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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Quadrol Polyol N,N,N`,N`-Tetrakis (2-Hydroxylpropyl) Ethylenediamine, Tetrahydroxypropylethylenediamine
- Physical state: viscous / colorless, clear liquid
- Analytical purity: 100.1 %
- Lot/batch No.: WPTB612C
- Date of production: 2006-06-22
- Storage condition of test material: ambient (room temperature); under N2

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, 97633 Sulzfeld, Germany
- Age at study initiation: 9-10 weeks
- Body weight: The weight variation of the animals used did not exceed 20 % of the mean weight of each sex.
- Housing: individually
- Diet (e.g. ad libitum): ad libitum; Kliba maintenance diet mouse/rat “GLP”
- Water (e.g. ad libitum): ad libitum, from water bottles
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 30-70 %
- Air changes (per hr): fully air-conditioned
- Photoperiod (hrs dark / hrs light): 12 h day/ 12 h night

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
demineralized
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
To prepare the solution, the appropriate amount of test substance was weighed out for each concentration. Then the vehicle (demineralized water) was added up to the desired volume, and subsequently mixed using a magnetic stirrer. The test substance dosing solutions were prepared at least once a week.


VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg bw
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: over night for a maximum of 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance in demineralized water (VE-Wasser) at room temperature for a period of 7 days was proven before the start of the administration period .
For homogeneity and concentration control analyses, duplicates of 6 samples of all concentrations were drawn at the start and towards the end of the administration period. Sampling was done under dosing conditions out of the beaker (2 samples from bottom, mid, and top of each concentration). One sample for each concentration and sampling time was analyzed; the other one was retained frozen until finalization of this report.
Duration of treatment / exposure:
The duration of treatment covered a 2 week premating period and mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period and 4 days of lactation in females. The male animals were treated for a total period of 30/31 days, the parental female animals were treated for a total period of 49 days.
Frequency of treatment:
daily
Details on study schedule:
Mating period started 13 days after beginning of the study. Mating was discontinued as soon as sperm was detected in the vaginal smear.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
10 males / 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
In a range finding study, ethylenediamine, +4PO was administered to groups of 3 male and female Wistar rats (Crl:WI[Han], supplied by Charles River Laboratories, in fully desalted water at dose levels of 1000, 300, 100 and 0 (vehicle only) mg/kg body weight/day. Apart from occasional salivation in one top dose (1000 mg/kg bw/day) male and 3 females, and a skin lesion on the head and neck in one top dose female, no test substance-related clinical findings were noted during the study. Neither food consumption nor body weights showed consistent test substance-related changes. Mating, conception and gestation (until
GD 15) were not affected by the test substance. Necropsy revealed no findings in male and female rats of all test groups. Thus, 100, 300 and 1000 mg/kg bw were selected as dose levels for the main study.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
- Time schedule: at least once daily
- Cage side observations: signs of morbidity, pertinent behavioral changes and signs of overt toxicity

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: weekly intervals
- Details of examination: Animals were transferred to a standard arena. Findings were ranked according to the degree of severity
- Parameters: abnormal behavior during “handling”, fur, skin, salivation, nose discharge, lacrimation, pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements, impairment of gait, activity/arousal level, feces (appearance/consistency), urine, other findings

BODY WEIGHT: Yes
- Time schedule for examinations: once weekly
- body weight changes were calculated

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule: once a week for male and female P animals except lactation period and females without positive avidence of sperm

FUNCTIONAL OBSERVATION BATTERY:
- Time schedule: once at the end of the administration period
- Details of examination: five animals per sex and group at the end of the administration period starting at about 10 am. The FOB started with passive
observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, when applicable.
- Parameters of passive home cage observations: posture, tremors, convulsions, abnormal movements, impairment of gait, other findings
- Parameters of open field observations: behavior when removed from cage, fur, skin, salivation, nose discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements, impairment of gait, activity/arousal level, feces (number of fecal pellets/appearance/consistency within two minutes, urine (appearance/quantity) within two minutes, number of rearings within two minutes
- Parameters of sensorimotor tests: approach response, touch response, vision (“visual placing response”), pupillary reflex, pinna reflex, audition (“startle response”), coordination of movements (“righting response”), behavior during “handling”, vocalization, pain perception (“tail pinch”), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test, other findings

MOTOR ACTIVITY MEASUREMENT:
- Time schedule: once at the end of the administration period
- Details of examination: Animals were placed in new clean polycarbonate cages for the time of measurement. Eighteen beams were allocated per cage. The number of beam interrupts was counted over 12 intervals, each lasting 5 minutes. During the measurements the animals received no food or water.
- Parameters: number of beam interrupts per interval

CLINICAL PATHOLODY:
- Time schedule: once on the day of sacrifice
- Details: Blood was taken from the retro-orbital venous plexus from fasted, anaesthesized animals.
- Hematology parameters: leukocyte count, erythrocyte count, hemoglobin, hematocrit, mean corposcular volume, mean corposcular hemoglobin, mean corposcular hemoglobin concentration, platelet count, differential blood count, reticulocytes, prothrombin time
- Parameters of clinical chemistry: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, γ-glutamyltransferase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium
Litter observations:
The following parameters were examined: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were sacrificed on study day 30 (after the end of the mating period)
- Maternal animals: All surviving animals were sacrificed on study day 49 (pups were raised for 4 days)

GROSS NECROPSY
- Animals were necropsied and assessed by gross pathology
- The following weight parameters were recorded: anesthetized animals, liver, kidneys, adrenal glands, testes, epididymides, seminal vesicle, prostate, ovaries, uterus, thymus, spleen, brain, heart, thyroid glands

HISTOPATHOLOGY
- The following organs and tissues were fixed in 4 % buffered formaldehyde solution: all gross lesions, brain, spinal cord (cervical, thoracic and lumbar cord), sciatic nerve, pituitary gland, salivary glands (glandula mandibularis and glandula sublingualis), thyroid glands/parathyroid glands, adrenal glands, prostate gland, seminal vesicles, coagulation glands, uterus, oviducts, vagina, female mammary gland, thymus, lymph nodes (axillary and mesenteric), spleen, trachea, lungs, heart, aorta, liver, pancreas, kidneys, esophagus, stomach (forestomach and glandular stomach), duodenum, jejunum (with Peyer’s patches), ileum, cecum, colon, rectum, urinary bladder, sternum with marrow, larynx, pharynx, nose (nasal cavity), bone marrow (femur), eyes with optic nerve, femur with knee joint, skin, skeletal muscle
- The following organs and tissues were wixed in Bouin's solution: testes, epididymides and ovaries
- Details of examination: Histotechnical processing and examination by light microscopy was performed for all fixed organs from control and high dose group, except for gross lesions and brain, were samples of all dose groups were assessed.
Postmortem examinations (offspring):
All surviving pups, all stillborn pups and those pups that died before schedule, were examined externally, eviscerated and their organs were assessed macroscopically.
Statistics:
Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), number of mating days, duration of gestation, number of pups delivered per litter, implantation sites, post implantation loss: two-sided DUNNETT-test for comparison of dose groups

Male and female mating index, male and female fertility index, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn
pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, number of litters with affected pups at necropsy: Pairwise comparison of dose groups using FISHER's EXACT test

Proportions of affected pups per litter with necropsy observations: Pairwise comparison of dose groups using the one-sided WILCOXON test

Feces, rearing, gripstrength of forelimbs and, hindlimbs, landing footsplay test, motor activity: Non-parametrical analysis using the two-sided KRUSKAL-WALLIS test; pairwise comparison with the control group using the two-sided WILCOXON test

Clinical pathology parameters, urine volume, urine specific gravity: non-parametric analysis using the two-sided KRUSKAL-WALLIS test; pairwise comparison with the control group using the two sided WILCOXON test

Organ weight parameters: non-parametric two-sided KRUSKAL-WALLIS test and pairwise comparison using the WILCOXON test
Reproductive indices:
MALE REPRODUCTION DATA
male mating index (%) = (number of males with confirmed mating / number of males placed with females) x 100
male fertility index (%) = (number of males proving their fertility / number of males placed with females) x 100

FEMALE REPRODUCTION AND DELIVERY DATA
female mating index (%) = (number of females mated / number of females placed with males) x 100
female fertility index (%) = (number of females pregnant / number of females mated) x 100
gestation index (%) = (number of females with live pups on the day of birth / number of females pregnant) x 100
post implantation loss (%) = (number of implantations - number of pups delivered / number of implantations) x 100
Offspring viability indices:
Live birth index(%) = (umber of liveborn pups at birth / total number of pups born) x 100
Viability index (%) = (number of live pups on day 4 after birth / number of liveborn pups on the day of birth) x 100

Results and discussion

Results: P0 (first parental animals)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
No test substance related mortalities were reported.
Clinical observations did not reveal any test substance-related abnormalities.

FUNCTIONAL OBSERVATION BATTERY:
No test substance-related abnormalities were reported.

MOTOR ACTIVITY:
No test substance-related observations concerning overall motor activity and single intervals

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Food consumption of all dose groups was comparable to the control groups. There were no significant differences among the dose groups.
Body weight and body weight gain of all substance-treated groups were in general comparable to concurent control group.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS):
No test substance-related abnormalities concerning reproduction and fertility.

CLINICAL PATHOLOGY
No treatment-related effects concerning hematology and clinical chemistry were observed.

ORGAN WEIGHTS (PARENTAL ANIMALS):
Absolute and relative organ weights did not show significant weight deviations.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no treatment-related gross lesions noted.

HISTOPATHOLOGY (PARENTAL ANIMALS)
1000 mg/kg bw: mainly moderate vacuolation was observed in the epithelial cells of choroid plexus of the lateral ventricles in the brain of parental animals (For details see section "any other information on results incl. tables")
There were no treatment-related findings in the other dose groups.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: reproductive performance and fertility
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: general systemic toxicity due to vacuolation of epithelial cells of the lateral ventricles of the brain

Results: F1 generation

Details on results (F1)

VIABILITY (OFFSPRING)
The mean number of delivered pups per dam and the rate of liveborn and stillborn pups were evenly distributed among the groups. The respective values reflect the normal range of biological variation in the strain used in this study. Viability index varied between 99-100 %. Sex ratio of F1 pups did not show biologically relevant differences between controls and test group.

CLINICAL SIGNS (OFFSPRING)
No treatment-related adverse clinical signs were recorded.

BODY WEIGHT (OFFSPRING)
Mean pup body weights and pup body weight changes in the substance treated groups were comparable to the control groups.

GROSS PATHOLOGY (OFFSPRING)
No test substance-related observations were reported.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: developmental toxicity

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Histopathology of adult animals:

Extent of vacuolisation in the epithelial cells of the choroid plexus of the lateral ventricles in the brain of parental animals:

    Male animals    Female animals  
 Dose group (mg/kg bw/day)  0 100 300 1000 0 100 300 1000
 Number of animals examined  10 10 10 10 10 10 10 10
 Vacuoles, choroid plexus 0 0 0 10 0 0 0 10

 minimal  

   1    2

 slight  

3 2

 moderate  

6 6

Applicant's summary and conclusion