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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-01-10 to 2012-12-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted in accordance with OECD/EU guidelines and principles of GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
(22 Mar 1996)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650: Combined Repeated Dose Toxicity Study With the Reproduction/Developmental Toxicity Screening Test (Jul 2000)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 11-12 weeks
- Weight at study initiation: M:303.1 - 337.2 g, F 209.3-237.3g
- Fasting period before study: no
- Housing: individually in Makrolon type M III cages, pregnant animals and their litters were housed together
- Diet (ad libitum): ground Kliba maintenance diet mouse/rat “GLP” meal
- Water (ad libitum): drinking water
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24°C
- Humidity: 30-70%
- Air changes: 15 times per hour
- Photoperiod: 12 hours light from 6.00 h to 18.00 h and 12 hours darkness from 18.00 h to 6.00 h

IN-LIFE DATES: From: 16 January 2012 To: M: 13 February 2012, F 07 March 2012

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance solutions in corn oil were prepared at the beginning of the administration period and thereafter in intervals, which took into account the analytical results of the stability verification.
For the preparation of the administration solutions the test substance (after melting by 70°C) was weighed in a graduated flask depending on the dose group, topped up with corn oil and subsequently thoroughly mixed by shaking until it was completely dissolved.

VEHICLE
- Justification for use and choice of vehicle: common vehicle in this kind of study
- Concentration in vehicle: 6.25, 12.5, 25 g/100mL
- Amount of vehicle: 4 mL/kg bw
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: from about 16.00 h until 07.00 - 09.00 h of the following morning for a maximum of 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical verifications of the stability of the test substance in corn oil for a period of 7 days at room temperature were carried out in a comparable batch prior to the start of the study.
Samples of the test substance solutions were sent to the analytical laboratory once at the beginning of the study for verification of the concentrations.

Determination of octadecylvinylether in corn oil
Method: GC, internal standard method
Apparatus: Agilent 6890 plus GC, Empower-Software (Waters)
Sample preparation: test item was dissolved in dichlormethane
GC conditions:
-Column: Optima 1, 100% Dimethylpolysiloxane, Length: 25 m, Internal diameter: 0.25 mm, Film thickness: 0.25 µm,
- Carrier gas: Nitrogen
- Oven temperature: 120°C to 240°C, 8°C/min, 240°C to 350°C, 40°C/min, 310°C, 20 min isothermal
- Injector temperature: 280°C
- Flow rate: 0.7 mL/min
- Detection: FID
- Detector temperature: 320°C
- Injection volume: 1.0 µL
Duration of treatment / exposure:
The treatment lasted up to one day prior to sacrifice (M: Day 29, F: Day 50)
Frequency of treatment:
Once daily
Details on study schedule:
After 2 weeks of premating treatment the F0 animals were mated to produce F1 generation pups. Mating pairs were from the same test group. Mating was discontinued as soon as sperm was detected in the vaginal smear. F0 animals were examined for their reproductive performance including determination of the number of implantation sites and the calculation of postimplantation loss for all F0 females.
The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1 and on PND 4. Their viability was recorded. At necropsy on PND 4, all pups were sacrificed with CO2, under isoflurane anesthesia, and examined macroscopically for external and visceral findings.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
250 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
500 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The aim of this study was to gain initial information on the possible effects of the test item on the integrity and performance of the male and female reproductive systems including gonadal function, mating behavior, conception, gestation and parturition. The study should also provide information about the general toxicological profile including target organs and no-observed-adverse-effect-level (NOAEL) after repeated oral administration.
- Rationale for animal assignment: random, according to body weight four days before the beginning of the administration period.
Positive control:
not applicable

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations included are any signs of morbidity, pertinent behavioral changes and signs of overt toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to the first administration and at weekly intervals during the administration period
- Parameters checked in table [No.1] were examined.

BODY WEIGHT: Yes
- Time schedule for examinations: once a week at the same time of the day

FOOD CONSUMPTION: Yes
- once a week for male and female parental animals
- of the F0 females with evidence of sperm was determined on gestation days (GD) 0, 7, 14 and 20
- of F0 females, which gave birth to a litter was determined on PND 1 and 4

WATER CONSUMPTION: No
Oestrous cyclicity (parental animals):
not examined
Sperm parameters (parental animals):
not examined
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, clinical observations, body weight, gross necropsy

GROSS EXAMINATION OF DEAD PUPS:
All stillborn pups and all pups that died before PND 4 were examined externally, eviscerated and their organs were assessed macroscopically.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals 28 days after the beginning of the administration
- Maternal animals: All surviving animals 50 and 51 days after the beginning of the administration. The females were allowed to deliver and rear their pups until day 4 after parturition.

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table No. 2 and 3 were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring was sacrificed at 4 days of age.
All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding.
Statistics:
- Food consumption (parental animals), body weight and body weight change: Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (twosided) for the hypothesis of equal means.

- Male and female mating indices, male and female fertility indices, gestation index, females with liveborn pups, females with stillborn pups, females with all
stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index: Pairwise comparison of each dose group with the control group using FISHER'S EXACT test for the hypothesis of equal proportions.

- Number of mating days: Pairwise comparison of the dose group with the control group using the WILCOXON-test (onesided) with Bonferoni-Holm-Adjustment for the hypothesis of equal medians.

- Feces, rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity: Non-parametric oneway analysis using KRUSKAL-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 (twosided) for the equal medians.

- Clinical pathology parameters: Non-parametric one-way analysis using KRUSKAL-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.

- 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 (twosided) for the equal medians.

* for p ≤ 0.05
** for p ≤ 0.01
Reproductive indices:
- Male mating index
- Male fertility index
- Female mating index
- Female fertility index
- Gestation index
Offspring viability indices:
- Live birth index
- Postimplantation loss

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY
There were no test substance-related or spontaneous mortalities in any of the groups. With one exception no clinical signs or changes of general behavior, which may be attributedt o the test substance, were detected in any male or female F0 generation parental animals during the whole study including gestation and lactation periods. Several male and female animals of all dose groups showed salivation after treatment during pre-mating, mating, gestation or post-mating. This transient salivation for a few minutes immediately after treatment was likely to be induced by the unpleasant taste of the test substance or by local irritation of the upper digestive tract. It is not considered to be a sign of systemic toxicity.

BODY WEIGHT AND WEIGHT GAIN
Mean body weights and mean body weight change of the male and female F0 generation parental animals in all test substance-treated groups (test groups 1 - 3) showed no decrease in comparison to the concurrent control group during the entire study period.

FOOD CONSUMPTION
Food consumption of the male and female F0 generation parental animals in all test substance-treated groups (test groups 1 - 3; 250, 500 and 1000 mg/kg bw/d) was comparable to the concurrent control group during the entire study period, with one exception. Regarding the statistically significantly increased food consumption of females (test group 3) during premating it was not likely but it could not be excluded that this finding was treatment related. However, this finding was not considered to be adverse.

ORGAN WEIGHTS
The decreased weights of the adrenal gland in female test group 1 (250 mg/kg bw/day) and 3 (1000 mg/kg bw/day) animals and the increase in relative weight of the epididymides of male test group 1 (250 mg/kg bw/day) animals did not show a dose-response relationship and was considered to be incidental.
Although the increase of the relative liver weight in female animals of test group 2 was not statistically significant, there was an increase in absolute weights in this group and the change in liver weights in female animals was dose-dependent. Therefore, the increase in liver weights of male and female animals of test groups 2 and 3 (500 and 1000 mg/kg bw/day) was considered to be treatment-related.
All other mean relative weight parameters did not show significant differences when compared to the control group 0.

GROSS PATHOLOGY
Treatment-related findings consisting of discoloration and prominent acinar pattern were seen in the liver. Light-brown discoloration was noted in 1/10 male test group 2 (500 mg/kg bw/day) and 2/10 male and 6/10 female test group 3 (1000 mg/kg bw/day) animals. Prominent acinar pattern was observed in one female animal of each test group 2 and 3.

HISTOPATHOLOGY: NON-NEOPLASTIC
Liver
Treatment-related fatty change of hepatocytes characterized by vacuoles of varying size was noted in many male and female animals of test groups 2 and 3 (500 and 1000 mg/kg bw/day, respectively) correlating partly to “light-brown discoloration” observed by gross examination. The distribution was different between the sexes: male animals showed centrilobular fatty change while female animals showed a differing pattern with dose: in test group 2, they showed a centrilobular or peripheral pattern whereas in test group 3, there was a peripheral or diffuse change.
Additionally, treatment-related minimal centrilobular hepatocellular hypertrophy was noted in 3/10 male test group 2 (500 mg/kg bw/day) and 4/10 male test group 3 (1000 mg/kg bw/day) animals.
The fatty change in test groups 2 and 3 (both sexes) as well as the centrilobular hypertrophy in test group 3 (male animals) were considered to have contributed to the increased liver weights in these groups.
Tigroid basophilic foci were observed in 4/10 female animals of test group 1 (250 mg/kg bw/day). They occurred singly and were very small, furthermore, there were none in the other treated test groups, and therefore they were considered to be incidental.
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.

REPRODUCTIVE PERFORMANCE
Male reproduction data:
For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100% in all groups including the controls.
Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter.
One high-dose male, one mid-dose male and two low-dose males did not generate implants in the mated females. Thus, the male fertility index ranged between 80% and 100% without showing any relation to dosing. This reflects the normal range of biological variation inherent in the strain of rats used for this study. The apparently infertile male rats of test groups 1-3 did not show relevant gross lesions.
Female reproduction and delivery data:
The female mating index calculated after the mating period for F1 litter was 100% in all test groups. The mean duration until sperm was detected (GD 0) varied between 1.8 and 3.1 days without any relation to dosing.
The fertility index varied between 80% in test group 1, 90% test groups 2 and 3 and 100% in control. These values reflect the normal range of biological variation inherent in the strain of rats used for this study. None of the non-pregnant females had any relevant gross lesions. The mean duration of gestation was similar in all test groups (i.e. between 22.2 and 22.5 days). The gestation index was 100% in all test groups.
Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the controls, taking normal biological variation into account (12.9 / 10.7 / 12.1 and 10.2 implants/dam in test groups 0-3 (0, 250, 500 and 1000 mg/kg body weight/day)). There were no statistically significant differences in post-implantation loss between the groups (4.9% / 7.7% / 5.6% / 11.9%), and the mean number of F1 pups delivered per dam remained unaffected (12.3 / 9.7 / 11.4 and 9.8 pups/dam at 0, 250, 500 and 1000 mg/kg bw/d). The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 100% (test group 3 and control), 99.1% (test group 2) and 97.9% (test group 1). Moreover, the number of stillborn pups was comparable between the groups.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: liver toxicity
Remarks on result:
other: Generation: P: general systemic toxicity (migrated information)
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed.
Remarks on result:
other: Generation: P: reproductive performance and fertility (migrated information)

Results: F1 generation

Details on results (F1)

LITTER DATA (OFFSPRING)
The viability index indicating pup mortality during lactation (PND 0 - 4) varied between 99.2% (test group 3) and 100% (test group 2, 1 and control) without showing any association to the treatment.
The mean number of delivered F1 pups per dam and the rates of liveborn and stillborn F1 pups were evenly distributed about the groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study.
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.

CLINICAL SIGNS (OFFSPRING)
There were no test substance-related adverse clinical signs observed in any of the F1 generation pups of the different test groups.

BODY WEIGHT (OFFSPRING)
No test compound-related influence on F1 pup body weights and pup body weight change were noted in all dose groups.
Two female runts were seen in the control. Five male and two female runts were seen in test group 2 (500 mg/kg bw/d). Two female runts were seen in test group 3 (1000 mg/kg bw/d).

GROSS PATHOLOGY (OFFSPRING)
No findings were observed at gross necropsy in any male or female pups of all test groups. One pup of the high dose group could not be assessed because it had been cannibalized.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Sex ratio of live F1 pups

PND 0

Test group 0

(0 mg/kg bw/d)

Test group 1

(250 mg/kg bw/d)

Test group 2

(500 mg/kg bw/d)

Test group 3

(1000 mg/kg bw/d)

Live males [%]

49.6

46.3

48.7

58.2

Live females [%]

50.4

53.7

51.3

41.8

PND 4

 

 

 

 

Live males [%]

49.6

46.3

48.7

58.8

Live females [%]

50.4

53.7

51.3

51.2

 

Applicant's summary and conclusion