Nickel bis(dibutyldithiocarbamate)

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / 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:

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

Test material information

Constituent 1

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 species / strain selection:

The rat was used because this species is considered suitable for this type of study, and is usually required by regulatory agencies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

- 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

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.

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

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 analyzing 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 analyzing one sample of each test dilution prepared on 3, 10 and 17 September 2018.
• Because the test substance was determined by analyzing 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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.2 mg/kg bw/day (actual dose received)

Remarks:

Group 2: Low dose

Doses / concentrations 2

Dose / conc.:

2 mg/kg bw/day (actual dose received)

Remarks:

Group 3: Mid dose

Doses / concentrations 3

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

Parental animals: Observations and 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). Subsequently, males were weighed weekly. 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: Males shortly prior to sacrifice, 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’.

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 all adult males and in two pups/litter collected on LD13.

Oestrous cyclicity (parental animals):

Estrous cycle evaluations
Vaginal smears to evaluate the estrous cycle length and normality were made daily in all females during the pre-treatment period until the start of the treatment period. Cyclicity during the first 14 days of the pre-treatment period was used as an allocation criterium in the study. Vaginal smears were also made and evaluated daily during the pre-mating period. In addition, the vaginal smears made during the mating period until confirmation of mating were evaluated for estrous cyclicity.
An additional vaginal smear was made at the day of sacrifice. The smears made at the day of sacrifice were stained but not analyzed and reported, because there was no need on the basis of inconclusive histopathology of uterus and vagina.

Sperm parameters (parental animals):

The following parameters were examined in P0 male parental generations:
Organ weights of the reproductive organs: epididymides, prostate (dorsolateral and ventral), seminal vesicles and coagulation glands, testes, levator ani plus bulbocavernosus muscle, complex (LABC muscle), cowper’s glands, glans penis.

Litter observations:

The following parameters were examined in F1 offspring:
The total litter size and numbers of each sex as well as the number and incidence of litters lost entirely, the number of stillbirths, live-, dead- and cannibilised pups, runts (pups that are less than the mean pup weight of the control group minus 2 SD) and grossly malformed pups were evaluated on days 0, 4, 7 and 13 of lactation. The alive pups were individually weighed on days 0, 4, 7 and 13 of lactation. Mean pup weight was calculated per sex and for both sexes combined per dose group. At lactation day 4, the anogenital distance (AGD) was measured of each pup before culling of the litter. The AGD was reported as such and corrected for the cube root of body weight. The total number of culled pups was recorded. Plasma samples were analyzed for Thyroxin (T4) hormone levels in two pups/litter collected on LD13. On lactation day 13 all surviving male pups were examined for the presence of nipples and/or areolas. Any abnormal behavior of pups was recorded on day 0, 4, 7 and 13 of lactation.

Postmortem examinations (parental animals):

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 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 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 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:
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.

Postmortem examinations (offspring):

SACRIFICE
- On lactation day 4 the litter size was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four pups per sex per litter. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment was conducted.
- Remaining pups were sacrificed on day 13 of lactation.

GROSS NECROPSY
A necropsy was performed on stillborn pups and pups dying during the study and any macroscopic abnormalities were recorded. At day 13 of lactation, pups were examined externally and killed by appropriate techniques. The thyroids were preserved of two pups per litter (the pups selected for blood collection). A number of these pups were examined macroscopically. The preserved thyroids were not examined microscopically.

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.

Reproductive indices:

See Table 4 in 'Any other information on materials and methods incl. tables'.

Offspring viability indices:

See Table 4 in 'Any other information on materials and methods incl. tables'.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 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

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.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Data on estrus cyclicity were comparable among the groups during the pre-treatment period. There were no statistically significant or treatment-related effects on estrus cyclicity during the pre-mating period.

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

Decreased weights of male reproductive organs (epididymides, prostate and seminal vesicles) were noted in high-dose males. These changes were not accompanied by histopathological alterations in these organs. In addition there was no functional loss, since mating and fertility indices were not affected in the high-dose group.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

See Tables 23 to 26 in 'Any other information on results incl. tables'.
Twelve females of each group were placed with males. There were no treatment-related effects on male and female mating index or fertility index. Except for two low-dose females and one high-dose female, all females were mated. Mating occurred within 4 days and there were no statistically significant differences in the mean number of pre-coital days (days until successful mating) among the groups. All mated females were pregnant. Dystocia occurred in the high-dose group. One pregnant high-dose female was found dead and eight pregnant females had to be sacrificed for humane reasons at parturition. Only two pregnant high-dose females delivered, mainly stillborn pups or pups that died soon after birth. 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. 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. 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. Viability indices and sex ratio were also comparable among these 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.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

0.2 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

haematology

organ weights and organ / body weight ratios

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

0.2 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

3

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity

Effect level:

>= 10 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No adverse effects were observed at the highest dose tested.

4

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity

Effect level:

>= 2 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: in the high-dose group (10 mg/kg-bw/day) insufficient animals were available for a meaningful evaluation.

Target system / organ toxicity (P0)

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

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

See Table 27 in 'Any other information on results incl. tables'.
The four live high-dose male pups from only one litter were cold on the day of delivery. In the other groups there were no treatment-related signs in pups during the lactation period, including the pups subjected to external examination on day 13 of lactation.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

See Tables 26 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. Viability indices and sex ratio were also comparable among these groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See Tables 28 to 30 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.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

See Table 33 in 'Any other information on results incl. tables'.
In the high-dose group insufficient pups were available for a meaningful evaluation. There were no statistically significant differences between the other groups in absolute or relative weights of the thyroid of pups on PND 13.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

MACROSCOPIC OBSERVATION OF DEAD PUPS:
Most of the (few) pups in the high-dose group were stillborn or died soon after birth. Macroscopic findings three dead pups in the control group and one dead pup in the mid-dose group indicated that these pups were liveborn but died after birth.

MACROSCOPIC OBSERVATIONS OF PUPS AT SACRIFICE ON PND 13:
In the high-dose group insufficient pups were available for a meaningful evaluation. Macroscopic observations of 2 pups/litter in the other groups at necropsy on PND 13 did not reveal treatment-related abnormalities.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

ANOGENITAL DISTANCE
See Table 31 in 'Any other information on results incl. tables'.
In the high-dose group insufficient pups were available for a meaningful evaluation. There were no statistically significant effects on anogenital distance or anogenital distance corrected for cube root of body weight.

NIPPLE RETENTION TIME
See Table 32 in 'Any other information on results incl. tables'.
There were no effects on nipple retention of male pups on PND 13.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

2 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

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

Table 23. 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	11^1
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 24 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 25. 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 26. 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 27: 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 28: 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 29. 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 30. 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 31. 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 32. 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 33. 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 34. 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 35: 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.
- In males, no adverse effects on reproductive organs or on reproductive performance parameters were observed in any dose group. Therefore, the NOAEL for reproduction for males was set at 10 mg/kg body weight/day.
- Because there were no relevant effects on reproductive organs or on reproductive performance parameters in the low- and mid-dose group, and because the reproductive effects in the high-dose group could not be studied, due to maternal toxicity, the NOAEL for reproduction in females 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. Male animals were sacrificed after 31 days of treatment. 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 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. Haematology 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 and 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.

• In males, no adverse effects on reproductive organs or on reproductive performance parameters were observed in any dose group. Therefore, the NOAEL for reproduction for males was set at 10 mg/kg body weight/day.

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