Registration Dossier

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 publication which meets basic scientific principles.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2000

Materials and methods

Principles of method if other than guideline:
Developmental toxicity studies in cynomolgus monkeys (Macaca fascicularis)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Test substance: Vitamin A palmitate (retinyl palmitate) (CAS No. 79-81-2), Arovit, 100000 IU per 2ml ampule
Coded: Ro 01-5852/000,
Supplier: Hoffmann-LaRoche Ltd, Basel (Switzerland);
Purity: 99.9%
Storage: 4°C, light protected

Test animals

Species:
monkey
Strain:
Macaca fascicularis
Details on test animals and environmental conditions:
TESTING FACILITIES
Studies were performed in two testing facilities, i.e. California Regional Primate Research Center (CRPRC), Davies, California and Shin Nippon Biomedical Laboratories Ltd (SNBL), Kagoshima, Japan.

TEST ANIMALS
- Weight at study initiation: 3-5 kg (CRPRC), 2-5 kg (SNBL).
- Housing: individually in aluminum (CRPRC) or stainless steel (SNBL) cages.
- Diet:
CRPRC: Purina Monkey Chow (25% protein) twice daily with fruit supplementation during the treatment period. Estimated daily vitamin A intake via food consumption was 1225 IU/kg body weight (140 g of food containing 35 IU/g vitamin A; average monkey weight 4.0 kg).
SNBL: Solid diet (110 g daily of feed containing 24 IU/g vitamin A, Harlan Teklad, Harlan Sprague–Dawley, Inc.) was provided. The estimated
daily vitamin A intake via food consumption was 750 IU/kg (assuming an average monkey weight of 3.5 kg).
- Water: ad libitum
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 (CRPRC), 26+/-2 (SNBL).
- Humidity (%): 60 (CRPRC), 50 +/-10 (SNBL).
- Air changes (per hr): 15 (SNBL).
- Photoperiod (hrs dark / hrs light): 12

Administration / exposure

Route of administration:
other: nasogatric intubation
Vehicle:
other: physiol. saline (SNBL), water (CRPRC)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Appropriate dosing volumes were calculated according to each animal’s most recent weight and
then administered to unfasted animals by nasogastric intubation under dimmed lighting conditions.
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
CRPRC:
2-h period mating with a fertile male on two alternate days during midcycle (between cycle days 10 and 14). The second (last) day of positive mating,
indicated by the presence of sperm in a vaginal smear, was considered gestational day (GD) 0. Pregnancy was confirmed by ultrasound on GD 12 to 16.
SNBL:
Mating for 3 days during midcycle (menstrual cycle days 12, 13, and 14); the median day of the mating period was designated as day 0 of pregnancy. Pregnancy. Confirmation of pregnancy without anesthesia via ultrasonography on GD 14 or 15.
Duration of treatment / exposure:
GD 16 - GD 27
Frequency of treatment:
once daily (twice daily for GD 26 and 27 for 20000/ 40000 IU dose group (CRPRC))
Duration of test:
GD 100 +/- 2 (Days of hysterotomies)
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 7500, 20000, 40000, 80000 IU/kg/d
Basis:
actual ingested
Remarks:
Doses / Concentrations:
0, 4.1, 11, 22, 44 mg/kg bw/d retinyl palmitate
Basis:
actual ingested
No. of animals per sex per dose:
Only female animals used
Control: 15 animals
7500 IU/kg/d: 25 animals
20000 IU/kg/d: 26 animals
40000 IU/kg/d: 8 animals
80000 IU/kg/d: 29 animals
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
CRPRC:
All females were observed twice daily for adverse physical and behavioral (e.g. lethargy, depression) signs.
Maternal weights were taken within 7 days before the first dosing and then weekly thereafter until hysterotomies on GD 100 +/- 2.
Food consumption was measured in a subset of control and treated animals during the period of dosing.


SNBL:
All females were observed twice daily for general condition during the treatment period and body weights were measured at regular intervals during
gestation. Food intake was observed daily during the treatment period.


Ovaries and uterine content:
No data
Fetal examinations:
Fetuses were weighed, sexed, measured, and examined for external malformations before evisceration. Selected organ weights were taken, and the skeletons were evaluated after staining with Alizarin Red S. The hearts were examined as described previously. At SNBL, fetal growth and development was monitored at specific timepoints (GD 20, 25, 30, 37, 44, 58, 72, and 86) using ultrasound. At CRPRC, fetal growth and viability was monitored 2 times weekly until GD 40, then every 2 weeks until pregnancy termination on unsedated hand-held animals.
Statistics:
Univariate and multivariate analysis of variance (ANOVA and MANOVA, Super ANOVA version 1.11, Abacus Concepts, Inc., Berkeley, CA, USA, 1989–1991) were used to analyze group differences in mean fetal weights and measurements. Because there were no significant differences in mean fetal weights (MANOVA P-value = 0.5838) or fetal measurements (MANOVA P-value = 0.2519) between the control groups from the CRPRC and SNBL studies, all fetal parameters from both studies were combined for statistical evaluation. The x2 statistic (Statview, version 4.01, Abacus Concepts, Inc., Berkeley, CA, USA, 1992) was used to analyze group differences in the combined rates of abortion, malformation, and variations from both the CRPRC and SNBL studies.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY
40 000 - 80 000 IU/kg: Mild to severe signs of hypervitaminosis A in 60% (22/37) of animals during and/or immediately after the period of treatment. 18/29 animals in the 80 000 IU/kg group showed symptoms of hypervitaminosis A.
7500 - 20 000 IU/kg: No signs indicative of vitamin A toxicity
Clinical findings: erythema (eyelids, forehead, or whole face), skin rash (face, chest, arms, and/or back), epistaxis, rhinorrhea, swollen eyelids, gingivitis, lip lesions, and alopecia.

BODY WEIGHT AND WEIGHT GAIN
Slight and transient decreases in mean maternal body weights in treated/control groups during and immediately after treatment period, beeing considered to be related to the stress of animal handling. Steady weight gain during the post-treatment period in all groups,but delayed in treated animals compared to the controls, especially at 40 000 and 80 000 IU/kg. Mean maternal weights by the end of the observation period comparable between treated and control groups.

FOOD CONSUMPTION
80 000 IU/kg: Moderate to severe decreases in food intake in the majority of animals (26/29). Normal appetite was restored in all affected animals after the cessation of treatment.
7500 - 40 000 IU/kg: No changes in food consumption compared to controls during treatment peroid







Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
7 500 other: IU/ kg bw/ day
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOAEL
Effect level:
20 000 other: IU/kg bw/day
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
Incidence abortions:
Controls: 7% (1/15)
7500 IU/kg: 4% (1/25)
20 000 IU/kg: 19% (5/26);
40 000 IU/kg, 38% (3/8)
80 000 IU/kg, 66% (19/29).

The majority of losses occurred early in pregnancy:
GD 18-30 (62%)
GD 31-45 (28%);
GD 46-91 (10%*),
* 1/3 aborted fetuses (40 000 IU/kg, GD 91) exhibited multiple craniofacial (abnormal ears with absent external auditory canals, open eyes, hypertelorism, mandibular hypoplasia, gingival hyperplasia, hypoplastic zygomatic processes and tympanic rings, abnormal middle ear bones), heart (transposition of great vessels), and thymus (hypoplasia) malformations.


Fetal body and organ weights:
No significant dose-related differences.
Decreases in mean spleen weight in the 7500 IU/kg group compared to the control group (isolated statistical finding with no biologic significance).

Fetal measurements.
80 000 IU/kg:
Significant differences in foot length, biparietal diameter, occipitofrontal diameter and head circumference compared to controls.


Incidence of malformations**
Controls: 0% (0/14)
7500 IU/kg: 0% (0/24);
20 000 IU/kg: 5% (1/22);
40 000 IU/kg, 33% (2/6);
80 000 IU/kg, 45% (5/11).

** three nonviable fetuses available for examination in addition to the viable fetuses examined at GD 100 +/-2 were included

Type of malformations:
Craniofacial region:
External ear malformation in 6/8 malformed fetuses. Defects were hypoplasia derived from the first pharyngeal arch (helix, crus of helix) or second pharyngeal arch (auricular tubercle, crura of antihelix, lobule, and antitragus), absent external auditory canals and accessory skin tag rostral to the ear.
Mandible (hypoplastic), zygomatic processes (hypoplastic, or abnormally shaped), tympanic rings (hypoplastic), and middle ear bones (abnormal malleus, incus, and/or stapes).
Eyes (open, hypertelorism, exophthalmia), maxilla (hypoplastic), nose (hypoplastic), palate (arched or cleft), and greater wing of the sphenoid bone
(hypoplastic).

Aditional findings in single animals:
Thymic hypoplasia, transposition of the great vessels, gingival hyperplasia, and fused coccygeal vertebrae, ventricular septal defect (VSD).


Incidence of variations.
Controls: 36% (5/14).
7500 IU/kg: 38% (9/24)
20 000 IU/kg: 43% (9/21)
40 000 IU/kg, 83% (5/6)
80 000 IU/kg, 60% (6/10)

Type of variations:
Axial skeletal variations, i.e. differences in rib count or length, including extra, rudimentary, shortened, and absent ribs/rib pairs. These types of variations are considered to be within the normal range of biologic variation for cynomolgus macaques. Non significant but dose-related incidence of these variations reflects the teratogenic and embryolethal potential of the test substance.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Dose Group (IU/kg/d) No. Animals treated No. Abortions Timing of abortions (GD) No. Malformations
Control 15 1 (7%) 25-30 0/14
7500 25 1 (4%) 27 0/24
20000 26 5 (19%) 18-46 1/22
40000 8 3 (38%) 19-91 2/6
80000 29 19 (66%) 20-60 5/11

All of the animals in the 40 000 IU/kg group exhibiting signs of vitamin A toxicity (n = 4) had an adverse pregnancy outcome, i.e. abortion or malformed fetus. Of a total of 29 animals in the 80 000 IU/kg group, 18 experienced symptoms of hypervitaminosis A and 17 had an abortion or a malformed fetus. Of the remaining 11 animals in the 80 000 IU/kg group with no adverse clinical signs, there were five abortions, two malformed fetuses, and four normal fetuses.

Dose Group (IU/kg/d)

 

Animal No. with malformations

 

Specific Malformations

Control

 

-

 

7500

 

-

 

20000

 

1

Hypoplastic zygomatic processes and tympanic rings; abnormal middle ear bones (malleus, incus, and/or stapes)

40000

 

1 (Aborted GD 91 fetus)

Abnormal ears with absent external auditory canals, open eyes, hypertelorism, mandibular hypoplasia, hypoplastic zygomatic processes and tympanic rings; abnormal middle ear bones; transposition of great vessels, thymic hypoplasia, gingival hyperplasia, fused coccygeal vertebrae

2

Skin tag rostral to right ear; abnormal middle ear bones

80000

 

1

Abnormal ear, exophthalmia, abnormal zygomatic processes, hypoplastic tympanic rings, nose, and maxilla; abnormal middle ear bones; arched palate

2

Abnormal ear, mandibular hypoplasia, abnormal zygomatic process

3

Abnormal ears, mandibular hypoplasia, abnormal zygomatic process, hypoplastic tympanic rings; abnormal middle ear bones

4

Abnormal tympanic rings

5

Abnormal ears, mandibular hypoplasia, hypoplatsic zygomatic processes, tympanic rings, and sphenoid bone; abnormal middle ear bones; cleft palate; ventricular septal defect

Applicant's summary and conclusion