

(g/kg

 bw) [G2]

0.0 mg/kg

Mean

365.67

5.847

2.813

0.1654

6.073

23.14

1.622

0.722

0.0441

9.988

3.333

2.897

3.516

2.9661

0.2970

0.3474

 

SD

16.21

0.291

0.086

0.0297

0.450

1.30

0.229

0.090

0.0153

0.767

0.375

0.490

0.637

0.3304

0.0603

0.0689

 

N

12

5

5

5

5

5

5

5

12

12

12

12

12

12

12

12

0.2 mg/kg

Mean

357.54

5.650

2.765

0.1454

5.491*

22.81

1.590

0.786

0.0471

9.765

3.276

2.767

3.939

2.8568

0.3153

0.3269

 

SD

16.20

0.301

0.179

0.0159

0.200

1.22

0.075

0.212

0.0103

0.795

0.267

0.508

0.542

0.2732

0.0410

0.0440

 

N

12

5

5

5

5

5

5

5

12

12

12

12

12

12

12

12

2.0 mg/kg

Mean

361.52

5.769

3.308*

0.1642

6.044

24.00

1.722

0.782

0.0485

9.886

3.329

2.658

3.655

3.0330

0.3212

0.3317

 

SD

15.79

0.185

0.199

0.0289

0.141

2.38

0.143

0.124

0.0180

0.834

0.232

0.395

0.521

0.3288

0.0592

0.0688

 

N

12

5

5

5

5

5

5

5

12

12

12

12

12

12

12

12

10.0 mg/kg

Mean

336.98**

6.102

4.226**

0.1680

5.814

28.84**

1.728

0.821

0.0483

10.477

3.192

1.950**

2.642**

2.9071

0.3089

0.3616

 

SD

19.59

0.429

0.402

0.0191

0.226

1.48

0.199

0.275

0.0126

1.185

0.222

0.321

0.600

0.3562

0.0956

0.0991

 

N

12

5

5

5

5

5

5

5

12

12

12

12

12

12

12

12

[G] - Ancova/Anova & Dunnett: * = p < 0.05; ** = p < 0.01
[G1] - Ancova/Anova & Dunnett(Log)

[G2] - Kruskal-Wallis & Dunnett on Ranks

 

Table 22. Relative organ weights -Day(s): 14 Relative to Littering (Litter: A)

Sex: Female

 

 

 

 

 

 

 

 

 

 

 

Terminal bw (g)

[G]

Brain
rel.wgt

(g/kg bw)
[G]

Heart rel.wgt

(g/kg bw) [G1]

Adrenals
rel.wgt

(g/kg

 bw) [G1]

Kidneys
rel.wgt

(g/kg

 bw) [G1]

Liver
rel.wgt

(g/kg bw) [G1]

Spleen
rel.wgt

(g/kg

 bw) [G1]

Thymus
rel.wgt

(g/kg

 bw) [G]

Thyroid
rel.wgt

(g/kg

 bw) [G]

Ovaries
rel.wgt

(g/kg

 bw) [G]

Uterus
rel.wgt

(g/kg

 bw) [G]

0.0 mg/kg

Mean

255.28

7.747

3.487

0.2909

6.264

34.65

2.119

0.680

0.0658

0.3148

1.874

 

SD

12.94

0.225

0.240

0.0312

0.189

2.84

0.111

0.095

0.0175

0.0308

0.178

 

N

12

5

5

5

5

5

5

5

12

12

12

0.2 mg/kg

Mean

252.33

7.911

3.303

0.2935

6.590

35.30

2.166

0.651

0.0579

0.3259

2.165

 

SD

8.49

0.588

0.449

0.0184

0.316

1.15

0.217

0.070

0.0086

0.0379

0.333

 

N

10

5

5

5

5

5

5

5

10

10

10

2.0 mg/kg

Mean

254.19

7.629

3.664

0.3740

6.407

36.58

2.032

0.847

0.0624

0.3386

2.033

 

SD

12.10

0.468

0.344

0.0997

0.900

5.99

0.436

0.275

0.0122

0.0500

0.325

 

N

12

5

5

5

5

5

5

5

12

12

12

10.0 mg/kg

Mean

244.65n

7.797n

7.396n

0.3784n

7.175n

40.26n

1.850n

1.190n

0.0619n

0.5034n

2.909n

 

SD

9.40

0.560

0.120

0.0174

0.073

0.88

0.224

0.174

0.0313

0.1379

1.418

 

N

2

2

2

2

2

2

2

2

2

2

2

[G] - Ancova/Anova & Dunnett: n - Inappropriate for statistics

[G1] - Kruskal-Wallis & Dunnett on Ranks: n - Inappropriate for statistics

Litter: A = First litter

Conclusions:
Based on the effects noted in the mid- and high-dose groups, the NOAEL for parental systemic effects was placed at 0.2 mg/kg body weight/day.
Executive summary:

In this GLP compliant OECD 422 study, the effect of the test substance on general toxicity, reproductive performance and development of pups was examined in four groups of 12 male and 12 female Wistar rats. The test substance was suspended in corn oil and administered by oral gavage at 0, 0.2, 2 and 10 mg/kg body weight/day. The content, and homogeneity of the test substance in the vehicle were confirmed by analysis. The test substance was administered daily, during a pre-mating period of 2 weeks, during mating, gestation and lactation. Male animals were sacrificed after 31 days of treatment. Parental female animals were sacrificed at day 14 of lactation.

Most pregnant high-dose females, however, had to be sacrificed for humane reasons at parturition. There were no treatment-related clinical signs in males or, until parturition, in females. At parturition, one high-dose female was found dead and eight high-dose females were humanely killed because of conditional decline. Signs noted prior to their death included respiratory distress, piloerection and soiled fur/perineum. Two other pregnant high-dose rats delivered, but had litters with mainly dead pups, and showed paleness, piloerection and a lower body temperature. The results of the neurobehavioral observations and motor activity assessment did not indicate any neurotoxic potential of the test substance in rats. Mean body weights and body weight gain were reduced in high-dose males and in high-dose females during gestation. Reductions in food consumption were noted in the high-dose group in both sexes. Hematology and clinical chemistry was conducted at scheduled sacrifice in 5 males/group and in 5 dams in the control-, low- and mid-dose groups. In mid- and high-dose males, red blood cell count, hemoglobin concentration and packed cell volume were decreased, and reticulocytes were increased. Prothrombin time was increased in high-dose males. Total white blood cell, lymphocyte and eosinophil counts were decreased and the percentage of monocytes was increased in high-dose males. In high-dose males, ASAT activity and urea, chloride and sodium concentrations were increased, while total protein, albumin, glucose and calcium concentrations were decreased compared to controls. The absolute and relative weights of the heart were increased in mid- and high-dose males. The absolute and relative weights of the heart were also considerably increased in the two surviving high-dose dams. The absolute and relative weights of the liver were increased, and those of the prostate and seminal vesicles were decreased in high-dose males. In addition, the absolute weight of the epididymides was decreased in high-dose males. Macroscopic examination at scheduled necropsy showed enlarged parathymic lymphnodes in 5/12 high-dose males and in 2/3 remaining high-dose females. Enlarged parathymic lymphnodes were also noted in 6/9 high-dose females that died or were killed in moribund condition. Microscopic examination was performed on males of the high-dose group and the control group. Because the female high-dose group was terminated untimely, microscopic examination was focussed on the next lower-dose group (mid-dose females) and the controls. Organs showing treatment-related microscopic findings were also examined in the intermediate- dose group(s). The following findings were noted:

• A dose-dependent degeneration of the heart muscle in mid- and high-dose males, characterized by the presence of fibrotic areas.

• Degeneration of the skeletal muscle accompanied by necrosis and mononuclear inflammation in high-dose males. In the mid-dose group, 1/5 males showed necrosis and mononuclear inflammation of the skeletal muscle.

• Hyperemia in the liver of high-dose males and mid-dose females.

• Perivascular inflammation and accumulation of alveolar macrophages in the lungs of mid- and high-dose males and mid-dose females. In addition, the incidence of alveolitis was increased in high-dose males.

• Vacuolation in the zona fasciculata of the adrenal gland in mid- and high-dose males.

• Increased incidence of histiocytosis in the parathymic lymphnodes in males and females of the high-dose group.

Results of T4 hormone analysis in male adult animals did not show any significant effects between the groups.

Conclusions:

• Based on the effects noted in the mid- and high-dose groups, the NOAEL for parental systemic effects was placed at 0.2 mg/kg body weight/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
0.2 mg/kg bw/day
Study duration:
subacute
Species:
rat
System:
musculoskeletal system
Organ:
heart
other: skeletal muscle

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

Combined repeated dose toxicity study with the reproduction / developmental toxicity screening test:

In this GLP compliant OECD 422 study, the effect of the test substance on general toxicity, reproductive performance and development of pups was examined in four groups of 12 male and 12 female Wistar rats. The test substance was suspended in corn oil and administered by oral gavage at 0, 0.2, 2 and 10 mg/kg body weight/day. The content, and homogeneity of the test substance in the vehicle were confirmed by analysis. The test substance was administered daily, during a pre-mating period of 2 weeks, during mating, gestation and lactation. Male animals were sacrificed after 31 days of treatment. Parental female animals were sacrificed at day 14 of lactation.

Most pregnant high-dose females, however, had to be sacrificed for humane reasons at parturition. There were no treatment-related clinical signs in males or, until parturition, in females. At parturition, one high-dose female was found dead and eight high-dose females were humanely killed because of conditional decline. Signs noted prior to their death included respiratory distress, piloerection and soiled fur/perineum. Two other pregnant high-dose rats delivered, but had litters with mainly dead pups, and showed paleness, piloerection and a lower body temperature. The results of the neurobehavioral observations and motor activity assessment did not indicate any neurotoxic potential of the test substance in rats. Mean body weights and body weight gain were reduced in high-dose males and in high-dose females during gestation. Reductions in food consumption were noted in the high-dose group in both sexes. Hematology and clinical chemistry was conducted at scheduled sacrifice in 5 males/group and in 5 dams in the control-, low- and mid-dose groups. In mid- and high-dose males, red blood cell count, haemoglobin concentration and packed cell volume were decreased, and reticulocytes were increased. Prothrombin time was increased in high-dose males. Total white blood cell, lymphocyte and eosinophil counts were decreased and the percentage of monocytes was increased in high-dose males. In high-dose males, ASAT activity and urea, chloride and sodium concentrations were increased, while total protein, albumin, glucose and calcium concentrations were decreased compared to controls. The absolute and relative weights of the heart were increased in mid- and high-dose males. The absolute and relative weights of the heart were also considerably increased in the two surviving high-dose dams. The absolute and relative weights of the liver were increased, and those of the prostate and seminal vesicles were decreased in high-dose males. In addition, the absolute weight of the epididymides was decreased in high-dose males. Macroscopic examination at scheduled necropsy showed enlarged parathymic lymphnodes in 5/12 high-dose males and in 2/3 remaining high-dose females. Enlarged parathymic lymphnodes were also noted in 6/9 high-dose females that died or were killed in moribund condition. Microscopic examination was performed on males of the high-dose group and the control group. Because the female high-dose group was terminated untimely, microscopic examination was focussed on the next lower-dose group (mid-dose females) and the controls. Organs showing treatment-related microscopic findings were also examined in the intermediate- dose group(s). The following findings were noted:

• A dose-dependent degeneration of the heart muscle in mid- and high-dose males, characterized by the presence of fibrotic areas.

• Degeneration of the skeletal muscle accompanied by necrosis and mononuclear inflammation in high-dose males. In the mid-dose group, 1/5 males showed necrosis and mononuclear inflammation of the skeletal muscle.

• Hyperemia in the liver of high-dose males and mid-dose females.

• Perivascular inflammation and accumulation of alveolar macrophages in the lungs of mid- and high-dose males and mid-dose females. In addition, the incidence of alveolitis was increased in high-dose males.

• Vacuolation in the zona fasciculata of the adrenal gland in mid- and high-dose males.

• Increased incidence of histiocytosis in the parathymic lymphnodes in males and females of the high-dose group.

Results of T4 hormone analysis in male adult animals did not show any significant effects between the groups.

Conclusions:

• Based on the effects noted in the mid- and high-dose groups, the NOAEL for parental systemic effects was placed at 0.2 mg/kg body weight/day.

Justification for classification or non-classification

The available data for subacute oral toxicity are reliable and suitable for classification purposes under Regulation 1272/2008. Based on the result of the available studies, classification for repeated oral toxicity is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.