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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and OECD guideline compliant study with well-characterized test material
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted in 1996 (This version does not include the endocrine disruptor parameters introduced with the update of 28 July 2015)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): Benzenamine, N-phenyl-,reaction products with 2,4,4-trimethylpentene
- Substance type: UVCB
- Physical state: Clear slightly yellow viscous liquid
- Analytical purity: 100% UVCB
- Purity test date: 2014 , study number 13L00223
- Lot/batch No.: 40401913D
- Expiration date of the lot/batch: 09 February 2016
- Stability under test conditions: stable
- Storage condition of test material: At room temperature in the dark

Test substance handling: Avoid temperatures <10°C, maximum temperature:30°C. In the case of crystallization, heat up the substance (temperature up to 30°C) until a clear solution is obtained
Specific Gravity / Density 0.975 g/cm3 (20°C)
pH 5.1-6.2 (1%(m), 20-25°C) (as suspension)
Stability at higher temperatures Maximum temperature: 30°C
Specific details on test material used for the study:
- Name of test material (as cited in study report): Benzenamine, N-phenyl-,reaction products with 2,4,4-trimethylpentene
- Substance type: UVCB
- Physical state: Clear slightly yellow viscous liquid
- Analytical purity: 100% UVCB
- Purity test date: 2014 , study number 13L00223
- Lot/batch No.: 40401913D
- Expiration date of the lot/batch: 09 February 2016
- Stability under test conditions: stable
- Storage condition of test material: At room temperature in the dark

Test substance handling: Avoid temperatures <10°C, maximum temperature:30°C. In the case of crystallization, heat up the substance (temperature up to 30°C) until a clear solution is obtained
Specific Gravity / Density 0.975 g/cm3 (20°C)
pH 5.1-6.2 (1%(m), 20-25°C) (as suspension)
Stability at higher temperatures Maximum temperature: 30°C

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 10-12 weeks
- Weight at study initiation: 353 g (males), 210 g (females)
- Fasting period before study: none
- Housing: groups of 5 animals/sex/cage (premating males and females and postmating males), otherwise single cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%,
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 13 March 2014 To: 1 May 2014

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) are be prepared daily within 6 hours prior to dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Test item is insoluble in water, but soluble in corn oil
- Amount of vehicle (if gavage): 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.


Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal
copulatory plug. This day was designated Day 0 post-coitum.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Duration of treatment / exposure:
28 days (males), ca 53 days (females)


Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
25, 75 and 225 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Range-finding standy
- Rationale for animal assignment (if not random): Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: mortality, clinical observations

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the first exposure and at the end of the study as part of the functional observation battery (FOB)

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of male and female animals were recorded shortly before the start of
administration of the test item at randomization and at the start of the study (day 0). Males
were weighed weekly until sacrifice. Females were weighed once per week during the premating period. Mated females were
weighed on days 0, 7, 14 and 21 during presumed gestation and on day 0 and 4 of lactation.
Non-mated females were weighed once per week after the mating period. In addition, the
animals were weighed on their scheduled necropsy date in order to calculate the correct organ to body weight ratios.


FOOD CONSUMPTION:
Feed consumption was measured per cage over weekly intervals during the study, with exemption of the mating period, during which no feed consumption was registered.


HAEMATOLOGY
Prior to sacrifice, 5 animals/sex/group were fasted overnight (water was freely available) and blood was taken from the aorta during necropsy, whilst under CO2/O2 anaesthesia. K3-EDTA was used as anticoagulant. In each sample the following determinations were carried out:
haemoglobin
packed cell volume
red blood cell count
reticulocytes
total white blood cell count
differential white blood cell counts (neutrophils, lymphocytes, eosinophils,
basophils, monocytes)
prothrombin time
thrombocyte count
mean corpuscular volume (MCV; calculated)
mean corpuscular haemoglobin (MCH; calculated)
mean corpuscular haemoglobin concentration (MCHC; calculated)
activated partial prothoplastin time (APTT)
red blood cell districution width (RDW)

CLINICAL CHEMISTRY
Prior to sacrifice, 5 animals/sex/group were fasted overnight (water was freely available) and blood was taken from the aorta during necropsy, whilst CO2/O2 anaesthesia. Blood was collected in heparinized plastic tubes and plasma was prepared by centrifugation. The following measurements were made in the plasma:
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,
phospholipids,
calcium (Ca),
sodium (Na),
potassium (K),
chloride (Cl),
inorganic phosphate (PO4),
bile acids


Thyroid hormone analyses:
Thyroid stimulating hormone (TSH) μIU/mL
total triiodothyronine (T3) ng/dL
total thyroxine (T4) μg/dL


NEURO-BEHAVIOURAL TESTING (FOB) AND SPONTANEOUS MOTOR ACTIVITY
During neuro-behavioural testing, the observer was unaware of the treatment of the animals. FOB and spontaneous motor activity were assessed in all study animals during the predose phase and in 5 animals/sex/group at the end of the study.


Sacrifice and pathology
GROSS NECROPSY AND HISTOLOGY OF PARENTAL ANIMALS
All surviving male and female parent animals were sacrificed by exsanguination from the abdominal aorta whilst under CO2/O2 anaesthesia at necropsy and then examined grossly for pathological changes. Male animals were sacrificed after the mating period. Female animals were sacrificed at or shortly after day 4 of lactation. A necropsy was performed on animals that died intercurrently (if not precluded by autolysis) or that had to be killed because they were moribund. Prior to preservation of organs/tissues, the following organ weights were recorded: adrenals, brain, heart, kidneys, liver, ovaries, prostate, seminal vesicles, spleen, thymus, thyroid, uterus. Samples of the following tissues and organs of all parent animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde; except for the testes which were preserved in Bouin's fixative:

- ovaries (after counting the corpora lutea)
- uterus (after counting of the implantation sites)
- testes
- epididymides
- seminal vesicles
- prostate
- all gross lesions

In addition of 5 animals/sex/group following organs were preserved:
adrenals
bone marrow (femur)
brain (including sections of cerebrum, cerebellum, medulla/pons)
caecum
cervix
clitorial gland
colon
coagulation gland
duodenum
eyes
femur including joint
heart
ileum
jejunum (including Peyer's patches)
kidneys
liver
lungs
mesenterial and axillary lymph nodes
ovaries
peripheral nerve (sciatic or tibial)
pituitary gland
preputial gland
prostate
rectum
seminal vesicles (including coagulation gland)
skeletal muscle (thigh)
spinal cord (cervical, mid-thoracic and lumbar)
spleen
stomach*
thymus
thyroid (including parathyroid)
trachea
urinary bladder
uterus
vagina
* Non glandular (“forestomach”) and glandular (fundus, pylorus) parts were examined
microscopically.

Microscopic examination was performed on the collected organs of all animals of the control (group 1) and high dose group (group 4). If treatment-related changes were observed in the high-dose group, the evaluation of these tissues/organs was extended to the intermediate-dose groups (2 and 3).
In addition, reproductive organs of males that failed to sire (did not mate or mated females were not pregnant) and females that were non-mated or non-pregnant, of the low- and mid-dose groups, were microscopically examined. Furthermore, organs showing gross lesions of animals of all groups were microscopically examined.


Sperm parameters (parental animals):
Parameters examined in male parental generations:
testis weight, epididymis weight, spermatogenesis.
Slides of the testes were made of all males of Groups 1 and 4 and stained with PAS/haematoxylin to assess spermatogenesis.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals [day 28, details see entries for repeated dose toxicity]
- Maternal animals: All surviving animals [between postnatal days 4 and 7]
GROSS NECROPSY AND HISTOLOGY OF PARENTAL ANIMALS
All surviving male and female parent animals were sacrificed by exsanguination from the abdominal aorta whilst under CO2/O2 anaesthesia at necropsy and then examined grossly for pathological changes. Male animals were sacrificed after the mating period. Female animals were sacrificed at or shortly after day 4 of lactation. A necropsy was performed on animals that died intercurrently (if not precluded by autolysis) or that had to be killed because they were moribund. Prior to preservation of organs/tissues, the following organ weights were recorded: adrenals, brain, heart, kidneys, liver, ovaries, prostate, seminal vesicles, spleen, thymus, thyroid, uterus. Samples of the following tissues and organs of all parent animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde; except for the testes which were preserved in Bouin's fixative:

- ovaries (after counting the corpora lutea)
- uterus (after counting of the implantation sites)
- testes
- epididymides
- seminal vesicles
- prostate
- all gross lesions
In addition of 5 animals/sex/group following organs were preserved:
adrenals
bone marrow (femur)
brain (including sections of cerebrum, cerebellum, medulla/pons)
caecum
cervix
clitorial gland
colon
coagulation gland
duodenum
eyes
femur including joint
heart
ileum
jejunum (including Peyer's patches)
kidneys
liver
lungs
mesenterial and axillary lymph nodes
ovaries
peripheral nerve (sciatic or tibial)
pituitary gland
preputial gland
prostate
rectum
seminal vesicles (including coagulation gland)
skeletal muscle (thigh)
spinal cord (cervical, mid-thoracic and lumbar)
spleen
stomach*
thymus
thyroid (including parathyroid)
trachea
urinary bladder
uterus
vagina
* Non glandular (“forestomach”) and glandular (fundus, pylorus) parts were examined
microscopically.

Microscopic examination was performed on the collected organs of all animals of the control (group 1) and high dose group (group 4). If treatment-related changes were observed in the high-dose group, the evaluation of these tissues/organs was extended to the intermediate-dose groups (2 and 3).
In addition, reproductive organs of males that failed to sire (did not mate or mated females were not pregnant) and females that were non-mated or non-pregnant, of the low- and mid-dose groups, were microscopically examined. Furthermore, organs showing gross lesions of animals of all groups were microscopically examined.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 5- 7 days of age.
- These animals were subjected to postmortem examinations (macroscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.]

Descriptions of all external abnormalities were recorded. The stomach of pups not surviving to the scheduled necropsy date were examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

Statistics:
Statistics
If the variables could be assumed to follow a normal distribution, the Dunnett-test (Ref. 2; many-toone t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Reproductive indices:
Mating index (%) Number of females mated x 100 / Number of females paired

Fertility index (%) Number of pregnant females x 100 / Number of females paired

Conception index (%) Number of pregnant females x 100 / Number of females mated

Gestation index (%) Number of females bearing live pups x 100 / Number of pregnant females

Duration of gestation Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
Percentage live females at first litter check: Number of live female pups at First Litter Check x 100 Number of live pups at First Litter
Check

Percentage live males at first litter check: Number of live male pups at First Litter Check x 100 / Number of live pups at First Litter Check

Percentage of postnatal loss: Number of dead pups before planned necropsy x 100 / Number of live pups at First Litter Check

Viability index: Number of live pups before planned necropsy x 100 / Number of pups born alive

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
liver
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
liver

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and spermatogenic staging profiles were normal for all males examined.

No clinical signs of toxicity were noted during the observation period.
Salivation was seen after dosing for animals at 75 and 225 mg/kg bw/day. This was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to possible irritancy/taste of the test substance. Alopecia was seen for one female at 75 mg/kg bw/day. This was the only other clinical sign noted and
was incidental in nature.No toxicologically relevant effects on reproductive parameters were noted with treatment up to 225 mg/kg bw/day.

The mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment. Actual numbers are provided in the table below.
There were 10, 10, 10 and 9 pregnant females in the control, 25, 75 and 225 mg/kg bw/day groups, respectively.
For female nos. 46 (Group 1) and 55 (Group 2), the number of pups were slightly higher than the number of implantations. This was considered caused by normal resorption of these areas as these enumerations were performed on Day 5 or 7 of lactation.

Effect levels (P0)

open allclose all
Dose descriptor:
NOEL
Remarks:
fertility
Effect level:
>= 225 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects on fertility observed at the highest tested dose.
Dose descriptor:
NOAEL
Remarks:
parental toxicity
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: adverse effects on liver at 75 and 225 mg/kg bw

Target system / organ toxicity (P0)

Critical effects observed:
yes
Lowest effective dose / conc.:
25 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

Gestation
The gestation index and duration of gestation were similar between all groups.

Parturition/maternal care
Female no. 50 (Group 1) was euthanized in extremis after having a prolonged parturition. No other signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth and
no deficiencies in maternal care were observed.

Early postnatal pup development
The number of dead pups at first litter check and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings. At 225 mg/kg bw/day the mean number of living pups at first litter check (7.9) was significantly lower
than for controls (10.9). Female no. 80 had only 4 pups, which contributed to this slightly low mean. Discounting her data, there were a mean of 8.4 pups/litter, which was also lower than controls and an effect of treatment could not be excluded.
The postnatal loss was also significantly higher for animals at 225 mg/kg bw/day with 11.3% of living pups lost (8 pups over 3 litters) than controls (no pups lost). High dose animals also had a correspondingly low viability index of 88.7%. Mortality
One pup was found dead at the first litter check and one pup was missing on postnatal Day 2 at 25 mg/kg bw/day. At 225 mg/kg bw/day, 7 pups (4 females, 3 males) went missing between Days 2 and 5 of lactation and 1 female pup was euthanized in extremis on lactation Day 1. Missing pups were most the missing pups at 225 mg/kg bw/day were from a single litter (no. 75; three other pups from this litter survived to the end of the necropsy period), two were from another litter (no. 74). The majority of the pups came from a single litter and there were no signs of ill health in other pups from this dose level. However, when taken together with the lower mean number of pups at first litter check, an effect of treatment could not be excluded. No toxicological relevance was attributed to the dead/missing pups at 25 mg/kg bw/day since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Clinical signs
Clinical signs noted for pups who went missing, died or were euthanized in extremis included cold, no
milk in the stomach, wounds (various body areas), pale and lean appearance. Incidental clinical signs
noted for pups surviving to the scheduled necropsy period included scabs (various body areas) and
pale appearance. The nature and incidence of these signs are not uncommon for pups of this age and
strain and were not considered to be treatment related or toxicologically relevant.

Body weights
Body weights of pups were unaffected by treatment up to 225 mg/kg bw/day.

Macroscopy
Sore on the throat region was seen for the pup that was euthanized in extremis. This was the only
macroscopic finding seen for pups that died before the scheduled necropsy. Scabbing on the chest
was the only finding noted for pups surviving to the scheduled necropsy. These findings were both
incidental in nature.

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Increase in postnatal pup mortality at 225 mg/kg bw., mostly in one animal and unlikely to be a true effect

Target system / organ toxicity (F1)

Critical effects observed:
not specified

Overall reproductive toxicity

Reproductive effects observed:
yes
Lowest effective dose / conc.:
225 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes

Any other information on results incl. tables

No toxicologically relevant effects on reproductive parameters were noted with treatment up to 225 mg/kg bw/day. The mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.

There were 10, 10, 10 and 9 pregnant females in the control, 25, 75 and 225 mg/kg bw/day groups, respectively. For female nos. 46 (Group 1) and 55 (Group 2), the number of pups were slightly higher than the number of implantations. This was considered caused by normal resorption of these areas as these enumerations were performed on Day 5 or 7 of lactation.

No toxicologically relevant effects on the gestation index and duration, parturition, maternal care or on most aspects of early postnatal pup development (clinical signs, body weight and macroscopy) were observed up to 225 mg/kg bw/day.

GROUP 1 CONTROL GROUP 2
25 MG/KG
GROUP 3
75 MG/KG
GROUP 4
225 MG/KG
Females paired 10 10 10 10
Females mated 10 10 10 10
Pregnant females 10 10 10 9
Non-pregnant female 0 0 0 1
Females killed in extremis 1 0 0 0
Females with living pups on Day 1 9 10 10 9
Mating index (%) 100 100 100 100
(Females mated / Females paired) * 100
Fertility index (%) 100 100 100 90
(Pregnant females / Females paired) * 100
Conception index (%) 100 100 100 90
(Pregnant females / Females mated) * 100
Gestation index (%) 90 100 100 100
(Females with living pups on Day 1 / Pregnant females) * 100

control 25 mg/kg  75 mg/kg  225 MG/KG
NECROPSY
Corpora Lutea
MEAN 13.2 13.7 12.1 12.2
ST.DEV 2.3 3.7 2.0 2.0
N 10 10 10 9
Implantations MEAN 10.9 11.5 10.5 9.2
ST.DEV 2.4 2.2 1.7 2.3
N 10 10 10 9

+/++ Steel-test significant at 5% (+) or 1% (++) level

  control 25 mg/kg bw 75 mg/kg bw 225 mg/kg bw
Total 9 10 10 9
DURATION OF GESTATION        
Days, MEAN (+) 21.3 21.2 21.1 21.1
ST.DEV. 0.5 0.6 0.6 0.8
N 9 10 10 9
DEAD PUPS AT FIRST LITTER CHECK        
LITTERS AFFECTED (#) 0 1 0 0
TOTAL no of dead pups 0 1 0 0
MEAN (+) 0 0.1 0 0
ST.DEV. 0 0.3 0 0
N 9 10 10 9
LIVING PUPS AT FIRST LITTER CHECK        
% of MALES / FEMALES )# 57/43 55/45 51/49 45/55
TOTAL   98 105 94 71
MEAN (+) 10.9 10.5 9.4 7.9+
ST.DEV. 2.6 2.5 2 1.8
N 9 10 10 9
Postnatal loss        
% of living pups 0 1 0 11.3
LITTERS AFFECTED (#) 0 1 0 3
Total no of pups lost (#) 0 1 0 8##
MEAN (+) 0 0.1 0 0.9
ST.DEV. 0 0.3 0 1.7
N 9 10 10 9
Viability Index (#) 100 99 100 88.7#

+/++ Steel-test significant at 5% (+) or 1% (++) level

Viability index = (Number of alive pups before planned necropsy / Number of pups born alive) *100

# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level

Number of pups at first litter check and postnatal loss for the high dose group (225 mg/kg bw)

Female no. Days of Gestation Dead males at first litter check Dead females at first litter check Living males at first litter check Living females at first litter check Living pups (total) Postnatal loss (males) Postnatal loss (females)
72 22 0 0 5 5 10 0 0
73 20 0 0 2 5 7 0 0
74 21 0 0 6 2 8 1 1
75 21 0 0 2 6 8 2 3
76 22 0 0 5 3 8 0 0
77 21 0 0 3 5 8 0 1
78 20 0 0 2 6 8 0 0
79 21 0 0 7 3 10 0 0
80 22 0 0 0 4 4 0 0
TOTAL   0 0 32 39 71 3 5
N 9 9 9 9 9 9 9 9
MEAN 21.1 0.0 0.0 3.6 4.3 7.9 0.3 0.6
ST.DEV. 0.8 0.0 0.0 2.3 1.4 1.8 0.7 1.0

Pup Bodyweights (g) during lactation

control 25 mg/kg bw 75 mg/kg bw 225 mg/kg bw
Postnatal Day 1 M MEAN 6.4 6.3 6.4 6.1
ST.DEV. 0.8 0.5 0.5 0.8
N 9 10 10 8
F MEAN 6.2 5.9 6.1 5.9
ST.DEV. 0.8 0.6 0.6 0.9
N 9 9 10 9
M+F MEAN 6.3 6.1 6.3 6.0
ST.DEV. 0.7 0.6 0.5 0.8
N 9 10 10 9
Postnatal Day 4 M MEAN 9.5 9.8 9.7 8.7
ST.DEV. 1.5 1.1 1.0 2.2
N 9 10 10 8
F MEAN 9.3 9.2 9.3 9.0
ST.DEV. 1.4 1.4 1.2 1.4
N 9 9 10 9
M+F MEAN 9.4 9.6 9.5 9.0
ST.DEV. 1.4 1.2 1.1 1.6
N 9 10 10 9

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Applicant's summary and conclusion