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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / 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:
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:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Crl:WI(Han)
- 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 (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least 5 days prior to start of treatment


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.9-22.0°C
- 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 mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: maximum of 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of post-coitum
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: individually
Analytical verification of doses or concentrations:
yes
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.
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:
0, 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.
Positive control:
None.

Examinations

Parental animals: Observations and 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: 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


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.
Sperm parameters (parental animals):
Parameters examined in 5 parental males of control and high dose groups:
staging of spermatogenesis
testis weight and epididymis weight were determined from all parental males
Litter observations:
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 abnormalities; possible cause of death was determined for pups born or found dead; the stomach was examined for the presence of milk
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals following completion of the mating period (a minimum of 28 days of dose administration)
- Maternal animals: All surviving animals on lactation Day 5

GROSS NECROPSY
- Gross necropsy according to OECD 422

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues according to OECD 422
Postmortem examinations (offspring):
SACRIFICE
Pups were killed on Day 5 of lactation or shortly thereafter.

GROSS NECROPSY
- Gross necropsy consisted of external examinations including presence of milk in the stomach.

HISTOPATHOLOGY / ORGAN WEIGTHS
no
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.
Reproductive indices:
Mating, fertility, and copulation/conception indices were calculated as follows:
Male (Female)
Mating Index (%) =No. of Males (Females) with Evidence of Mating (or Confirmed Pregnant)/Total No. of Males (Females) Used for Mating) x 100

Male Fertility Index (%) =(No. of Males Siring a Litter/Total No. of Males Used for Mating) x 100

Male Copulation Index (%) =(No. of Males Siring a Litter/ No. of Males with Evidence of Mating (or Females with Confirmed Pregnancy)) x 100

Female Fertility Index (%) =(No. of Females with Confirmed Pregnancy/ Total No. of Females Used for Mating) x 100

Female Conception Index (%) =(No. of Females with Confirmed Pregnancy/ (No. of Females with Evidence of Mating (or Confirmed Pregnancy)) x 100


Offspring viability 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: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

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

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reduced number of live pups and reduced number of implantation sites.
Dose descriptor:
NOAEL
Remarks:
breeding toxicity
Effect level:
15 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: deficiencies in maternal care.

Results: F1 generation

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: absence of adverse effects in pups.

Overall reproductive toxicity

Reproductive effects observed:
not specified

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:

 Reproduction 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.

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.