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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
Study period:
22nd October to 30th December 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
CAPA 3050
IUPAC Name:
CAPA 3050
Constituent 2
Chemical structure
Reference substance name:
ε-Caprolactone, oligomeric reaction products with propylidynetrimethanol
EC Number:
500-099-5
EC Name:
ε-Caprolactone, oligomeric reaction products with propylidynetrimethanol
Cas Number:
37625-56-2
Molecular formula:
(C6H14O3)x.C6H10O2 (x=0-6)
IUPAC Name:
ε-Caprolactone, oligomeric reaction products with propylidynetrimethanol
Constituent 3
Reference substance name:
2-Oxepanone, polymer with 2-ethyl-2-(hydroxymethyl)-1, 3-propanediol
IUPAC Name:
2-Oxepanone, polymer with 2-ethyl-2-(hydroxymethyl)-1, 3-propanediol
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): CAPA 3050
- Substance type: Oligomer
- Physical state: Liquid
- Analytical purity: 100%
- Lot/batch No.: WCB000999
- Expiration date of the lot/batch: 23th August 2015
- Stability under test conditions: Stable for the duration of the study
- Storage condition of test material: Ambient
Specific details on test material used for the study:
Identification: Capa 3050
Alternative Name: 2-Oxepanone, polymer with 2-ethyl-2-(hydroxymethyl)-1,
3-propanediol
CAS Number: 37625-56-2
EC Number: 500-099-5
Batch (Lot) No.: WCB000999
Receipt Date: 31 March 2014
Expiration Date: 26 August 2015
Physical Description: Liquid
Purity: 100 %
Storage Conditions: Ambient
Supplier: Perstorp

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
Source: Charles River, Margate, Kent, UK
Age at study initiation: 9 weeks
Weight at study initiation: 200-262 g
Fasting period before study: not applicable
Housing: Individual in plastic cages with solid bottoms and bedding material
Diet: ad libitum
Water: ad libitum
Acclimation period: 3-5 days

ENVIRONMENTAL CONDITIONS
Temperature (°C): 19-23
Humidity (%): 40-85
Air changes (per hr): 10 (minimum)
Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES
From: 24th October 2014
To: 12th November 2014

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% hydroxypropylmethylcellulose, 0.1% Tween 80 in water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The control item, 0.5% HPMC, 0.1% Tween 80 in Milli-Q water was prepared weekly, stored at 2-8°C, and dispensed daily. The prepared control item was stirred for at least 30 minutes before dosing and continuously during dosing.Test item dosing formulations were prepared based on a method established at the Test Facility at appropriate concentrations to meet dosage level requirements. The required amount of test item was weighed into a pre-labelled container according to instructions from the formulation computerised system (Dispense 8). The appropriate amount of the vehicle was added to the container and the formulation was mixed by means of magnetic stirring and high shear mixing until a homogenous formulation was obtained. The dosing formulations were prepared weekly, stored at 2-8°C, and dispensed daily. The dosing formulations were stirred for at least 30 minutes before dosing and continuously during dosing. Details of the preparation and dispensing of the test item have been retained in the study records.VEHICLE- Justification for use and choice of vehicle (if other than water): the vehicle was water-based, with the addition of 0.5% HPMC and 0.1% Tween 80 to ensure homogeneity of the dosing solution- Concentration in vehicle: 0, 10, 30, 100 mg/mL- Amount of vehicle (if gavage): 10 mL/kg bw (constant volume)Milli-Q water (batches MILLIQ297, MILLIQ304 and MILLIQ311; expiry dates 31 Oct 2014, 07 Nov 2014 and 14 Nov 2014 respectively), Methocel E4M Premium HPMC (batch DT357129, expiry date 17 Aug 2016) and Polysorbate 80 Ph Eur (batches BCBK8291V and BCBN2111V; expiry dates 01 Nov 2014 and 31 Mar 2016) were used during control item preparation on this study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulation analysis on this study was performed using a validated analytical procedure (Charles River Study 432576). Dosing formulation samples were analysed by UPLC with UV detection for the determination of CAPA 3050. Duplicate top, bottom and middle samples were taken from each formulation pepared for Weeks 1 and 2. Triplicate back-up samples were also taken for the verification of results out of specification.
Details on mating procedure:
Female rats were provided time-mated and were received in the testing facility on Day 1-3 of gestation.
Duration of treatment / exposure:
The test and control items were administered to the appropriate animals by once daily oral gavage from Days 6-19 of gestation. The dose for each animal was based on the most recent body weight measurement.
Frequency of treatment:
Daily (Day 6-19 of gestation)
Duration of test:
Rats were terminated on Day 20 of gestation
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
24 time-mated females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected in agreement with the Sponsor following review of data from a preliminary prenatal developmental toxicity study using this test item (Charles River Study No. 496926). In that study, there was no adverse maternal toxicity at dose levels up to 1000 mg/kg bw/d however; a reduction in foetal weight of approximately 10% was noted at 1000 mg/kg bw/d. Therefore, 1000 mg/kg bw/d was chosen as the high dose level in this study to further explore toxicity.- Rationale for animal assignment: On arrival from the suppliers, the animals were allocated to cages on racks. Animals were allocated by the use of randomly sequenced numbers, in such a way that each full rack of cages contained similar numbers of representatives from all groups. It was ensured that females inseminated by the same male did not appear in the same group. Two extra females were assigned to a separate group. The disposition of all animals was documented in the study records.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS:
Time schedule: All animals were checked early morning and as late as possible each day for viability.

DETAILED CLINICAL OBSERVATIONS:
All animals were examined daily from the start of dosing; animals were removed from the cage for examination

BODY WEIGHT:
Time schedule for examinations: Pre-test – once (Day 4 of gestation); dosing period – daily on days 6 – 20 of gestation

FOOD CONSUMPTION:
Daily from Day 4 of gestation (first measured quantity given on Day 3 of gestation). Food consumption was quantitatively measured for individual animals

WATER CONSUMPTION:
Time schedule for examinations: Regularly during the study. Water consumption was monitored by visual inspection of the water bottles. It was considered that there were no intergroup differences therefore water consumption was not assessed quantitatively.

POST-MORTEM EXAMINATIONS:
Sacrifice on gestation Day 20- Organs examined: All adult animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.:
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: YesExaminations included:- Gravid uterus weight: Yes. The gravid uterus was weighed (with the exception of Animal 27, group 2, 100 mg/kg bw/d, which was not weighed in error.- Number of corpora lutea: Yes- Number of implantations: Yes- Number of early resorptions: Yes- Number of late resorptions: YesEach implant was classified as being live, or a dead fetus (dead full term foetus that showed no sign of maceration), or a late embryonic death (macerated tissue identifiable as an embryo or foetus, with recognizable external features such as tail, limbs, mouth and nares present; attached to distinct identifiable placentae), or an early embryonic death (discrete, formless, discoloured tissue mass attached to the internal uterine wall; may have been of varying size).
Fetal examinations:
- External examinations: all per litter
Soft tissue examinations: half per litter
Skeletal examinations: Yes: half per litter
Statistics:
In order to assist interpretation, tests were applied to determine the statistical significance of observed differences between Control and groups receiving test item. Unless otherwise stated, all statistical tests were two-sided and performed at the 5% significance level using in house software. Pairwise comparisons were only performed against the control group. Body weight and food consumption data was analysed for homogeneity of variance using the ‘F-Max' test. If the group variances appeared homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher’s F protected LSD method via Student's t test i.e. pairwise comparisons were made only if the overall F-test was significant. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remained heterogeneous, then a Kruskal-Wallis nonparametric ANOVA was used and pairwise comparisons were made using chi squared protection (via z tests, the non-parametric equivalent of Student's t test).
Indices:
Not relevant to the study type
Historical control data:
Not reported; not required for interpretation of any study findings

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
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
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
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Details on maternal toxic effects:
There were no premature deaths during the study. There were no clinical observations which were considered to be related to treatment with Capa 3050. All clinical observations were considered to be incidental background findings commonly observed in this species at Charles River, Edinburgh. Group mean body weights were similar between all groups throughout the study. Group mean food consumption was similar between all groups throughout the study. It was noted that there were occasional low consumption values in individual animals across all groups; however, these did not follow a treatment related pattern and were noted to be transient. These were therefore considered to be incidental and unrelated to treatment with Capa 3050. There were no gross necropsy findings which were considered to be related to treatment with Capa 3050. All findings were considered to be incidental background findings commonly observed in this species at Charles River, Edinburgh.

Effect levels (maternal animals)

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

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
A slightly lower mean litter mean foetal weight was seen at 1000 mg/kg bw/d but was considered to be incidental as it was associated with a slightly higher litter size and weight in this group.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
effects observed, non-treatment-related
Description (incidence and severity):
Slightly higher litter size and weight in this group were observed at 1000 mg/kg bw/d but are not related to treatment.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/d, there were increased incidences of cervical remnant of thymus, incompletely ossified thoracic vertebral centrum, retarded sternebrae; and minimally kinked ribs. These findings could indicate a delay in foetal development; however, all incidences were within the historical control ranges at Charles River Edinburgh, and for cervical remnant of thymus, incompletely ossified thoracic vertebral centrum and retarded sternebrae, the increases were minor and generally did not correlate in individual foetuses and/or litters. Findings are often associated with low foetal weight and, as the foetuses with these findings were the smallest within their litters, it was considered that these findings were unlikely to be treatment-related. Additionally, kinked ribs are known to resolve as the foetus develops, therefore, this finding is considered not to be detrimental to the foetus and it is considered that the other findings would also likely resolve. All findings were, therefore, considered unlikely to be adverse.
Details on embryotoxic / teratogenic effects:
Pregnancy performance was similar between all groups. Slight intergroup variations were considered to be incidental and too small to be attributed to treatment with Capa 3050. At 1000 mg/kg bw/d, slightly increased incidences of cervical remnant of thymus, thoracic vertebral centrum incompletely ossified and retarded sternebrae were noted when compared to controls and other groups. In one foetus at 300 and 1000 mg/kg bw/d, bent scapula with minimally shortened humerus was noted, which was also associated with minimally kinked ribs in these foetuses. The type and distribution of foetal abnormalities and skeletal ossification parameters at 100 mg/kg bw/d was similar to controls.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

No unscheduled deaths occurred and no signs of toxicity were observed during the study period. Group mean bodyweights and food consumption were unaffected by treatment. Gross necropsy of dams did not reveal any treatment-related findings. Litter parameters were comparable in all groups; slight variations were seen between groups but were not considered to be an effect of treatment. Mean foetal weight was slightly (~5.5%) lower at 1000 mg/kg bw/d; mean foetal weight at 100 and 300 mg/kg bw/d was comparable to controls. The slightly lower mean foetal weight at 1000 mg/kg bw/d reflects to a certain extent the higher litter size in this group (14.3) compared to controls (13.8); calculation of mean total litter weight does not reveal any dose-response relationship.

Maternal findings and litter parameters

Dose level (mg/kg bw/d)

0

100

300

1000

Terminal bodyweight (g)

383

374

384

376

Weight gain (g)

129

123

125

121

Mated (#)

24

24

24

24

Pregnant (#)

24

24

23

24

Corpora lutea (#)

14.3

14.0

13.9

14.8

Litter size (#)

13.9

13.6

13.4

14.3

Live implants (#)

13.1

12.0

12.8

13.8

Dead implants (#)

0.8

1.6

0.6

0.5

Early embryonic deaths (#)

0.7

1.4

0.6

0.5

Late embryonic deaths (#)

0.1

0.2

-

-

Foetal weight (g)

4.00

4.07

3.94

3.78

Litter weight (g)

55.6

55.4

52.8

54.1

 

The incidence and type of malformations did not reveal any relationship to treatment with CAPA 3050. A slightly increased incidence of cervical remnant of thymus was noted in foetuses from the highest dose level; this contributed to a slightly higher incidence of total visceral variations in this group. The incidences of other visceral variations were comparable in all groups. The total number of foetal skeletal variations was comparable in all groups. At 1000 mg/kg bw/d, slightly increased incidences of thoracic vertebral centrum incompletely ossified and retarded sternebrae were noted when compared to Controls and other groups. The type and distribution of foetal abnormalities and skeletal ossification parameters at 100 mg/kg bw/d was similar to Controls.

Foetal findings

Dose level (mg/kg bw/d)

0

100

300

1000

Total malformations (#)

-

1 (1)

2 (2)

1 (1)

Total visceral variations (#)

19 (11)

21 (14)

11 (9)

26 (13)

Cervical remnant of thymus (#)

1 (1)

3 (2)

2 (2)

7 (5)

Total skeletal variations (#)

22 (15)

19 (14)

21 (13)

24 (14)

Vestigial supernumerary rib(s) on 1st lumbar vertebra (#)

9 (7)

18 (7)

5 (5)

22 (10)

14 complete rib(s) (#)

-

-

-

1 (1)

Incomplete ossification: thoracic vertebral centra

16 (11)

12 (11)

11 (9)

22 (11)

Retarded sternebrae: 0 (#)

75 (23)

78 (22)

60 (17)

41 (17)

Retarded sternebrae: 1 (#)

55 (23)

52 (19

57 (20)

67 (22)

Retarded sternebrae: 2 (#)

26 (15)

13 (9)

24 (12)

53 (20)

Retarded sternebrae: >2 (#)

3 (3)

2 (2)

6 (5)

6 (4)

foetal incidence (litter incidence)

Applicant's summary and conclusion

Conclusions:
No evidence of teratogenicity was seen in this study. Based on the results of this study, it was concluded that 1000 mg/kg bw/d was the maternal no-observed-effect level (NOEL) and the foetal no-observed-adverse-effect-level (NOAEL).
Executive summary:

The objective of this study was to determine the potential toxicity of CAPA 3050 when administered during the period of organogenesis to pregnant rats. Groups of 24 time-mated female Sprague-Dawley rats were gavaged with the test material (in 0.5% aqueous hydroxypropylmethylcellulose, 0.1% Tween 80) at dose levels of 0, 100, 300 and 1000 mg/kg bw/d on Days 6-19 of gestation, where the day of detection of mating was designated Day 0. Animals were regularly monitored for clinical signs of toxicity, body weights and food consumption and were killed on Day 20 of gestation for examination of pregnancies and embryofoetal development. Dosing with CAPA 3050 at dose levels up to 1000 mg/kg bw/d was not associated with any maternal treatment-related clinical observations, body weight or food consumption effects or gross necropsy findings. Pregnancy performance was similar between all groups. A slightly lower mean litter mean foetal weight was seen at 1000 mg/kg bw/d but was considered to be incidental as it was associated with a slightly higher litter size and weight in this group. At 1000 mg/kg bw/d, there were increased incidences of cervical remnant of thymus, incompletely ossified thoracic vertebral centrum, retarded sternebrae; and minimally kinked ribs. These findings could indicate a delay in foetal development; however, all incidences were within the historical control ranges at Charles River Edinburgh, and for cervical remnant of thymus, incompletely ossified thoracic vertebral centrum and retarded sternebrae, the increases were minor and generally did not correlate in individual foetuses and/or litters. Findings are often associated with low foetal weight and, as the foetuses with these findings were the smallest within their litters, it was considered that these findings were unlikely to be treatment-related. Additionally, kinked ribs are known to resolve as the foetus develops, therefore, this finding is considered not to be detrimental to the foetus and it is considered that the other findings would also likely resolve. All findings were, therefore, considered unlikely to be adverse. Based on the results of this study, it was concluded that 1000 mg/kg bw/d was the maternal NOEL and the developmental NOAEL.