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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 04 - October 14, 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
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): VC
- Substance type: organic
- Physical state: solid (under storage conditions)
- Test material was warmed at 25°C in a warming bath to produce a liquid before use.
- Analytical purity: >99.9%
- Lot/batch No.: 0801102
- Expiration date of the lot/batch: 31 December 2008
- Stability under storage conditions: stable
- Storage condition of test material: In refrigerator (2-8°C) protected from light and under nitrogen. Avoid contact with air and water.

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approximately 10 weeks
- Weight at study initiation: males 292-301 g (mean range) females 201-208 g (mean range)
- Fasting period before study: not applicable
- Housing: premating: in groups of 5 animals/sex/cage in Macrolon cages (MIV type); mating: females were caged together with males on a one-to-one basis in Macrolon cages ((MIII type); post-mating: males were housed in their home cage (Macrolon cages, MIV type) with a maximum of 5/cage, females were individually housed in Macrolon cages (MIII type); lactation: offspring was kept with the dam until termination. General: sterilised sawdust as bedding material.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: At least 5 days prior to start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.9-22.0
- Humidity (%): 30-81
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test material was warmed at approximately 25 °C in a warming bath to produce a liquid before use. Formulations (w/w) were prepared daily within 4 hours prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for specific gravity of the test substance (1.36) and vehicle (1.036).

VEHICLE
- Justification for use and choice of vehicle (if other than water): based on trial formulations performed at NOTOX
- Concentration in vehicle: 0, 7.2, 24, 71 mg/mL
- Amount of vehicle (if gavage): 2 mL/kg bw
Details on analytical verification of doses or concentrations:
Sampling and analysis of formulations (duplicate samples) prepared on one day during the treatment period (20 May 2008) was performed according to:
group 1: accuracy
group 2: accuracy, homogeneity and stability
group 3: accuracy
group 4: accuracy, homogeneity and stability
Duration of treatment / exposure:
Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 40 to 44 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. Pups were exposed only indirectly via lactation.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 4 hours difference between the earliest and latest dose.
Doses / concentrations
Remarks:
Doses / Concentrations:
15, 50, 150 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: : Dose levels are based on results of a 5-day dose range finding study with two groups of 3 females dosed for 5 days.
- Parturition F0: the females were allowed to litter normally. Day 1 of lactation is defined as the day when a litter was found completed (i.e. membranes, placentas cleaned up, nest built up and/or feeding of pups started).
- Lactation Fo: deficiencies in maternal care, such as inadequate constructions or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding, were recorded.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: parental animals at least twice daily; offspring were checked on day 1 of lactation and daily thereafter.
- Cage side observations: Mortality/Viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily (parental animals and offspring)

BODY WEIGHT: Yes
- Time schedule for examinations: Parental animals: males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4. Offspring: live pups were weighed during lactation on Days 1 and 4.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Weekly; food consumption was not recorded during the breeding period
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post-mortem examination.
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes, overnight
- How many animals: 5/sex/dose
- Parameters examined: White blood cells, Differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haemotocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets, Prothrombine time, Activated Partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post-mortem examination.
- Animals fasted: Yes, overnight
- How many animals: 5/sex/dose
- Parameters examined: Alanine aminotransferase, Aspartate aminotransferase, alkaline phosphatase, Total protein, Albumin, Total Bilirubin, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potasiium, Chloride, Calcium, Inorganic Phosphate

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
Functional observations: Yes
The following teste were performed in the first five mated males/group and the first five females/group with live offspring:
- hearing ability, pupillary reflex, static righting reflex and grip strength
- motor activity test (recording period: 12 hours during overnight for individual animals, using a computerised monitoring system. females were caged with their offspring)
The assigned males were tested during Week 4 of treatment and the assigned females were tested during lactation (all before blood sampling). In order to avoid hypothermia of pups, dams were removed from the pups for not more than 30-40 minutes.

GROSS PATHOLOGY: Yes
Parental animals
All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs.
From 5 surviving animals/sex/group the following tissues and organs were collected:
Adrenal glands, Aorta, Brain (cerebellum, mid-brain, cortex), Caecum, Cervix, Clitoral gland, Colon, Coagulation gland, Duodenum, Epididymides, (Eyes with optic nerve (if detectable) and Harderian gland), (Female mammary gland area), (Femur including joint), Heart, Ileum, Jejenum, Kidneys, (Larynx), (Lacrimal gland, exorbital), Liver, Lung (infused with formalin), Lymph nodes (mandibular, mesenteric), (Nasopharynx), Oesophagus, Ovaries, Pancreas, Peyer's patches (jejenum, ileum if detectable), Pituitary gland, Preputial gland, Prostate gland, Rectum, (Salivary glands, mandibular, sublingual), Sciatic nerve, Seminal vesicles, (Skeleton muscle), (Skin), Spinal cord (cervical, midthoracic, lumbar), Spleen, Sternum with bone marrow, Stomach, Testes, Thymus, Thyroid including parathyroid (if detectable), (Tongue), Trachea, Urinary bladder, Uterus, Vagina, All gross lesions.

Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

The following organ weights (and terminal body weight) were recorded: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Spleen, Testes, Thymus


HISTOPATHOLOGY: Yes
Of the selected 5 males/group of the control and high dose group, additional slides of the testes were prepared to examine staging of spermatogenesis.
The following slides were examined by a pathologist:
- all tissues collected at the scheduled sacrifice from control and high dose animals
- the additional slides of the testes of the selected 5 males/group of control and high dose groups
- the preserved organs and tissues of the animals of all dose groups which died spontaneously or were killed in extremis
- all gross lesions of all animals (all dose groups)
- the reproductive organs (cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testis, uterus and vagina) of all animals that failed to mate, conceive, sire or deliver healthy offspring

Due to possible treatment-related changes at the high dose group, histological examination was extended to liver, mesenteric lymph node, spleen, thymus and stomach for males and females and to mandibular lymph node for males of the selected animals of the low and mid dose groups.

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes / No / No data
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
Fetal examinations:
- External examinations: Yes: all per litter
Pups were killed by decapitation on day 5 of lactation or shortly thereafter.
All offspring was sexed and externally examined. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated.
Statistics:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one-t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the contro 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.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
No statistical analysis was performed on histopathology findings.
Indices:
Mean Live Litter Size = Total No. of Viable Pups on PND 0 / No. of Litters with Viable Pups PND 0

Postnatal Survival Between Birth and PND 0 or PND 4 (% Per Litter) = ((Sum of (Viable Pups Per Litter on PND 0 or PND 4 /No. of Pups Born Per Litter)) / No. of Litters Per Group) x 100

Postnatal Survival for All
Other Intervals (% Per Litter) = (Sum of (Viable Pups Per Litter at End of Interval N/Viable Pups Per Litter at Start of Interval N) / No. of Litters Per Group) x 100

Where N = PND 0-1 and 1-4

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: clinical signs, increased liver weight, histopathological changes in liver, stomach and thymus, deficiencies in maternal care

Details on maternal toxic effects:
At 50 mg/kg bw/day, animals showed clinical signs, increased liver weight (m), microscopic changes in liver (m), stomach (f) and thymus (f), and deficiencies in maternal care.
At 150 mg/kg bw/day, animals showed clinical signs, decreased body weight (gain), several haematological and clinical biochemistry changes, stomach and liver abnormalities at macroscopic examination, increased liver and kidney weights, microscopic changes in liver, stomach, thymus, spleen, mesenteric lymph nodes (m/f) and mandibular lymph nodes (m). High dose females showed a reduced number of living pups, a reduced number of implantation sites and deficiencies in maternal care.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: other:

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: absence of adverse effects in pups.

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
In a repeated dose toxicity study with reproduction/developmental toxicity screening, animals showed clinical signs, increased liver weight (m), microscopic changes in liver (m), stomach (f) and thymus (f), and deficiencies in maternal care at 50 mg/kg bw/day.
At 150 mg/kg bw/day, animals showed clinical signs, decreased body weight (gain), several haematological and clinical biochemistry changes, stomach and liver abnormalities at macroscopic examination, increased liver and kidney weights, microscopic changes in liver, stomach, thymus, spleen, mesenteric lymph nodes (m/f) and mandibular lymph nodes (m). High dose females showed a reduced number of living pups, a reduced number of implantation sites and deficiencies in maternal care.
Based on the observed effects in mid and high dose animals, the parental NOAEL was established to be 15 mg/kg bw/day.
The NOAEL for reproduction was established to be 50 mg/kg bw/day, based on reduced number of live pups and reduced number of implantation sites in high dose females. Based on observed deficiencies in maternal care in mid- and high- dose females, the NOAEL for breeding was set at 15 mg/kg bw/day.
In the absence of effects on pups, The NOAEL for developmental toxicity was established to be at least 150 mg/kg bw/day.
Executive summary:

Developmental toxicity of vinylene carbonate was studied in rats in dosed by oral gavage to 0, 15, 50 or 150 mg/kg bw/day for a minimum of 28 days in a combined repeated dose toxicity study with reproduction/developmental screening test according to OECD 422.

At 50 mg/kg bw/day, animals showed clinical signs, increased liver weight (m), microscopic changes in liver (m), stomach (f) and thymus (f), and deficiencies in maternal care.

At 150 mg/kg bw/day, animals showed clinical signs, decreased body weight (gain), several haematological and clinical biochemistry changes, stomach and liver abnormalities at macroscopic examination, increased liver and kidney weights, microscopic changes in liver, stomach, thymus, spleen, mesenteric lymph nodes (m/f) and mandibular lymph nodes (m). High dose females showed a reduced number of living pups, a reduced number of implantation sites and deficiencies in maternal care.

Based on the observed effects in mid and high dose animals, the parental NOAEL was established to be 15 mg/kg bw/day.

In the absence of effects on pups, The NOAEL for developmental toxicity was established to be at least 150 mg/kg bw/day.