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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented study report according to OECD guideline 414: GLP .

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Details on test animals and environmental conditions:
Identification: MCP2484
Alternative name: SYNESSTIC 12
Action: Base oil
Description: Amber liquid
Storage conditions: At ambient temperature
Supplier: Sponsor
Batch number: E09K005
Date of receipt: 7 June 2010
Quantity received: 3 x 1L
Expiry date: 31 December 2011

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Females were treated from Day 6 to Day 19 after mating. Animals received the test material or vehicle control formulations orally at a volume-dose of 4 mL/kg bodyweight, using a suitably graduated syringe and a rubber catheter inserted via the mouth into the stomach.
All animals were dosed in sequence of cage-number within each group, once each day at approximately the same time as the previous day, seven days per week. The volume administered to each animal was calculated from the most recently recorded bodyweight. Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The homogeneity and stability of the test material in the liquid matrix was demonstrated as 15 days at 2-8°C (refrigerated) and 24 hours when stored at ambient temperature. During the first and last weeks of treatment, the test formulations were analysed for achieved concentration of the test substance. Four samples (nominally 1 mL accurately weighed) were taken from all groups. Two assays from each group were analysed and the remainder were retained refrigerated (nominally 2-8 ºC) as contingency.
Details on mating procedure:
Crl: CD (SD) rats (a total of 90 females) were received from Charles River (UK) Ltd. They were ordered at approximately 65 days of age and within a weight range of 227 to 254g. On receipt animals appeared healthy. Within 24 hours of arrival, a representative sample of 15 animals was weighed and these animals were within the weight range. The animals were checked twice daily for health and general condition and were allowed to acclimatise to the conditions described below for five days before they were paired on a 1:1 basis with stock males from the same source. Daily checks were made after pairing for evidence of mating, including ejected copulation plugs in cage trays and the presence of sperm in a vaginal smear. Animals were allocated to study on Day 0 of gestation, when positive evidence of mating was detected. Only females with a sperm positive vaginal smear or at least two copulation plugs were selected. Females were allocated to group and cage position in sequence of mating, thus ensuring that animals mated on any one day were evenly distributed amongst the groups. The sequence of allocation was controlled to prevent stock males from providing more than one mated female in each treatment group.
Duration of treatment / exposure:
Females were treated from Day 6 to Day 19 (inclusive) after mating
Frequency of treatment:
All animals were dosed in once each day at approximately the same time as the previous day, seven days per week.
Duration of test:
Study initiation:
(Protocol signed by Study Director)
3 September 2010
Experimental start date:
(Animal arrival)
22 September 2010
Pairing commenced: 27 September 2010
Treatment commenced: 4 October 2010
Necropsy completed: 21 October 2010
Experimental completion date:
01 December 2010
Study completion: 20 January 2011
No. of animals per sex per dose:
20 females/dose
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each adult female was recorded on Days 0, 3 and 6-20 after mating.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- The weight of food supplied to each adult female, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 inclusive after mating.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. After ventral midline incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ. Any abnormal position, morphology or interaction was recorded. External and cut surfaces of the organs and tissues were examined as appropriate. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative.

Ovaries and uterine content:
The following reproductive assessment was made for all animals after termination:
The gravid uterus was weighed before removal of the fetuses; this weight included the weight of the cervix, uterus, fallopian tubes and ovaries.
For each animal, the number of corpora lutea in each ovary and the number of implantation sites, the number and distribution of resorption sites (classified as early or late) and live and dead fetuses were recorded for each uterine horn. The retained tissues were checked before disposal of the carcass.
Fetal examinations:
All fetuses and placentae were dissected from the uterus and weighed individually. Fetuses were individually identified within the litter, using a coding system based on their position in the uterus. Each fetus and placenta was externally examined and any abnormalities were recorded. The sex of each fetus was recorded. Approximately half of the fetuses in each litter were eviscerated and their skeletons were fixed in Industrial Methylated Spirit, prior to processing and staining with Alizarin Red and Alcian Blue. The remaining fetuses were fixed whole in Bouin’s fluid. Free-hand serial sections were prepared from the Bouin’s fixed fetuses and were examined under the microscope for visceral abnormalities. Fetuses stained with Alizarin Red and Alcian Blue were assessed for skeletal and cartilage development and abnormalities.

Findings observed were classified, according to severity and incidence, as:
Major abnormalities: normally rare, definitely detrimental to normal subsequent development, possibly lethal, e.g. ventricular septal defect
Minor abnormalities: minor differences from normal that are detected relatively frequently considered to have little detrimental effect and may be a transient stage in development e.g. bipartite centrum, dilated urether.
Variants: alternative structures or stages of development occuring regularly in the control population, e.g. number of ribs, incomplete ossification of 5th and 6th sternebrae.
In the Liberate Fetal Pathology Appendix, observations on repeated structures like ribs, vertebrae and sternebrae are reported as the first and last affected element, in the form “5th 13th bilateral ribs”, which should be interpreted as “5th to 13th bilateral ribs
Statistics:
Statistical analyses were applied where there was indication of possible meaningful intergroup differences. All statistical analyses were carried out using the individual animal (or litter) as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis, and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.
The following data types were analysed at each timepoint separately:
Bodyweight, using absolute weights and gains over appropriate study periods;
Gravid uterine weight, adjusted bodyweight and bodyweight change;
Food consumption, using means over appropriate study periods;
Litter size and survival indices;
Fetal, placental and litter weight.

The following sequence of statistical tests was used for bodyweight, food consumption, litter size and survival indices and fetal placental and litter weight data: A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. The F1 approximate test was applied. If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, suggesting that the dose-response was not monotone, Dunnett's test was performed instead. A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations.. The H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied. If the H1 approximate test for monotonicity of dose-response was not significant at the 1% level, Shirley's test for a monotonic trend was applied. If the H1 approximate test was significant, suggesting that the dose-response was not monotone, Steel's test was performed.
Indices:
Individual values are presented for the numbers of corpora lutea, implantations, resorptions (early, late and total) and live fetuses (male, female, total) and sex ratio (percentage male) for litters at Day 20 of gestation. Prenatal losses are separated into pre- and post implantation phases. Pre-implantation loss was considered to reflect losses due to non-fertilisation of ova but may include very early post-implantation deaths (i.e. those occurring during the first two to three days post-implantation), in addition to true pre-implantation loss. It was calculated from the formula:
(Number of corpora lutea – Number of Pre-implantation loss (%) = implantations)
Number of corpora lutea
x 100
Where the number of implantations exceeded the number of corpora lutea observed,
pre-implantation loss was assumed to be zero (i.e. no pre-implantation loss was considered to
have occurred).
Post-implantation loss was considered to exclude the first two to three days post-implantation
as deaths occurring at this stage are considered to leave no remains visible at Day 20 of
gestation. It was calculated from the formula:
Post-implantation loss (%) = (Number of implantations – Number of live fetuses) Number of implantations x 100
All group values and SD (as appropriate) were calculated from the individual litter values.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Gross pathological findings:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
Maternal clinical condition, bodyweight, food consumption and macroscopic evaluation was unaffected by treatment with MCP2484 upto 1000 mg/kg/day when compared with Control animals.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in litter size and weights:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg/day there was a slightly higher incidence of fetuses/litters with incomplete
ossification of cranial centres and haemorrhages in the abdominal cavity and a low incidence
of 13/14, 14/14 ribs compared to concurrent Control, all of which are considered of no
biological significance.
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Embryo fetal survival growth and external morphology were unaffected by treatment with MCP2484 upto 1000 mg/kg/day.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
reduction in number of live offspring
changes in litter size and weights
changes in postnatal survival

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Oral administration of MCP 2484, (a base oil), to Crl:CD(SD) rats during the organogenesis phase of embryo fetal development resulted no adverse effects being observed at dose levels upto 1000 mg/kg/day. It was concluded from this study that the dosage of 1000 mg/kg/day was the maternal noobserved- adverse-effect-level (NOAEL) and 1000 mg/kg/day was the no-observed-adverseeffect- level (NOAEL) for embryo fetal survival and development. These findings do not warrant classification of the test material for developmental/reproductive toxicity under the new Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP), under the Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations, or under the Globally Harmonized System of Classification and Labelling of Chemicals (GHS).
Executive summary:

The influence of MCP2484 (a base oil) on embryo-fetal survival and development in Crl:CD(SD) rats was assessed following oral administration during and after the organogenesis phase of pregnancy (Days 6-19 after mating). Three groups of twenty female rats received MCP2484 by gavage at doses of 100, 300 or 1000 mg/kg/day from Day 6 to 19 after mating, inclusive. A similarly constituted Control group received the vehicle, corn oil, at the same dose volume throughout the same period. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination. During the study, clinical condition, dosing signs, bodyweight and food consumption were monitored. Adult females were examined macroscopically at necropsy on Day 20 after mating and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal and cartilaginous examination.

Maternal clinical condition, bodyweight, food consumption and macroscopic evaluation was unaffected by treatment with MCP2484 upto 1000 mg/kg/day when compared with Control animals. Embryo fetal survival growth and external morphology were unaffected by treatment with MCP2484 upto 1000 mg/kg/day.

It was concluded from this study that the dosage of 1000 mg/kg/day was the maternal no observed- adverse-effect-level (NOAEL) and 1000 mg/kg/day was the no-observed-adverse effect- level (NOAEL) for embryo fetal survival and development