Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: 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

Test material

Constituent 1
Chemical structure
Reference substance name:
Nickel bis(dibutyldithiocarbamate)
EC Number:
237-696-2
EC Name:
Nickel bis(dibutyldithiocarbamate)
Cas Number:
13927-77-0
Molecular formula:
C18-H36-N2-Ni-S4
IUPAC Name:
Nickel(II) Dibutyldithiocarbamate
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
other: Wistar Han IGS rats (Crl:WI (Han))
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): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 03 Aug 2018 to 15 Oct 2018

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on 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.
Details on mating procedure:
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: one week
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged individually
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
Doses / concentrationsopen allclose all
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).

Examinations

Maternal examinations:
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:
The animals were sacrificed by exsanguination from the abdominal aorta whilst under CO2/O2 anaesthesia and then subjected to macroscopic examination for pathological changes. 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 1 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 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 1 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:

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:
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. Pooled blood samples were collected by decapitation from the surplus pups (pooled per litter) at culling on lactation day 4 and plasma was stored in a freezer at ≤-18° C for possible future determination. Individual blood samples were collected from the heart whilst under CO2/O2 anaesthesia from two pups per litter at sacrifice on lactation day 13, and plasma was stored in a freezer at ≤-18° C.

Hormone determinations (T4)
Plasma samples were analysed for Thyroxin (T4) hormone levels in two pups/litter collected on LD13.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
Statistics:
The statistical procedures for analysis of data are described in Table 3 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.
Indices:
See Table 4 in 'Any other information on materials and methods incl. tables'.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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.
Dermal irritation (if dermal study):
not examined
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 6 in 'Any other information on results incl. tables'.
Mean body weights were statistically significantly reduced in high-dose females on day 7 and 20 of gestation. In addition, body weight gain was statistically significantly reduced 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 females of the low- and mid-dose groups.
Statistically significantly lower body weight gains noted in females prior to the start of the 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 7 to 9 in 'Any other information on results incl. tables'.
Statistically significantly reductions in food consumption were noted in high-dose females during premating, gestation and (in two remaining rats only) lactation.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
See Tables 10 and 11 in 'Any other information on results incl. tables'.
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. There were no statistically significant differences in white blood cell counts in females of the low- and mid-dose groups.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
See Table 12 in 'Any other information on results incl. tables'.
There were no treatment-related differences in clinical chemistry data in females of the low- and mid-dose groups. Results obtained in the high-dose female group are not discussed because the measurements were performed in one dam only.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
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 13 and 14 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 also considerably increased in the two surviving high-dose females (no statistics applied).

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 2/3 surviving high-dose females. Red discoloration was observed incidentally.
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 low-dose group, 4/12 females showed minimal histiocytosis. In the mid-dose group 3/12 females showed minimal histiocytosis. In the high-dose group the three surviving females showed mild to moderate histiocytosis of the parathymic lymphnodes.

Liver:
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 5/5 mid-dose females. In addition, minimal to mild accumulation of alveolar macrophages was noted in 4/5 mid-dose females. Apart from the incidental occurrence of alveolar macrophages, these findings were not observed in the control group or the low-dose group. Occasional occurrence of alveolar macrophages 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:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
See Table 18 in 'Any other information on results incl. tables'.
There were no statistically significant differences between the low- and mid-dose groups and the controls in the number of implantation sites and the number of pups delivered. Consequently, no statistically significant differences were observed on post-implantation loss between the control and these treatment groups. The number of implantation sites and the number and appearance of the pups from females that were humanely killed were not noticeably affected by the test substance.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
not specified
Dead fetuses:
effects observed, treatment-related
Description (incidence and severity):
See Table 18 in 'Any other information on results incl. tables'.
Only two pregnant high-dose females delivered, mainly stillborn pups or pups that died soon after birth. In the control, low- and mid-dose groups, all pregnant females had viable litters and there were no stillborn pups. Hence the live birth index was 100% in these groups.
Changes in pregnancy duration:
effects observed, non-treatment-related
Description (incidence and severity):
See Table 16 in 'Any other information on results incl. tables'.
There were no adverse changes in the duration of gestation. In the low- and mid-dose groups, the mean duration of gestation was slightly, though statistically significantly reduced. There was, however, no dose-response relationship and all individual rats (except for one) in the low- and mid-dose group showed a completely normal gestation length of 22 days. Therefore the statistical significance obtained in these groups is considered not to represent a test substance-related effect on gestation length.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
See Table 16 in 'Any other information on results incl. tables'.
All mated females were pregnant.
Other effects:
not examined

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
Systemic toxicity
Effect level:
0.2 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
Reproductive toxicity
Effect level:
2 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: in the high-dose group (10 mg/kg-bw/day) insufficient pups were available for a meaningful evaluation.

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
See Tables 20 to 22 in 'Any other information on results incl. tables'.
In the high-dose group insufficient pups were available for a meaningful evaluation. In the low- and mid-dose groups, mean male, female and total pup weight/litter were statistically significantly lower than in controls on day 7 of lactation. Mean pup weight/litter was also statistically significantly lower than in controls in female pups only on day 13 of lactation. The difference with the controls were not dose-related and only slight (≤8%). Therefore these findings were not considered to be adverse.
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
See Tables 16 and 17 in 'Any other information on results incl. tables'.
Only two pregnant high-dose females delivered, mainly stillborn pups or pups that died soon after birth. Only two male pups from one high-dose litter survived until necropsy on day 13. In the control, low- and mid-dose groups, all pregnant females had viable litters and there were no stillborn pups. Hence the live birth index was 100% in these groups. Viability indices were also comparable among these groups.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
See Table 18 in 'Any other information on results incl. tables'.
Sex ratio was comparable among the control, low- and mid-dose groups. In the high-dose group insufficient pups were available for a meaningful evaluation.
Changes in litter size and weights:
effects observed, non-treatment-related
Description (incidence and severity):
In the high-dose group insufficient pups were available for a meaningful evaluation.
In the low- and mid-dose groups, mean male, female and total pup weight/litter were statistically significantly lower than in controls on day 7 of lactation. Mean pup weight/litter was also statistically significantly lower than in controls in female pups only on day 13 of lactation (Tables 20 to 22 in 'Any other information on results incl. tables'). The difference with the controls was not dose-related and only slight (≤8%). Therefore these findings were not considered to be adverse.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
See Table 18 in 'Any other information on results incl. tables'.
Viability indices were comparable among the control, low- and mid-dose groups. In the high-dose group insufficient pups were available for a meaningful evaluation.
External malformations:
no effects observed
Description (incidence and severity):
See Table 19 in 'Any other information on results incl. tables'.
The appearance of the pups from females that were humanely killed were not noticeably affected by the test substance.
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
not examined

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Remarks:
Developmental toxicity
Effect level:
2 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
other: in the high-dose group (10 mg/kg-bw/day) insufficient pups were available for a meaningful evaluation.

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Table 1. 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 2. 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 3. 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 4. 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 5. 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 6. 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 7. 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 8. 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 9. 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 10. 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 11. 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 12. 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 13. 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 14. 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

Table 15. Mating report

Sex: Both

(Litter: A)

0.0 mg/kg

0.2 mg/kg

2.0 mg/kg

10.0 mg/kg

Females Placed with Males

N+ve

12

12

12

12

Females Mated

N+ve

12

10

12

11

Females Not Mated

N+ve

0

2

0

1

Female Mating Index

%

100.0

83.3

100.0

91.7

Males Placed with Females

N+ve

12

12

12

12

Males Mated

N+ve

12

10

12

11

Males Not Mated

N+ve

0

2

0

1

Males that became sire

N+ve

12

10

12

111

Male Mating Index

%

100.0

83.3

100.0

91.7

Male Fertility Index

%

100.0

83.3

100.0

91.7

Pre-coital time (days) [k]

Mean

2.4

2.3

2.6

2.4

 

SD

1.4

0.8

0.9

1.3

 

N

12

10

12

11

Day 1-4 [f]

N+ve

12

10

12

11

 

%

100.0

100.0

100.0

100.0

Day 5-7 [f]

N+ve

0

0

0

0

Day 1-7 [f]

N+ve

12

10

12

11

 

%

100.0

100.0

100.0

100.0

Litter: A = First litter

Female mating index: no. of females mated * 100/no. of females placed with males

Male mating index: no. of males mated * 100/no. of males placed with females

Male fertility index: no. of males that became sire *100/no. of males placed with females

1 Including the males that succesfully mated, but for which the females died or were humanely killed at parturition

[k] - Kruskal-Wallis & Wilcoxon

[f] - Chi-Squared & Fisher's Exact

 

Table 16. Pregnancy report

Sex: Female

(Litter: A)

0.0 mg/kg

0.2 mg/kg

2.0 mg/kg

10.0 mg/kg

Females Pregnant [f]

N+ve

12

10

12

11

Females Not Pregnant

N+ve

0

2

0

1

Pregnant, Found Dead

N+ve

0

0

0

1

Pregnant, Killed Moribund

N+ve

0

0

0

8

Females Completing Delivery

N+ve

12

10

12

2

Females with Liveborn [f]

N+ve

12

10

12

2n

Female Fecundity Index

%

100.0

100.0

100.0

100.0

Female Fertility Index

%

100.0

83.3

100.0

91.7

Gestation Index

%

100.0

100.0

100.0

NR

Gestation Days [k]

Mean

22.5

21.9*

22.0*

23.5n

 

SD

0.7

0.3

0.0

0.7

 

N

12

10

12

2

Females with Stillborn Pups [f]

N+ve

0

0

0

2n

 

%

0.0

0.0

0.0

100.0n

Females with all Stillborn Pup [f]

N+ve

0

0

0

0n

 

%

0.0

0.0

0.0

0.0n

[f] - Chi-Squared & Fisher's Exact: ** = p < 0.01;

NR - Not Relevant

[k] - Kruskal-Wallis & Wilcoxon: * = p < 0.05; ** = p < 0.01; n - Inappropriate for statistics

Litter: A = First litter

Female fecundity index: no. of females pregnant * 100 /no. of females mated

Female fertility index: no. of females pregnant *100/no. of females placed with males

Gestation index: no. of females with a viable litter *100/no. of females pregnancy

 

Table 17. Delivery report

Sex: Female

(Litter: A)

0.0 mg/kg

0.2 mg/kg

2.0 mg/kg

Pups Delivered (Total) [k]

Mean

12.3

12.8

12.3

 

SD

1.8

1.5

2.2

 

Sum

147

128

148

Liveborn [f]

Sum

147

128

148

Live Birth Index (%)

 

100.0

100.0

100.0

Stillborn day 0 [f]

Sum

0

0

0

Stillborn Index (%)

 

0.0

0.0

0.0

[k] - Kruskal-Wallis & Wilcoxon

[f] - Chi-Squared & Fisher's Exact

Litter: A = First litter

Live birth index: no. of liveborn pups *100/no. of total pups delivered

Stillborn index: no. of stillborn pups *100/no. of total pups delivered

Table 18. Litter report

Sex: Female

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

0.0 mg/kg

0.2 mg/kg

2.0 mg/kg

Live Pups/Litter day 0 [k]

Mean

12.1

12.8

12.3

 

SD

1.7

1.5

2.1

 

N

12

10

12

 

Sum

145

128

147

Live Pups/Litter day 4 Pre [k]

Mean

11.8

12.8

12.2

 

SD

1.6

1.5

1.9

 

N

12

10

12

 

Sum

142

128

146

Culled pups

Sum

46

48

50

Live Pups/Litter day 4 Post [k]

Mean

8.0

8.0

8.0

 

SD

0.0

0.0

0.0

 

N

12

10

12

 

Sum

96

80

96

Live Pups/Litter day 7 [k]

Mean

8.0

8.0

8.0

 

SD

0.0

0.0

0.0

 

N

12

10

12

 

Sum

96

80

96

Live Pups/Litter day 13 [k]

Mean

8.0

8.0

8.0

 

SD

0.0

0.0

0.0

 

N

12

10

12

 

Sum

96

80

96

Dead, Missing, Cannibalized d0-d4

 

5

0

2

Dead, Missing, Cannibalized d5-d7

 

0

0

0

Dead, Missing, Cannibalized d8-d13

 

0

0

0

No. of litters lost entirely d0-d4 [f]

N+ve

0

0

0

 

%

0.0

0.0

0.0

No. of litters lost entirely d5-d7 [f]

N+ve

0

0

0

 

%

0.0

0.0

0.0

No. of litters lost entirely d8-d13 [f]

N+ve

0

0

0

 

%

0.0

0.0

0.0

No. of litters lost entirely d0-d13 [f]

N+ve

0

0

0

 

%

0.0

0.0

0.0

Viability Index 0-4 (%)

 

97

100

99

Viability Index 4-13 (%)

 

100

100

100

Live Males on Day 0

Mean

5.8

6.3

6.0

 

SD

2.2

1.8

2.3

 

N

12

10

12

 

Sum

70

63

72

Sex Ratio Day 0 - Males (%)

 

48.3

49.2

49.0

Live Males on Day 13

Mean

3.8

4.0

4.2

 

SD

0.5

0.0

0.7

 

N

12

10

12

 

Sum

45

40

50

Sex Ratio Day 13 - Males

 

46.9

50.0

52.1

Implantation Sites Total

Mean

12.6

13.5

12.9

 

SD

1.2

1.6

1.8

 

N

12

10

12

 

Sum

151

135

155

No. of lost implantations

Sum

4

7

7

Post-Implantation Loss % [k]

Mean

3.0

5.1

4.6

 

SD

5.9

4.8

9.6

[k] - Kruskal-Wallis & Wilcoxon

[f] - Chi-Squared & Fisher's Exact

Litter: A = First litter

Viability index 0-4: no. of live pups on day 4 *100/no. of liveborn pups

Viability index 4-13: no. of live pups on day 13 * 100/no. of live pups post cull

Sex ratio day n: no. of live male pups on day n *100/ no. of live pups on day n

Post-implantation loss: no. of implant sites - no. of liveborn *100/no. of implant sites

 

Table 19. Pup clinical observations

Exam Type: Pup Necropsy

0.0 mg/kg

0.2 mg/kg

2.0 mg/kg

10.0 mg/kg

Number of Litters Examined:

12

10

12

2

Number of Pups Examined:

145

128

147

4

Abdomen
Blue/grey

Pups N

1

0

0

0

 

Litters N

1

0

0

0

Leg

Swollen (anterior left)

Pups N

0

0

1

0

 

Litters N

0

0

1

0

Skin

Haematoma

Pups N

2

1

14

1

 

Litters N

1

1

2

1

Tail
Kink

Pups N

0

1

0

0

 

Litters N

0

1

0

0

General
Cold

Pups N

0

0

0

4

 

Litters N

0

0

0

1

Table 20. Pup body weight per litter -Day(s) Relative to Littering (Litter: A)

 

 

Mean Male
Pup BW /L

(g)

[G]

Mean Male
Pup BW /L

(g)

[G1]

Mean Male
Pup BW /L

(g)

[G]

Mean Male
Pup BW /L

(g)

[G]

0

4

7

13

0.0 mg/kg

Mean

6.30

11.48

18.59

33.37

 

SD

0.53

1.06

1.14

2.35

 

N

12

12

12

12

0.2 mg/kg

Mean

6.11

10.64

17.23*

32.16

 

SD

0.41

0.88

1.15

1.48

 

N

10

10

10

10

2.0 mg/kg

Mean

6.12

10.57

17.24*

31.23

 

SD

0.28

0.51

1.10

2.60

 

N

12

12

12

12

10.0 mg/kg

Mean

5.53n

9.05n

13.70n

25.20n

 

SD

.

.

.

.

 

N

1

1

1

1

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

/L = per Litter

 

Table 21. Pup body weight per litter -Day(s) Relative to Littering (Litter: A)

 

 

Mean Female
Pup BW /L

(g)

[G]

Mean Female
Pup BW /L

(g)

[G1]

Mean Female
Pup BW /L

(g)

[G]

Mean Female
Pup BW /L

(g)

[G]

0

4

7

13

0.0 mg/kg

Mean

6.05

11.11

17.96

32.69

 

SD

0.54

1.17

1.33

2.18

 

N

12

12

12

12

0.2 mg/kg

Mean

5.75

10.20

16.62*

31.37

 

SD

0.40

0.72

0.77

1.09

 

N

10

10

10

10

2.0 mg/kg

Mean

5.82

10.17*

16.51**

30.40*

 

SD

0.26

0.50

1.01

2.54

 

N

12

12

12

12

10.0 mg/kg

Mean

.

.

.

.

 

SD

.

.

.

.

 

N

.

.

.

.

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

/L = per Litter

 

Table 22. Pup body weight per litter -Day(s) Relative to Littering (Litter: A)

 

 

Mean Total
Pup BW /L

(g)

[G]

Mean Total
Pup BW /L

(g)

[G1]

Mean Total
Pup BW /L

(g)

[G]

Mean Total
Pup BW /L

(g)

[G]

0

4

7

13

0.0 mg/kg

Mean

6.15

11.27

18.22

32.98

 

SD

0.50

1.08

1.16

2.22

 

N

12

12

12

12

0.2 mg/kg

Mean

5.92

10.40

16.93*

31.77

 

SD

0.41

0.79

0.91

1.19

 

N

10

10

10

10

2.0 mg/kg

Mean

5.97

10.36

16.88**

30.82

 

SD

0.24

0.49

1.02

2.54

 

N

12

12

12

12

10.0 mg/kg

Mean

5.53n

9.05n

13.70n

25.20n

 

SD

.

.

.

.

 

N

1

1

1

1

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

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

Litter: A = First litter

/L = per Litter

 

Table 23. Pup anogenital distance -Day(s) Relative to Littering (Litter: A)

 

 

 

 

 

 

 

Mean Male
Pup BW /L

(g)

[G]

Mean Male
Pup AGD /L

(mm)

[G]

AGDcorrected
for BW - M
(mm/g3)

[G]

Mean Female
Pup BW /L

(g)

[G]

Mean Female
Pup AGD /L

(mm)

[G]

AGDcorrected
for BW - F
(mm/g3)

[G]

4

4

4

4

4

4

0.0 mg/kg

Mean

11.48

6.218

2.759

11.11

3.656

1.642

 

SD

1.06

0.570

0.219

1.17

0.321

0.144

 

N

12

12

12

12

12

12

0.2 mg/kg

Mean

10.64

5.944

2.707

10.20

3.551

1.639

 

SD

0.88

0.159

0.073

0.72

0.263

0.122

 

N

10

10

10

10

10

10

2.0 mg/kg

Mean

10.57

6.102

2.784

10.17*

3.716

1.716

 

SD

0.51

0.392

0.201

0.50

0.292

0.131

 

N

12

12

12

12

12

12

10.0 mg/kg

Mean

9.05n

6.500n

3.120n

.

.

.

 

 

SD.

.

.

.

.

.

 

N

1

1

1

.

.

.

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

Litter: A = First litter

/L = per Litter

 

Table 24. Pup nipple retention - Day(s) Relative to Littering (Litter: A)

 

 

Mean Male
Pup NR /L

[k]

13

0.0 mg/kg

Mean

0.0

 

SD

0.0

 

N

12

0.2 mg/kg

Mean

0.0

 

SD

0.0

 

N

10

2.0 mg/kg

Mean

0.0

 

SD

0.0

 

N

12

10.0 mg/kg

Mean

0.0n

 

SD

.

 

N

1

[k] - Kruskal-Wallis & Wilcoxon: n - Inappropriate for statistics

Litter: A = First litter

/L = per Litter

Table 25. Pup organ weights - Day(s) Relative to Littering (Litter: A)

 

 

 

 

 

 

 

 

 

 

Mean Pup
TermBW /L

(g)

[G]

Mean Pup (M)
TermBW /L

(g)

[G]

Mean Pup (F)
TermBW /L

(g)

[G1]

Mean Pup
Thyroid wgt

(g)

[G1]

Mean Pup (M)
Thyroid wgt

(g)

[G1]

Mean Pup (F)
Thyroid wgt

(g)

[G1]

Mean Pup
Thyroid relw
(g/kg
body wgt)
[G2]

Mean Pup (M)
Thyroid relw
(g/kg
body wgt)
[G1]

Mean Pup (F)
Thyroid relw
(g/kg
body wgt)
[G2]

13

13

13

13

13

13

13

13

13

0.0 mg/kg

Mean

33.096

33.567

32.625

0.0049

0.0051

0.0047

0.1483

0.1517

0.1449

 

SD

2.490

2.729

2.597

0.0015

0.0015

0.0016

0.0472

0.0450

0.0540

 

N

12

12

12

12

12

12

12

12

12

0.2 mg/kg

Mean

32.160

32.650

31.670

0.0055

0.0061

0.0049

0.1707

0.1876

0.1537

 

SD

1.235

1.436

1.490

0.0012

0.0015

0.0014

0.0396

0.0464

0.0447

 

N

10

10

10

10

10

10

10

10

10

2.0 mg/kg

Mean

30.575

30.933**

30.217

0.0047

0.0044

0.0050

0.1532

0.1410

0.1653

 

SD

2.526

2.779

2.658

0.0015

0.0020

0.0015

0.0480

0.0576

0.0548

 

N

12

12

12

12

12

12

12

12

12

10.0 mg/kg

Mean

25.200n

25.200n

.

0.0029n

0.0029n

.

0.1156n

0.1156n

.

 

 

SD.

.

.

.

.

.

.

.

.

 

N

1

1

.

1

1

.

1

1

.

[G] - Kruskal-Wallis & Dunnett on Ranks: ** = p < 0.01; n - Inappropriate for statistics

[G1] - Ancova/Anova & Dunnett: n - Inappropriate for statistics

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

Litter: A = First litter

 

Table 26. Hormone determinations pups (T4) -Day: 13 Relative to Start Date

Sex: Male

 

T4
(ng/ml)

[G]

0.0 mg/kg

Mean

603.46

 

SD

135.86

 

N

9

0.2 mg/kg

Mean

571.57

 

SD

59.90

 

N

6

2.0 mg/kg

Mean

600.21

 

SD

134.61

 

N

10

10.0 mg/kg

Mean

703.60n

 

SD

122.61

 

N

2

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

 

Table 27. Hormone determinations pups (T4) -Day: 13 Relative to Start Date

Sex: Female

 

T4
(ng/ml)

[G]

0.0 mg/kg

Mean

551.91

 

SD

132.49

 

N

8

0.2 mg/kg

Mean

584.06

 

SD

52.32

 

N

8

2.0 mg/kg

Mean

585.57

 

SD

74.95

 

N

9

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

Applicant's summary and conclusion

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.
Because there were no relevant effects on reproductive performance or on developmental parameters in the mid-dose group, the NOAEL for reproduction and development was placed at 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. Pups were sacrificed at day 13 of lactation. 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, 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 females during gestation. Reductions in food consumption were noted in the high-dose group. Hematology and clinical chemistry was conducted at scheduled sacrifice in 5 dams in the control-, low- and mid-dose groups. The absolute and relative weights of the heart were considerably increased in the two surviving high-dose dams. Macroscopic examination at scheduled necropsy showed enlarged parathymic lymphnodes 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. 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:

• Hyperemia in the liver of mid-dose females.

• Perivascular inflammation and accumulation of alveolar macrophages in the lungs of mid-dose females.

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

Results of T4 hormone analysis in male and female pups on PND 13 did not show any significant effects between the groups. There were no treatment-related effects on estrus cyclicity during the pre-mating period, or in the number of pregnant females, pre-coital time or mating indices. In the high-dose females that were humanely killed, the number of implantation sites and the number and appearance of the pups were not noticeably affected. In the remaining (low- and mid-dose) groups there were no adverse changes in the duration of gestation, the number of implantation sites and the number of pups delivered. In these groups, there were no relevant or treatment-related changes in pup observations, pup sex and pup survival, pup anogenital distance, nipple retention, pup thyroid weight or macroscopy. Slight (≤8%) and not dose-related reductions in total pup weight in the low- and mid-dose groups on day 7 of lactation, and in female pup weight in the mid-dose group on day 13 of lactation were not considered to be adverse.

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.

• Because there were no relevant effects on reproductive performance or on developmental parameters in the mid-dose group, and because due to maternal toxicity the reproductive performance or developmental parameters in the high-dose group could not be studied, the NOAEL for reproduction and development was placed at 2 mg/kg body weight/day. 

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