Registration Dossier

Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented publication which meets basic scientific principles.

Data source

Reference
Reference Type:
publication
Title:
Vitamin A teratogenicity and risk assessment in the macaque retinoid model.
Author:
Hendrickx AG et al.
Year:
2000
Bibliographic source:
Reprod Toxicol 14: 311-323

Materials and methods

Objective of study:
toxicokinetics
Principles of method if other than guideline:
Other: no data
GLP compliance:
not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Test substance: Vitamin A palmitate (retinyl palmitate)
retinyl palmitate (CAS No. 79-81-2), coded Ro 01-5852/000, supplier: Hoffmann-LaRoche Ltd, Basel (Switzerland); according to the authors, purity was 99.9%
Radiolabelling:
not specified

Test animals

Species:
monkey
Strain:
Macaca fascicularis
Sex:
female
Details on test animals and environmental conditions:
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: oral (nasogastric intubation)
Vehicle:
other: physiol. saline (SNBL), water (CRPRC)
Details on exposure:
Vitamin A (retinyl palmitate) was administered by nasogastric intubation  at  dose levels of 7500 IU/kg (4.1 mg/kg), 20000 IU/kg (11 mg/kg), 40000  IU/kg (22 mg/kg), and 80000 IU/kg (ca. 44 mg/kg) during early  pregnancy(i.e. between gestational days (gd) 16 and 27). 
The animals  given one or two doses during this period of gestation. Control monkeys  were administered the vehicle 
(physiological saline or distilled water,  depending on the performing institute). Hysterectomies were performed on  gd 100 +/- 2, and the fetuses were examined.
Duration and frequency of treatment / exposure:
Vitamin A (retinyl palmitate) was administered by nasogastric intubation  at 
dose levels of 7500 IU/kg (4.1 mg/kg), 20000 IU/kg (11 mg/kg), 40000  IU/kg 
(22 mg/kg), and 80000 IU/kg (ca. 44 mg/kg) during early  pregnancy (i.e. between
 gestational days (gd) 16 and 27). The animals  given one or two doses during 
this period of gestation. Control monkeys  were administered the vehicle 
(physiological saline or distilled water,  depending on the performing institute)
. Hysterectomies were performed on  gd 100 +/- 2, and the fetuses were examined.
Doses / concentrations
Remarks:
Doses / Concentrations:
Males: no males included
Females: approx. 4.1, 11, 22, 44 mg/kg (7500, 20000, 40000, 80000 IU/kg)
No. of animals per sex per dose:
Only female animals used
Experiment 1: n=5 controls, n=4 per dose group
Experiment 2: n=8
Experiment 3: n=6 per group
Control animals:
yes
Positive control:
no data
Details on study design:
These investigations were part of a teratogenicity study in cynomolgus  monkeys 
(Macaca fascicularis) . The studies were carried out at the California Regional Primate Research  
Center (CRPRC), Davis, California, and the Shin Nippon Biomedical  Laboratories, Ltd., Kagoshima, Japan.
Details on dosing and sampling:
For kinetic analysis, blood samples (1-2 ml plasma) were obtained under yellow light before dosing (0 h), and 1, 2, 3, 4.5, and 8 h after dosing  
(experiments 1 and 2) or at 0, 1, 2, 3, 4.5, 8, and 24 h before the next dose (Experiment 3).
Statistics:
The AUC for each treatment group was evaluated by descriptive statistics  
(mean, SD).

Results and discussion

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
Four  metabolites of  Vitamin A, the retinoic acids (RA) all-trans RA,  13-cis-RA, all-trans-4-oxo-RA, and 13-cis-4-oxo-RA 
were measured simultaneously using a validated reversed-phase HPLC/column switching  assay. 

Any other information on results incl. tables

Experiment 1:
-------------
Retinoid plasma concentrations indicated a dose-related increase in AUC  0-8 on 

the last day of sampling (gd 27) for the four metabolites  generated. Elevated 

pre-dose plasma concentrations of retinol and the RA  metabolites were observed 

in the one 20000-IU/kg-female that had a  malformed fetus.

Table: mean +/- SD AUC 0-8 values of retinoids in maternal plasma on gd 27
Retinoids:
R = retinol
RE = retinyl esters
AT-RA = all-trans-retinoic acid
13C-RA = 13-cis-retinoic acid
ATO-RA = all-trans-4-oxo-retinoic acid
13C-RA = 13-cis-4-oxo-retinoic acid

              control      20000 IU      40000 IU
retinoid       (n=5)         (n=4)         (n=4)
-----------------------------------------------------
R            7.8+/-1.5    17.4+/-5.6    17.4+/-2.7
RE           5.4+/-2.3     293+/-153     327+/-140
AT-RA       15.2+/-1.0    67.0+/-36.0    152+/-108
13C-RA      24.7+/-5.7     271+/-86.2    380+/-293
ATO-RA       5.9+/-4.1    46.6+/-15.6   61.8+/-41.5
13CO-RA     15.7+/-4.3     328+/-146     411+/-223

Experiment 2:
-------------
The AUC 0-8 for retinol and the four retinoic acid metabolites were  higher on 

gd 16 than on gd 27. The fetal outcome was related to kinetic  parameters. The 

plasma levels for retinol and the four metabolites were consistently higher on  

gd 16 in the six females that had an adverse pregnancy outcome (n= 5 abortions 

and n = 1 moderately malformed  fetus). In contrast, intermediate or low values 

for these parameters were  observed in the two females with a mildly  malformed 

fetus or with a  normal fetus, respectively.

AUC 0-8 of retinoids in maternal plasma

  80000 IU/kg)
N=8 GD16 GD27
R          Mean 28.9 15.4
  SD 11.8 10.7
RE Mean 322 353
  SD 181 539
AT-RA      Mean 6100 45.6
  SD 3610 37.8
13C-RA     Mean 1096 168
  SD 595 99.7
ATO-RA   Mean 1105 32
  SD 638 16.7
13CO-RA     Mean 619 170
  SD 309 111


Experiment 3:
-------------
There was a dose-related increase in AUC 0-24 of the four retinoic acid  

metabolites on gd 16. The AUC 0-24 values for these compounds were  consistently

 higher on gd 16 than on gd 27 in the 20000-IU/kg-group.  There were no 

comparable changes between the first and last day of  treatment in the 

7500-IU/kg-group. The AUC 0-24 of retinol in the two  treatment groups was only 

slightly higher than in the control group and  did not change with dose and time.

 In contrast, the AUC 0-24 of retinyl  esters was 30 times higher after Vitamin 

A treatment compared with  controls although there was no consistent pattern 

with dose or time.

AUC 0 -24 of retinoids in maternal plasma

  Group 1 (control) Group 2 (7500 IU/kg) Group 3 (20000 IU/kg)
N=6 GD16 GD27 GD16 GD27 GD16 GD27
R          Mean 31.1 34.8 45.8 45.9 45.0 31.4
  SD 7.2 9.8 8.5 9.4 6.0 8.7
RE Mean 1.37 2.6 55.5 71.1 88.2 47.1
  SD 0.8 0.8 40.4 48.5 66.1 27.4
AT-RA      Mean 60.3 65.0 265 238 1790 353
  SD 7.8 9.8 205 150 1717 390
13C-RA     Mean 47.1 46.1 239 286 893 399
  SD 14.9 11.1 92.5 60.1 597 227
ATO-RA   Mean 3.5 2.9 73.8 78.5 430 142
  SD 8.6 4.2 32.5 27.7 371 115
13CO-RA     Mean 9.2 17.6 193 274 765 427
  SD 13.1 9.6 113 139 567 284

Applicant's summary and conclusion