Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
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
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 oral 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
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

Observations and examinations performed and frequency:
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.
Sacrifice and pathology:
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

Offspring: 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. Description of all external abnormalities were recorded, if possible, defects or case of death were evaluated.

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.
Other examinations:
None.
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.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
see table
One low dose female was killed in extremis on Day 18 of the study. This death is considered not substance-related.
Slight hunched posture was observed in only one low-dose female, in one mid-dose female and 2 mid-dose males, and in all high dose animals. Slight piloerection was noted for all animals in all dose groups. Piloerction was observed half an hour after dosing and lasted for only several hours. In low dose animals, some animals showed piloerection only on one day during the study period. In the absence of other effects this observed piloerction is considered not adverse.
Slight to moderate salivation was observed in all dose groups. However, since this effect was observed directly after dosing, and stopped after some time, it is considered to be a physiological response to the taste of the formulation and not a sign of systemic toxicity, and is therefore considered not toxicologically relevant.

BODY WEIGHT AND WEIGHT GAIN
see table
Decreased body weights and body weight gain were observed in high dose animals. Statistically significant decreased body weights were observed from Day 8 onward in males and on Day 4 of lactation in females.

FOOD CONSUMPTION
see table.
During days 11-17 post coitum, slightly increased food consumption (absolute and relative) was observed in high dose females. This slight increase during this period is considered not toxicologically relevant.

HAEMATOLOGY
see table
Statistically significant effects were confined to high dose animals. In males , an increased % of reticulocytes was observed. In females, decreases were observed in mean corpuscular volume, in mean corpuscular haemoglobin and in activated partial thromboplastin time, and increases were observed in white blood cell number, and platelet numbers.

CLINICAL CHEMISTRY
see table
Statistically significant effects were confined to high dose animals, except for potassium. Decreases were observed in total protein and in albumin (m/f) and in creatinine (f) , and increases were observed in total bilirubin (m/f), urea (m), potassium (m/f), alkaline phosphatase (f) and cholesterol (f).
Potassium levels were increased in males of all dose groups, but these values were within the normal range of variation, and therefor considered not toxicologically relevant.

ORGAN WEIGHTS
see table
Liver weights were statistically significant increased in mid- and high dose males (rel) and in high dose females (abs/rel). Kidney weights were statistically significant increased in high dose males (rel) and in females (abs/rel).
Spleen weights were non-statistically significant increased in mid- and high dose males (rel) and in mid- and high dose females (abs/rel).
The observed increases in relative weights of brain, heart and testes in high dose males are caused by the lower terminal body weight, and are considered not relevant.

GROSS PATHOLOGY
see table
In high dose animals, abnormalities were observed in liver and stomach.

HISTOPATHOLOGY: NON-NEOPLASTIC
see table
Microsopical examination showed changes in liver (m), stomach (f) and thymus (f) of mid-dose animals and changes in liver, stomach, thymus, spleen and mesenteric lymph nodes in both sexes and in mandibular lymph nodes in males.
The observed hepatic changes were bile duct hyperplasia, centriacinar individual cell necrosis (mid dose males and high dose male and females), periacinar hepatocytic hypertrophy and centriacinar fibrosis.
In high dose animals atrophy was observed in spleen, thymus and mandibular and mesenteric lymph nodes, with changes in thymus also observed in mid-dose females, accompanied by an increase in severity.
Ulceration of the keratinized stomach (limiting ridge) was observed in high dose animals. Acanthosis of the keratinized stomach and submucosal oedema were present in mid-dose females and in high dose animals. Submucosal oedema in the glandular stoamch was observed in mid- and high dose females.
There were no variations in the subjectively judged stages of testicular spermatogenesis.
The observed squamous carcinoma in a mid-dose male is an isolated observation, and considered not substance-related.

FUNCTIONAL OBSERVATIONS
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals.
During lactation, a slight increase in total low sensor count was observed in mid-and high dose females.

REPRODUCTION
No treatment related findings were observed for mating performance, fertility parameters, duration of gestation, number of dead pups and number of corpora lutea. In the high dose group, the number of pups was decreased (9.7 pups/litter vs 12.1 pups/litter in high dose and control groups, respectively. This was confirmed at necropsy at which the number of implantation site statistically significant was reduced (9.7 vs 13.9 in high dose and control groups, respectively).

BREEDING DATA
Deficiencies in maternal care were noted in mid- and high dose females. Postnatal loss and viability index were similar for the control and treated groups.

PUP DEVELOPMENT
Mean body weight and development of pups was similar in control and treated groups.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Remarks:
parental toxicity
Effect level:
15 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: clinical signs, increased liver weight (m), microscopic changes in liver (m), stomach (f) and thymus (f)
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
50 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: reduced number of live pups and reduced number of implantation sites.
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
150 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: absence of adverse effects in pups.
Dose descriptor:
NOAEL
Remarks:
breeding toxicity
Effect level:
15 mg/kg bw/day (nominal)
Sex:
female
Basis for effect level:
other: deficiencies in maternal care.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

0

15

50

150

dr

m

f

m

f

m

f

m

f

 Mortality

1/5

Clin. Signs

- Slight hunched posture

1/10

2/10

1/10

10/10

10/10

m

- Slight piloerection

10/10

10/10

10/10

10/10

10/10

10/10

- Slight to moderate salivation

6/10

3/10

10/10

6/10

10/10

10/10

f

Body weight (gain)

ds

ds

Food consumption

No toxicologically relevant effects

Functional observation

- motor activity, total low sensor count (females lactation)

9102 ± 2271

9143 ± 6019

13776 ± 7737

14730 ± 5331

Haematology

- Reticulocytes

is

- MCV

ds

-

ds

- White blood cell number

is

- Platelet number

is

- APTT

ds

 Clin. Biochemistry

- Total prot.

ds

ds

- Albumin

ds

ds

- Bilirubin

is

is

- Urea

is

- Creatinine

ds

- AP

is

-Cholesterol

is

- Potassium

is

is

is

is

 Pathology

-Macroscopic examination

liver

- accentuated lobular patters

2/10

- pale liver

1/10

- lobe infraportalis reduced

3/10

stomach

-greenish or yellowish hard nodules at forestomach

2/10

- irregular surface forestomach

1/10

-irregular surface limiting ridge

1/10

- Organ weight

- Liver

isr

isr

isa/r

m,f

- Kidneys

isr

isa/r

- Spleen

ir

ia/r

ir

ia/r

- Brain

isr

f

- Heart

isr

- Testes

isr

Microscopic examination

Liver

- bile duct hyperplasia

5/6

5/6

- centriacinar individual cell necrosis

2/6

5/6

5/6

m

-periacinar hepatocytic hypertrophy

1/6

3/6

2/6

m

- centriacinar fibrosis

2/6

3/6

Lymph nodes

-mandibular, atrophy

1/6

5/6

-mesenteric, lymphoid

atrophy

1/6

1/7

2/7

5/6

4/6

f

spleen

- lymphoid atrophy

5/6

5/6

- diminished extramedullary haemopoiesis

1/7

3/6

1/7

1/6

2/7

5/6

1/6

stomach

- ulceration keratinized stomach (limiting ridge)

2/6

4/6

- acanthosis keratinized stomach

1/7

3/7

2/6

3/6

f

- submucosal oedema keratinized stomach

1/7

4/7

2/6

5/7

f

- submucosal oedema glandular stomach

2/7

4/6

f

thymus

- atrophy

1/7

1/7

1/7

3/7

4/6

5/6

m

severity

f

-minimal

1/1

1/1

1/1

1/3

4/4

-mild

2/3

1/5

-moderate

4/5

Reproduction/development

Reproduction

- number of living pups (mean)

- number of implantation sites

12.1

13.9

11.2

12.8

9.7

9.7

Breeding data

- deficiencies in maternal care

2/10

8/10

f

- Pup development

No toxicologically relevant effects

i/is: increased/increased statistically significant

d/ds: decreased/decreased statistically significant

a: absolute

r: relative

dr: dose related

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.

Classification:
In the absence on more information on time-dependent dose effects of Vinylene carbonate, Category 2 for the oral route is proposed, since within the guidance values of 10 < concentration ≤ 100 mg/kg bw/day in a combined repeated dose toxicity study with reproduction/developmental screening effects on liver (increased liver weight and liver cell necrosis in 2/6 males) were observed in males exposed for 28 days, whereas these effects in females exposed for 44 days were observed at a higher dose. Also the more severe effects seen at 150 mg/kg bw/day (males and females) are within the range of guidance values as given for a 28-day repeated dose toxicity study. In addition, the effect levels (50/150 mg/kg bw/d) are outside the range for a Category 1 classification (90-day: concentration ≤ 10 mg/kg bw/d; 28-day: concentration ≤ 30 mg/kg bw/d).
In conclusion, the substance should be classified as STOT Re Category 2 with Hazard Statement H373.
Executive summary:

Repeated dose 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 15 mg/kg bw/day the observed incidental salivation in a few animals and piloerection during a short period after dosing were not accompanied by other effects in this group. In the absence of other effects, these observations are considered not adverse/toxicological relevant.

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.