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Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 January 2002 - 08 February 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
Adopted: 22" January 2001.
Deviations:
yes
Remarks:
Due to the pilot status of this study a reduced number of animals is used.
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
A mixture mainly based on: 2,3-dihydro-6-(2-hydroxy-2-methyl-1-oxopropyl)-1,1,3-trimethyl-3-[4-(2-hydroxy-2-methyl-1-oxopropyl)phenyl]-1H-indene; 2,3-dihydro-5-(2-hydroxy-2-methyl-1-oxopropyl)-1,1,3-trimethyl-3-[4-(2-hydroxy-2-methyl-1-oxopropyl)phenyl]-1H-indene
EC Number:
402-990-3
EC Name:
A mixture mainly based on: 2,3-dihydro-6-(2-hydroxy-2-methyl-1-oxopropyl)-1,1,3-trimethyl-3-[4-(2-hydroxy-2-methyl-1-oxopropyl)phenyl]-1H-indene; 2,3-dihydro-5-(2-hydroxy-2-methyl-1-oxopropyl)-1,1,3-trimethyl-3-[4-(2-hydroxy-2-methyl-1-oxopropyl)phenyl]-1H-indene
Cas Number:
163702-01-0
Molecular formula:
C26H32O4
IUPAC Name:
Reaction mass of 2-hydroxy-1-{3-[4-(2-hydroxy-2-methylpropanoyl)phenyl]-1,1,3-trimethyl-2,3-dihydro-1H-inden-5-yl}-2-methylpropan-1-one and 2-hydroxy-1-{1-[4-(2-hydroxy-2-methylpropanoyl)phenyl]-1,3,3-trimethyl-2,3-dihydro-1H-inden-5-yl}-2-methylpropan-1-one
Test material form:
solid
Remarks:
Form specified in RSS.
Specific details on test material used for the study:
Identity ESACURE KIP 150
Description Solid (at temperatures below 35°C)
Purity 99.999% (calculated as 100% - % of dichloromethane content)
Storage At room temperature, protected from light (uv)
Stability of the pure test item Stable under storage conditions
Stability in the vehicle Stable for at least 6 hours
Safety precautions Fioutine hygienic procedures will be applied to ensure personnel security.











Test animals

Species:
rat
Strain:
Wistar
Remarks:
HanBrl:WlST (SPF)
Details on test animals or test system and environmental conditions:
TEST SYSTEM
Species Hat HanBrl:WlST (SPF)
Rationale Specified by the international guidelines as a recommended test system
Source RCC Ltd, Biotechnology & Animal Breeding Division, wolferstrasse 4, CH-4414 Fijllinsdori / Switzerland
Acclimatization Ten days minimum under test conditions with an evaluation of the health status.
Number of animals 20 mated females, 5 per group
Age at pairing 11 weeks minimum
Body weights 211 - 255 grams (day 0 post coitum)
identification Individual animal number tattooed on the pinnae (day 0 post coitum)

HUSBANDRY
Conditions
The animals were housed under standard laboratory conditions: air-conditioned with 10—15 air changes per hour; the environment monitored continuously with hourly recordings of temperature (target range 22 ± 3°C) and relative humidity (target range 30 - 70%), 12 hours artificial fluorescent light/ 12 hours dark with background music played at a centrally defined low volume for at least 8 hours during the light period.

Accommodation
The animals were housed individually in Makrolon cages (type-3) with wire mesh tops and standardized granulated softwood bedding (Lignocel, Schill AG, CH-4132 Muttenz/ Switzerland).

Diet
Pelleted standard Kliba 3433 rat/mouse maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst/ Switzerland) was available ad libitum (Batch Nos. 77/01 and 119/01).

Water
Tap water in bottles was available ad libitum.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
Identity Corn oil
Supplier Carl Roth GmbH
Batch No. 02938667
Corn oil was used as the vehicle for the test item in the dose groups and was administered as the control item to the females of the control group.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration, homogeneity and stability of the test item/vehicle mixtures was determined during the first and last week of the administration period. Samples were taken after preparation and again 6 hours later. On each occasion samples (appr. 2 g per sample) were taken from the top, middle and bottom of each formulation. Each dose sample was individually identified. The samples were frozen (approx. -20°C) for analysis by (HPLC) by:
Dr. C. Knuppe, 900 Environmental Chemistry & Pharmanalytics Division, CH 4452 Itingen/ Switzerland.

The results of the analyses are attached (Attachment Vl, pp. 140 - 154)

The overall mean concentrations of the homogeneity samples were found to be 91.0%, 92.5%, and 101.2% of the nominal concentrations of dose group 2 (2 mg/ml), dose group 3 (20 mg/ml), and dose group 4 (50 mg/ml), respectively. The individual concentrations varied in the range from -5% to +2% of the mean concentrations. Therefore, the test item was found to be homogeneously distributed in the vehicle.
Details on mating procedure:
After acclimatization, the females were housed with the males (one male : one female) in special automatic mating cages, i.e. with synchronized timing to initiate the nightly mating period, until evidence of copulation was observed. This system reduces the variation in the copulation times of the different females. The females were removed and housed individually if:

a) the daily vaginal smear was sperm-positive, or
b) a copulation plug was observed.

This day was designated day 0 post coitum.
The male rats used for mating were in the possession of RCC. The fertility of these males was proved and continuously controlled.
Duration of treatment / exposure:
From day 6 through to day 20 post coitum.
Frequency of treatment:
Once daily.
Duration of test:
21 days.
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
Mated rats were assigned to the different groups using a computer-generated random algorithm.

All animals received a dose volume of 5mn body weight with a daily adjustment of the individual volume to the actual body weight. Control animals were similarly dosed with the vehicle alone.

Oral intake is one possible route of human exposure. International guidelines recognize the efficacy of oral administration.

Examinations

Maternal examinations:
Mortality rate The animals were checked at least twice daily for any modalities.

Signs and/or symptoms The animals were observed at least twice daily for signs of reaction to treatment and/or symptoms of ill health.

Food consumption Food consumption was recorded for the following periods: days 0-3. 3-6. 6-9, 9-12, 12-15. 15-18 and 18- 21 post coitum (see Data Compilation and Data Processing).

Body weight Body weights was recorded daily from day 0 until day 21 post coitum (see Data Compilation and Data Processing).









Fetal examinations:
TERMINATION OF THE STUDY

On day 21 post coitum, the females were killed by CO2 asphyxiation and the fetuses removed by Caesarean section.

CAESAREAN SETION AND POST MORTEM EXAMINATION

Postmortem examination, including gross macrosco-pic examination of all internal organs, with emphasis on the uterus, uterine contents, position of fetuses in the uterus and number of corpora lutea, was performed and the data recorded. The uteri (and contents) of all females with live fetuses were weighed at necropsy on day 21 post coitum to enable the calculation of the corrected body weight gain (see Data Processing).

Additionally, livers of females were weighed at necropsy. The fetuses were removed from the uterus, sexed, weighed individually, examined for gross external abnormalities, killed by subcutaneous injection of sodium pentobarbitone (Narcoren®) and allocated to one of the following procedures:

1) Microdissection technique (sectioning/dissection technique). Half of the fetuses from each litter were fixed in Bouin's fixative (one fetus per container). They were examined by a combination of serial sections of the head and microdissection of the
thorax and abdomen. This included detailed examination of the major blood vessels and sectioning of the heart and kidneys. After examination the sections were preserved in a solution of glycerine/ethanol (one fetus per container). Descriptions of any abnormalities and variations were recorded.

2) The remaining fetuses were eviscerated and with the exception of over the paws, the skin was removed and discarded. Carcasses were processed through solutions of ethanol. glacial acetic acid with Alcian blue (for cartilage staining), potassium hydroxide with Alizarin red S (for clearing and staining ossified bone) and agueous glycerin for preservation and storage. The skeletons were examined and all abnormal findings and variations recorded. They were preserved individually.
Statistics:
The following statistical methods were used to analyse body weights, food consumption and reproduction data:

- Means and standard deviations of various data were calculated and included in the report.
- If the variables could be assumed to follow a normal distribution, the Dunnett many-one t-test, based on a pooled variance estimate, was used for intergroup comparisons (i.e. single treatment groups against the Control group).
- The Steel test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- Fisher's Exact test for 2x2 tables was applied if the variables could be dichotomized without loss of information.

All methods of analysis and the results are included in the report.
Historical control data:
Attachment V in 'attached background material'

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No mortalities or clinical symptoms were noted in any female throughout the study.
Mortality:
no mortality observed
Description (incidence):
No mortalities or clinical symptoms were noted in any female throughout the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weight gain was not affected by treatment with the test item.

Also, corrected body weight gain (corrected for uterus weight) gave no indication for any test item-related findings. The slightly decreased corrected body weight gain noted in group 4 was considered to be incidental.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Mean food consumption during the treatment period was clearly reduced in group 4 (-10.6% compared to vehicle controls). Food consumption in groups 2 and 3 was similar to that of vehicle controls.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Mean liver weights were slightly but dose-dependently increased in groups 3 and 4 (17.6 and 17.9 9 compared to 15.8 g in vehicle controls). Liver weights in group 2 were similar to those in vehicle controls (15.4 g).

The increase in female liver weights in groups 3 and 4 was possibly test item-related since the liver is a known target organ of the test item.
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
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

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
food consumption and compound intake
organ weights and organ / body weight ratios

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
No test item-related effects on fetal body weights were noted.

Fetal body weights were slightly increased in group 4 (5.0 9 versus 4.8 g in vehicle controls, combined litter-based data for male and female fetuses), but this increase was considered to be incidental.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No test item-related effects on the sex ratio of fetuses were noted in any group.
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No abnormalities were noted during external examination of fetuses
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were 8 fetuses with abnormal skeletal findings as follows:

None in group 1 (0/32), one in group 2 (1/35), one in group 3 (1/32) and six in group 4 (6/34). The sternebral and vertebral abnormalities noted in one fetus in group 2, one fetus in group 3 and two fetuses in 4 were common abnormal findings that are frequently seen in control rat fetuses of this strain. The incidence in this study was considered to be unrelated to treatment with the test item.

The abnormal finding of fusion of two components of the zygomatic arch noted in one fetus in group 3 and four fetuses (from 2 litters) in group 4, is also occasionally seen in control rat fetuses of this strain. The increased incidence in group 4 noted could well be due the low number of fetuses examined in this pilot study.

Examination of the stage of skeletal development revealed a slightly delayed ossification of limb structures in group 4 and to a lesser extent group 3. Also in group 4. a slight delay in ossification of bones of the skull was noted.

Furthermore the incidence of rudimentary supernumerary ribs was increased in group 4.
Visceral malformations:
no effects observed
Description (incidence and severity):
The frequency and type of common abnormal findings (mainly comprising elongated thymus, small additional liver lobes in the median cleft, median liver lobe with displaced cleft and left- sided umbilical artery) was similar in all groups.

There was no indication for any test item-related effects.
Other effects:
no effects observed
Description (incidence and severity):
During cartilage examination only one fetus with an abnormal cartilage finding (abnormally shaped cartilaginous parts of thoracic vertebral bodies 12 and 13) was noted in group 4.
This isolated finding was considered to be unrelated to treatment with the test item.

Examination of fetal cartilages for the stage of development gave no indication for any test item-related effects.
When calculated on an individual basis, two statistically significant differences at level 1% being due to a higher stage of development of cartilaginous sternebrae were noted in both groups 2 and 4. NO differences in the stage of development were noted in group 3.

When calculated on a litter basis, no significant differences in the stage of development of cartilages between groups treated with the test item and vehicle controls were noted.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
skeletal malformations

Fetal abnormalities

Abnormalities:
effects observed, non-treatment-related
Localisation:
skeletal: skull
skeletal: supernumerary rib

Overall developmental toxicity

Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
In order to detect effects on embryonic and fetal development in pregnant rats, Esacure KIP 150 was administered orally by gavage once daily from day 6 through to day 20 post coitum at dose levels of 10, 100 and 250 mg/kg body weight/day.
In females treatment with the test item caused increased liver weights at 100 and 250 mg/kg/day, and clearly reduced food consumption at 250 mg/kg/day.

At 250 mg/kg body weight/day an increased incidence of skeletal variations was noted. Also the stage of skeletal development was slightly decreased at 100 and 250 mg/kg body weight/day.

Due to the low number of litters examined in this pilot study it could not finally be concluded, whether these findings were incidental or caused by treatment with the test item.

Based on these data the following dose levels are considered appropriate for a consecutive main study: 10, 50 and a range between 100 and 250 mg/kg body weight/day, choosing 250 mg/kg body weight/day as a conservative approach, while 10 mg/kg body weight/day is expected to be the NOAEL.
Executive summary:

Fetal development when administered orally by gavage once daily to mated female rats from day 6 through to day 20 post coitum, inclusive.

Each group consisted of 5 mated female rats. Esacure KIP 150 was administered once daily at dose levels of:

Group 1: 0 mg/kg body weight/day (vehicle control)

Group 2: 10 mg/kg body weight/day

Group 3: 100 mg/kg body weight/day

Group 4: 250 mg/kg body weight/day

A standard dose volume of 5 ml/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (corn oil).

All females were sacrificed on day 21 post coitum and the fetuses were removed by Caesarean section. Examination of dams and fetuses was performed in accordance with international recommendations.

The following results were obtained:

MATERNAL DATA

General Tolerability

No mortalities or clinical symptoms were noted in any female throughout the study.

Mean liver weights were slightly but dose-dependently increased at 100 and 250 mg/kg/day (17.6 and 17.9 9 compared to 15.8 g in vehicle controls). This increase was possibly test item-related since the liver is a known target organ of the test item.

Food Consumption and Body Weights

Mean food consumption during the treatment period was clearly reduced at 250 mg/kg/day (-10.6% compared to vehicle controls). Food consumption at 10 and 100 mg/kg/day was not affected by treatment with the test item.

Mean body weight gain was not affected by treatment with the test item at any dosage employed.

Reproduction Data

No differences that were considered to be test item related were noted among the relevant mean reproduction data (post-implantation loss, number of fetuses per dam).

FETAL DATA

All results obtained are based on a low number of fetuses due to the pilot design of the study.

No test item-related effects on fetal body weights or fetal sex ratios were noted.

During external and visceral examination of fetuses no abnormalities that were considered to be attributable to treatment with the test item were noted.

At 250 and 100 mg/kg/day fusion of two of the components of the zygomatic arch were noted in four and one fetuses, respectively. Although this finding is occasionally noted in control rat fetuses of this strain. it cannot be finally concluded whether the increased incidence noted in this study is incidental or test item-related.

Examination of the stage of skeletal development revealed a slightly delayed ossification of limb structures at 250 mg/kg/day and to an even lesser extent at 100 mg/kg/day. Also at 250 mg/kg/day, a slight delay in ossification of bones of the skull was noted. Furthermore the incidence of rudimentary supernumerary ribs was increased at 250 mg/kg/day.

During cartilage examination no test item related findings were noted.