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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 Jun 2015 - 17 May 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solidified melt
Details on test material:
Name of test material (as cited in study report): Octadecyl vinyl ether
Analytical purity: pure
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 20150128
- Purity : 85.5 area-%


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: guaranteed for the study period


Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Crl:WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services
GmbH, Sulzfeld, Germany
- Age at study initiation: 42 ± 1 days
- Weight at study initiation: male: 153.6; female: 127.9
- Housing: The animals were housed together (5 animals per cage) in polysulfonate cages supplied by
TECNIPLAST, Hohenpeißenberg, Germany (floor area about 2065 cm2). Dust-free wooden bedding was used in this study
- Diet: ad libitum, ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland
- Water: ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C,
- Humidity (%): 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours (12 hours light from 06.00-18.00 h, 12 hours dark from 18.00-06.00 h

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route was selected since this was proven to be suitable for the detection of a toxicological hazard
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was applied as a solution. To prepare this solution, the appropriate amount of test substance
was weighed out depending on the desired concentration. Then, corn oil was filled up to the desired volume,
subsequently mixed by a magnetic stirrer on a hotplate at 50°C.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance in corn oil at room temperature for a period of 7 days was proven before the start of the study.
Homogeneity as well as concentration control of the test substance in in corn oil were performed in all concentrations at the
beginning of the study and towards the end of the administration period
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
800 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
On day of arrival, the animals were subjected to an acclimatization period during which they
received ground diet and drinking water ad libitum. Prior to the first detailed clinical
observation, the animals were distributed according to weight among the individual test
groups, separated by sex. The weight variation of the animals used did not exceed 20 percent
of the mean weight of each sex. The list of randomization instructions was compiled with a
computer.
The test substance was administered daily for 13 weeks. Control animals received only the
vehicle. All remaining animals were sacrificed after a fasting period (withdrawal of food) of at
least 16 hours.
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays
and public holidays. If animals were in a moribund state, they were sacrificed and necropsied
- All animals were checked daily for any abnormal clinically signs before the administration as well as within 2 hours
and within 5 hours after the administration. Abnormalities and changes were documented for each animal.

DETAILED CLINICAL OBSERVATIONS: Yes
- Abnormal behavior when handled, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions,
abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmos, feces (appearance/ consistency), urine, pupil size

BODY WEIGHT: Yes
- Body weight was determined before the start of the administration period in order to randomize the animals.
During the administration period the body weight was determined on day 0 (start of the administration period)
and thereafter at weekly intervals. The difference between the body weight on the respective day of weighing
and the body weight on day 0 was calculated as body weight change

OPHTHALMOSCOPIC EXAMINATION: Yes
- Prior to the start of the administration period on day -1 the eyes of all animals and on study day 85 the eyes
of the control and high-dose animals were examined for any changes using an ophthalmoscope after administration
of a mydriatic agent

HAEMATOLOGY: Yes
- Time schedule for collection of blood: study days 92 and 93
- Anaesthetic used for blood collection: Yes : isoflurane
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined:
leukocyte count (WBC), erythrocyte count (RBC), hemoglobin (HCT), hematocrit (HCT), mean corpuscular volume (MCV),
mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT),
differential blood count, reticulocytes (RET), prothrombin time (Hepato Quick´s test) (HQT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: study days 92 and 93
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined:
alanine aminotransferase (ALT), aspartate aminotransferase (AST), Alkaline phosphate (ALP), γ-Glutamyltransferase (GGT),
sodium (Na), potassium (K), Chloride (Cl), inorganic phosphate (INP), calcium (Ca), urea (UREA), creatinine (CREA),
glucose (GLUC), total bilirubin (TBIL), total protein (TPROT), albumin (ALB), globulins (GLOB), triglycerides (TRIG), cholesterol (CHOL).

URINALYSIS: Yes
- Time schedule for collection of urine: day 85
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined:
pH, protein (PRO), glucose (GLU), ketones (KET), urobilinogen (UBG), bilirubin (BIL), blood, specific gravity (SP.GR),
sediment, color, turbidity (COL, TURB), volume (VOL)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the administration period
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity, motor activity,
other: home cage observation; open field observation

IMMUNOLOGY: No

OTHER:

ESTROUS CYCLE DETERMINATION:
Estrous cycle length and normality were evaluated daily for all female animals for a minimum
of 3 weeks prior to necropsy.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Organ weights:
anesthetized animals, adrenal glands, brain, cauda epididymis, epididymides, heart, kidneys, liver, ovaries,
pituitary gland, prostate, seminal vesicles incl. coagulating glands, spleen, testes, thymus, thyroid glands, uterus with cervix


HISTOPATHOLOGY: Yes
all gross lesions, adrenal glands, aorta, bone marrow (femur), brain, cecum, cervix, coagulating glands, colon, duodenum,
esophagus, extraorbital lacrimal glands, eyes with optic nerve (modified Davidson’s solution), femur with knee joint, harderian glands,
heart, ileum, jejunum (with Peyer’s patches), kidneys, larynx, left epididymis (modified Davidson’s solution),
left testis (modified Davidson’s solution), liver, lungs, lymph nodes (mesenteric and axillary lymph nodes),
mammary gland (male and female), nose (nasal cavity), ovaries, oviducts, pancreas, parathyroid glands, pharynx,
pituitary gland, prostate, rectum, salivary glands (mandibular and sublingual glands), sciatic nerve, seminal vesicles, skeletal muscle,
skin, spinal cord (cervical, thoracic and lumbar cord), spleen, sternum with marrow, stomach (forestomach and glandular stomach),
thymus, thyroid glands, trachea, urinary bladder, uterus, vagina.

Other examinations:
Sperm Parameters:
Sperm motility, sperm morphology, sperm head count cauda epididymis and testis
Statistics:
Body weight/change:
A comparison of each group with the control group was performed using
DUNNETT's test (two-sided) for the hypothesis of equal means

Feces, rearing, grip strength forelimbs, grip strength hindlimbs, footsplay test, motor activity, estrous cycle:
Non-parametric one-way analysis using KRUSKA-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05,
a pairwise comparison of each dose group with the control group was performed using WILCOXON test (two-sided) for the equal
medians

Blood parameters:
For parameters with bidirectional changes:
Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05,
a pairwise comparison of each dose group with the control group was performed.

Urinalysis parameters (apart from pH, urine volume, specific gravity, color and turbidity):
Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the
hypothesis of equal medians. In case of exactly the same numbers of the dose group and the control,
no statistical test is performed.

Urine pH, volume and specific gravidity:
Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less
than 0.05, a pairwise comparison of each dose group with the control group was performed
using WILCOXON-test (two-sided) for the hypothesis of equal medians.

Sperm analysis parameters:
Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) with
Bonferroni-Holm adjustment for the hypothesis of equal medians; If only control and one dose group are measured,
WILCOXON-test (one-sided) without adjustment were used.

Weight parameters:
Non-parametric one-way analysis using KRUSKALWALLIS test (two-sided). If the resulting p-value
was equal or less than 0.05, a pairwise comparison of each dose group with the control group was
performed using WILCOXON-test (two-sided) for the equal medians.





Results and discussion

Results of examinations

Details on results:
Clinical examinations and detailed clinical observations:
Slight and moderate salivation shortly after treatment was observed in 8 male and 8 female animals of test
group 3 (800 mg/kg bw/d) on several days of the study. Additionally, 3 male animals of test group 3 (800 mg/kg bw/d) ploughed
nose-first into bedding after application.
From the temporary, short appearance immediately after dosing (or shortly before) it was concluded that both types of
findings were induced by a bad taste of the test substance or local affection of the upper digestive tract.
No test substance-related effects were obtained in test group 1 and 2 (50 and 200 mg/kg bw/d).

Food consumption
No test substance-related findings were observed.

Water consumption
No test substance-related, adverse changes with regard to water consumption were
observed

Mortality
No animal died prematurely in the present study.

Body weight data
No test substance-related changes of mean body weights and mean body weight change values in all
male animals and in female animals of test group 2 (200 mg/kg bw/d) were observed.
Mean body weights and mean body weight change values were significantly increased in female animals
of test group 3 (800 mg/kg bw/d) from study day 21 onwards. The maximum body weight value was observed on
study day 91 by +10% and the maximum body weight change value was observed on study day 28 by +29%.
In female animals of test group 1 (50 mg/kg bw/d) the mean body weight value as well as body weight change value
were significantly decreased on study days 21, 28 and 49.
With regard to these changes, a clear dose-response relationship did not occur over the complete course of treatment.
Furthermore, the control animals were at the lower end of the historical control data (min. 221 g) whereas the female animal of test groups 1 and 3 were at the upper limit, but still within the historical range.
Therefore, these changes were assessed as being incidental and not related to treatment.

Functional observation battery:
Home cage observations, open field observations, sensorimotor tests/reflexes, quantitative parameters:
No test-substance related effects were observed.

Motor activity measurement:
Regarding the overall motor activity as well as single intervals, no test substance-related deviations to the control animals
were noted for male and female animals of test groups 1-3 (50, 200 and 800 mg/kg bw/d).
The single interval No. 10 in test group 1 (50 mg/kg bw/d) was significantly decreased. This finding was assessed to be incidental and
not related to treatment as no dose-response relationship occurred and only one individual value was changed.

Ophthalmological examinations:
No treatment-related findings were observed.

Estrous cycle:
No test substance-related effects on estrous cycle length and the number of cycles were obtained.

Hematology
After the administration period in male animals of test groups 2 and 3 (200 and 800 mg/kg bw/d) mean corpuscular volume (MCV) was decreased,
but the means were within the historical control range (MCV 47.8-51.1 fL).
In females of test group 3 (800 mg/kg bw/d) absolute monocyte and eosinophil cell counts were increased.

Clinical chemistry
After the administration period in rats of both sexes of test group 3 (800 mg/kg bw/d) γ- glutamyl transferase (GGT)
activities were increased. GGT activities were already higher in females of test group 2 (200 mg/kg bw/d) but the means
were within the historical control range (GGT 0-8 nkat/L). Therefore, in this test group the change was regarded as incidental
and not treatment-related.
In females of test group 3 (800 mg/kg bw/d) total protein, globulin and cholesterol values were increased. Cholesterol values were also
increased in male animals of test group 3 (800 mg/kg bw/d). Globulins and cholesterol values were already higher compared
to controls in females of test group 2 (200 mg/kg bw/d).
Albumin levels were increased in male rats of test group 3 (800 mg/kg bw/d), but compared to historical controls, the mean
of test group 3 was within whereas the study control mean was below the historical control range (albumin 33.42-41.09 g/L).
Therefore, the higher albumin levels in males of test group 3 were regarded as an incidental finding because of low
study control values.
In males of test group 1 (50 mg/kg bw/d), globulin levels were decreased and chloride values were increased, but both parameters
were not dose-dependently changed. Therefore, these alterations were regarded as incidental and not treatment-related.
In females of test group 3 (800 mg/kg bw/d) inorganic phosphate levels were higher compared to controls, but the mean was within
the historical control range (inorganic phosphate 1.14-1.55 mmol/L). Therefore, this alteration was regarded as incidental and not
treatment-related.

Urinalyses
No treatment-related, adverse changes among urinalysis parameters were observed.

Sperm parameters
Concerning motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as
sperm head counts in the testis and in the cauda epididymidis no treatment-related effects were observed.

Pathology
Regarding pathology, the liver was the target organ. Males and females of test group 3 (800 mg/kg bw/d) revealed
a diffuse hepatocellular hypertrophy which correlated with the enlargement observed by gross pathology in females
and with the increased liver weight in both sexes. Furthermore, hepatocytes with vacuoles were detected histopathologically, which
could be shown to be neutral fat storage within the hepatocytes. This correlated with macroscopic finding of light brown discoloration.
For test group 3 (800 mg/kg bw/d) males and females the hypertrophy and fatty change was regarded to be treatment-related and
adverse in combination with clinical pathology findings. In test group 2 (200 mg/kg bw/d) only the centrilobular hypertrophy in a
single female and the mean liver weight increase were regarded to be treatment-related and adverse, as also clinical pathology
parameters were changed in this test group. The fatty change occurred in different animals than the one with
hypertrophy. These animals did not reveal other findings than the minimal fatty change, which was regarded to be treatment-related
but not adverse.
In the lungs some animals revealed histiocytes within the alveoli which occasionally showed not only a foamy cytoplasm
(as normally observed in alveolar histiocytes) but clear vacuoles of variable size. With the ORO stain and electron microscopic
evaluation it could be demonstrated to be neutral fat. As no other changes were observed in the lungs this was not
regarded to be an adverse finding. Furthermore, it could not be excluded that part of the gavage fluid was aspired.
All other findings occurred either individually or were biologically equally distributed over control and treatment groups.
They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no adverse effects observed
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The oral administration of Octadecylvinylether by gavage to male and female Wistar rats for 3 months caused test substance-related,
adverse signs of systemic toxicity at a dose level of 200 (females only) and 800 mg/kg bw/d (both sexes).
Therefore, under the conditions of the present study the no observed adverse effect level (NOAEL) was 200 mg/kg bw/d for male
and 50 mg/kg bw/d for female Wistar rats.