Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Remarks:
This experimental study was performed only for one constituent (2,2'-[ethylenebis(oxymethylene)]bisoxirane, CAS 2224-15-9, EC 218-746-2) of the UVCB substance.
Adequacy of study:
supporting study
Study period:
1 November 2010 - 11 January 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

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:
March 22, 1996
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in the closed bottel at room temperature in the dark
- Stability under test conditions: verified at the end of the study

Test animals

Species:
rat
Strain:
other: Crl:CD (SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc., Yokohama, Japan
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 10 wks
- Weight at study initiation: (P) Males: 352 - 442 g; Females: 203 - 300 g
- Fasting period before study: fasting only before termination for dams (at lactation Day 4)
- Housing: Two animals were housed in suspended wire-mesh cage during quarantine/ acclimation period. Animals were then individually housed in the same cages during the in-life phase of the study. At the GD 18, dams were transfered to the plastic cages with autoclaved flake bedding and stayed there until Day 4 post-portum. Dams were thereafter housed in the wire-mesh cage.
- Use of restrainers for preventing ingestion (if dermal): no
- Diet (e.g. ad libitum): ab libitum with CRF-1 by Oriental Yeast Co., Ltd., Itabashi-ku, Japan
- Water (e.g. ad libitum): tap water, ab libitum
- Acclimation period: at least 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.5 - 24.7 °C
- Humidity (%): 41.3 - 58.4%
- Air changes (per hr): 12 times
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: 2 November 2010 to 15 January 2011

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dosing solution was prepared more than once a week, storaged in the brown bottle in a refrigerator and used until 7 days. In case of dosing, the dosing solution was used within 6 hours after taking from the refrigerator. The stability of the dosing solution was verified for 7 days in the refrigerator and 6 hours at the room temperature.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: maximum 14 days
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): At the GD 18, dams were transfered from the wire-mesh cage to the plastic cages with autoclaved flake bedding and stayed there until Day 4 post-portum.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
males and females in the main group: 28 days (14 day prior to mating and 14 days thereafter)
dams: 42-46 days; (from 14 days before mating to day 5 of lactation)
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
12.5 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
In the main groups:
6 males and 5 females for control, 12.5, 50, and 200 mg/kg bw/day

In the satellite groups:
6 males for control, 12.5, 50, and 200 mg/kg bw/day
5 females for control, and 200 mg/kg bw/day

In the mating groups:
12 females for control, 12.5, 50, and 200 mg/kg bw/day
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The preliminary test was performed with 0, 100, 200 and 500 mg/kg bw/day in 5 males and 5 females for 14 days. All males in 500 mg/kg bw/day were dead, and adverse effects were found in male 100 and 200 mg/kg bw/day groups. 4 females were dead or life threadning and several adverse effects were observed in female 100 and 200 mg/kg bw/day groups. According to these results, the highest dose was set as 200 mg/kg bw/day.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: before administration and after administration (between 1 and 183 min) during administration period, once a day during recovery period

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before administration, Day 7, 14, 21 and 27 for males and females in the main groups, before administration, Day 7, 14, Days of Gestation (GD) 1, 8, 15 and Days of Lactation 4
- Detailed clinical observation checked:
(Observation of animals in cages) posture, palpebral closure, excessive grooing, repetitive circling, bitting behavior, clonic convulsions, tonic convulsions,
(Observation of animals on observer's palm) ease of removal from cage, ease of handling, muscle tone, fur conditions, mucous memranes, lacrimation, salivation, piloerection, pulpi size, and respiration,
(Open-field test) frequency of urination, frequency of defecaton, frequency of rearing, frequency of grooming, gait, palpebral closure, consciousness, behavioral abnormalities, and righting reflex

SENSORY REACTIVITY: only for the males and females in the main groups
- Time schedule: on Day 27 after FOB
- Checked parameters:
pupillary reflex, approaching behavior, response to touch, auditory reflex, and pain reflex

GRIP STRENGTH: only for the males and females in the main groups
- Time schedule: on Day 27 after FOB
- Checked parameters:
Forelimb and hindlimb

SPONTANEOUS MOTOR ACTIVITY: only for the males and females in the main groups
- Time shcedule: on Day 26
- Checked parameters:
Ambulator counts for 10 minutes between 60 and 120 minutes after administration
Vetical counts for 10 minutes between 60 and 120 minutes after administration

BODY WEIGHT: Yes
- Time schedule for examinations: Day 1, 4, 8, 11, 15, 18, 22, 25 and 28 diring the administrataion period, and Day 1, 4, 8, 11 and 14 during the recovery period for males and females in the main groups,
Day 1, 4, 8, 11, 15, 18, 22, 25, 29, 32, 36, 39, 43, 46, 50, and 53 for females non-pregnant dams inspite mating
Day 1, 4, 8, 11, 15 and 18 during the administration period before pregnancy, GD 0, 7, 14 and 20, ad Days of lactation 0 and 4

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
- Time schedule for examinations:
Day 2, 5, 9, and 12 during the administration period, Day 2, 5, 9 and 12 during the recovery period for males
Day 2, 5, 9, 12, 16, 19, 23 and 26 during the administration period, and Day 2, 5, 9 and 12 during the recovery period for females in main test
Day 2, 5, 9, and 12 before mating, GD 2, 9, 16, and 20 and Day of lactaion 2 in dams

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations:
Day 2, 5, 9, and 12 during the administration period, Day 2, 5, 9 and 12 during the recovery period for males
Day 2, 5, 9, 12, 16, 19, 23 and 26 during the administration period, and Day 2, 5, 9 and 12 during the recovery period for females in main test
Day 2, 5, 9, and 12 before mating, GD 2, 9, 16, and 20 and Day of lactaion 2 in dams

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected for hematological evaluations from all animals on the day of scheduled (one day after the last administration day and last day of the recovery period)
- Anaesthetic used for blood collection: Yes (Pentobarbital-Na). While the animals were under pentobarbital-Na anesthesia a syringe and needle were used to collect blood samples from the abdominal vena cava to sample collection tubes containing EDTA
- Animals fasted: Fasting overnight
- How many animals: All
- Parameters checked: Red blood cell, hemoglobin, hematocrit, MCV, MCH, MCHC, platelet, reticulocyte, PT (prothlombin time), APTT (activated partial thromboplastin time), fibrinogen, white blood cell, differential leukocyte

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected for clinical chemistry evaluations from all animals on the day of scheduled
- Animals fasted: Fasting overnight
- How many animals: All
- Parameters checked: AST (aspartate aminotransferase), ALT (alanine aminotransferase), ALP (alkaline phosphatase), gamma-GT (γ-glutamyltransferase), total protein, albumin, A/G ratio, total bilirubin, urea nitrogen, creatinine, glucose, total cholesterol, triglyceride, sodium, potassium, chloride, calcium and inorganic phosphorus.

URINALYSIS: Yes
- Time schedule: On Day 23 during the administration period but just before the administration on that day and Day 12 during the recovery period, frish urine was collected by using the urine collection cage by fasting animals. No detailed information for fasting is available. Thereafter, the 24 h urine was collected without fasting.
- Parameters checked: color, pH, protain, glucose, ketone body, bilirubin, occult blood, urobilinogen, and urinary sediments

HORMONE CONCENTRATION:
- Time schedule:
- Parameters checked: T3, T4 and TSH




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, body weight, 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 on Day 28 during the administration period and on Day 14 during the recovery period
- Female animals (main study): All surviving animals on Day 28 during the administration period and on Day 14 during the recovery period
Maternal animals: on Day 4 of lactation
Maternal non pregnant females: on Day 53


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

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated below were prepared for microscopic examination and weighed, respectively.
Organ weights: Brain, pituitary, salivary glands, thyroids, thymus, heart, liver, spleen, kidneys, adrenals, testes, epididymides, ventral prostate, seminal vesicles, ovaries, and uterus
Histopathological findings: Heart, lung, trachea, liver, pancreas, sublingual gland, submandibular gland, esophagus, stomach, duodenum, jejunum, ileum, Peyer's patch, cecum, colon, rectum, thymus, spleen, mandibular lymph node, mesenteric lymph node, kidney, urinary bladder, testis, epididymis, ventral prostate, seminal vesicle, coagulating gland, pituitary, adrenal, thyroid, parathyroid, cerebrum, cerebellum, pons, spinal cord, sciatic nerve, eyevall, harderian gland, sternal bone, femoral bone, sternal bone marrow, gemoral bone marrow, muscke (retus femoris) mammary gland, ovary, uterus, vagina, stomach,
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 4 days of age.
- These animals were subjected to postmortem examinations (macroscopic) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
Statistics:
The mean and standard deviation was calculated for body weight, food consumption, water consumption, frequency of urination, frequency of defecaton, frequency of rearing, frequency of grooming, gait, spontaneous motor activity, urine volume, specific gravity, parameters of hematological findings, paameteres of clinical biochemistry, hormone concentrations (T3, T4 and TSH), organ weight including relative organ weight, number of estrous cases before pairing, number of conceiving days, length of gestation, number of corpora lutea, number of implantation scars, number of pups born, nuber of live pups born, number of stillbirths, and number of live pups on Day 4 of lactation. Then, Bartlett test was used to analysis the variance. In case of equal variance, Dunnett's test was applied between the dosing groups and the control group. In case of unequal variance, Steel's test was applied.

After mean values and standard deviation was calculated for implantation index, delivery index, birth inndex, live birth index, viability index, sex ratio and external abnormalities, Steel's test was applied.

In the recovery period, body weight, food consumption, water consumption, urine volus, specific gravity, parameters of hematological and clinical biochemistry findings, and organ weight including relative organ weight of females were tested by F-test to analyse the variance between the 200 mg/kg bw/day group and control group. In case of equal variance, Student's t test was applied. In case of unequal variance, Aspinn-Welch's t test was applied.

After mean value and range was calculated for parameters of FOB (except for frequency of urination, frequency of defecaton, frequency of rearing and frequency of grooming) and sensitivity test, Steel's test was applied.

Fischer's exact test was applied for gestation index.

Steel's test and Cochran Armitage test was applied for the histopathological findings in stomach.


Reproductive indices:
copulation index
fertility index
implantation index
gestation index
delivery index
Offspring viability indices:
birth index
live birth index
viability index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Salivation was observed in the males and females in the 200 mg/kg bw/day main groups and in the 200 mg/kg bw/day maternal groups. Since this change transiently appeared after the administration and no supporting changes were found in the organ weight of salivary gland and histopathological changes, this effect was regarded as non-treatment-related effect.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the male 200 mg/kg bw/day group, significant decreased body weight was observed between Day 8 in the administration period and Day 11 in the recovery period in comparison with the control group. In the male 50 mg/kg by/day group, the body weight was significantly decreased between Day 25 and 39 in the administration period in comparison with the control group.
In the female 200 mg/kg bw/day group, the body weight was significantly decreased between Day 8 and 15 in the administration period.
In the maternal 200 mg/kg bw/day group, the body weight was significantly decreased between Day 8 and 11 in the administration period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In the male and female 200 mg/kg bw/day main group, the food consumption was significantly decreased on Day 5, 9, and 12 and on Day 5 and 9 during the administration period in compared with the control group, respectively.
In the maternal female 200 mg/kg bw/day group, the food consumption was significantly decreased on Day 5 in the administration period.
In the maternal female 50 mg/kg bw/day group, the food consumption was significantly decreased on Day 2 of lactation. However, this change was not a toxicological change because this could be caused by the decreased pups.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
In the female 200 mg/kg bw/day main group, the water consumption was significantly increased on Day 16, 19 and 23 in compared with the control group. Reason?
In the maternal female 50 mg/kg bw/day group, the water consumption was significantly decreased on Day 2 of lactation in compared with the control group. However, this change could be caused by the decreased number of pups. This was not a toxicological effect.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
(At the end of the administration period)
The platelet and lympocyte were significantly increased in the female 50 and 200 mg/kg bw/day main groups. Reticulocyte and PT were significantly increased in the female 200 mg/kg bw/day main group.
Neutrophil was significantly decreased in the 50 and 200 mg/kg bw/day female main group in comparison with the control group.


(At the end of the recovery period)
The RBC and hemoglobin were significantly decreased in compared with the control group in the 200 mg/kg bw/day male main group. This change was not regarded as a treatment-related effect because this was not observed at the end of the administration period. Eosinophil was significantly increased in compared with the control group in the 50 mg/kg bw/day male main group. This was also not regarded as a treatment-related effect due to lack of the dose dependency.
MCV was significantly increase din the female 200 mg/kg bw/day main group in compared with the control group. Since this change was not observed at the end of the administration period, this was not caused by the test material.

Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
(At the end of the administration period)
Triglyceride was significantly decreased in male 50 and 200 mg/kg bw/day main group in compared with the control group.
ALT was significantly decreased in the male 200 mg/kg bw/day main group in compared with the control group. This was not regarded as a treatment-related effect because this change was within the historical background data at the laboratory (ALT: 27.6 ± 4.1 (IU/L)].
Calcium was significantly increased in the male 50 mg/kg bw/day main group in compared with the control group. This was not regarded as a treatment-related effect because this change was within the historical background data at the laboratory (Ca: 9.3 ± 0.3 (mg/dL)) and dose dependency was not observed.

(At the end of the recovery period)
Inorganic phosphorus was significantly increased in the male 50 mg/kg bw/day main group. This was not regarded as a treatment-related effect because this change was within the historical background data at the laboratory (Inorganic phosphorus: 7.4 ± 0.5 (mg/dL)) and the dose dependency was not observed.

No changes were found in the females at the end of the administration and recovery period.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
(FOB)
According to the open field test, the significant increased frequency of urination was observed on Day 27 in the male 50 mg/kg bw/day main group and on Day 21 in the female 12.5 mg/kg bw/day main group. This change was not regarded as a treatment-related change due to lack of the dose dependency.
According to the open field test, the frequency of rearing was significantly decreased on Day 7 in the maternal female 50 and 200 mg/kg bw/day groups. This change was no a toxicological effect because it was transiently observed.

According to the results of spontaneous motor activity, the vertical counts were significantly increased in the female 50 mg/kg bw/day main group at the end of the administration period. This change was not regarded as a treatment-related effect due to lack of the dose dependency.
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):
Hyperplasia of squamous epithelium in forestomach was observed in male and female 200 mg/kg bw/day main group (mild: 6/6 male and 5/5 female) at the end of the administration period. This change was significant and dose dependency was also confirmed. Chronic ulcer in glandular stomach was observed in male and female 200 mg/kw bw/day main group (slight: 4/6 and 3/5 female) at the end of the administration period.

Hyperplasia of squamous epithelium in forestomach was observed in male and female 200 mg/kg bw/day main group (slight: 4/6 male and 3/5 female) at the end of the recovery period. Scar glandular mucosa in stomach was observed in the male 50 mg/kg bw/day main group (slight: 2/6), male 200 mg/kg bw/day main group (slight: 4/6), and female 200 mg/kg bw/day main group (slight: 5/5 significantly increased).

No changes were determined in the ovary, uterus, vagian and mammary gland of parental female 200 mg/kg bw/day group.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
No changes were found in the number of conceiving days and copulation index.
5 and 12 females were not pregnant in 50 and 200 mg/kg bw/day group, individually. One female in the 12.5 mg/kg bw/day did not copulate.
Fertility index was sifnificantly decreased in 50 and 200 mg/kg bw/day groups in compared with the control group.
The length of gestation was significantly prolonged in 50 mg/kg bw/day group in compared with the control group.
Corpora lutea, implantation scars and implantation index were significantly decreased in the 50 mg/kg bw/day group in compared with the control group.
Since three dams didn't deliver pups, the gestation index was significantly decreased in the 50 mg/kg bw/day group in compared with the control group.
All pups in one dam in the 50 mg/kg bw/day group were dead on Day 2 of lactation (is this relevant for F1???)


Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
12.5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
The number of pups born and live pups born were significantly decreased in the 50 mg/kg bw/day group on Day 0 of lactation in compared with the control group.
The number of live pups on Day 4 of lactation was significantly decreased in the 50 mg/kg bw/day group in comapred with the control group. The viability index showed decreasing tendency in the 50 mg/kg bw/day group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weight in males and females at Day 0 and 4 of lactation, and mean pup weight weight at Day 0 and 4 of lactation were significantly increased in the 50 mg/kg bw/day group in compared with the control group.
The mean litter weight at Day 0 and 4 of lactation were significantly decreased. This change was related with the decreased number of live pups.
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:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The reason for dead was unknown in the pups of 50 mg/kg bw/day group because they were eaten by dams.
Histopathological findings:
not examined
Other effects:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
12.5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain

Applicant's summary and conclusion