Confidential

Safety conclusion: No evidence of increased calcium excretion or hypercalcemia was

C.8 Proposed ADI, Margin of Safety, and Conclusion on Safe Use

Calcifediol is a metabolite of vitamin D3 which is formed in the liver upon oral ingestion of
vitamin D3 (from food or from supplement) or upon UV-light induced synthesis of vitamin D3
in skin (Christakos et al., 2016). Vitamin D3 is essential for humans. When requirements are
not met serious health problems such as rickets in infants or osteoporosis in adults can occur.
Indeed, aspects of the Western lifestyle have raised concerns about inadequate exposure to
vitamin D3 (Krzyscin et al., 2016).
Animals and humans are exposed to vitamin D3 and consequently also to calcifediol due to
UV-light exposure. The synthesis of vitamin D3 from UV-light and consequent vitamin D3
status was recently re-investigated in humans and food-producing animals. In a very recent
article, a daily supplementation with 50 µg vitamin D3 throughout the entire year was
estimated to achieve the same exposure as compared to solar vitamin D3 synthesis in
ancestral humans (see section C.1.1 of this Notice.).
Calcifediol is the major metabolite of vitamin D3 in blood with a relatively long half-life and
is therefore used as a biomarker for vitamin D3 status.
To conclude, calcifediol is an endogenous substance for animals and human. It is therefore a
known substance to human beings and present in food. See sections C.1.1 and C.2 of this
notice.
Calcifediol administered in corn oil is of moderate acute oral toxicity with an LD50 above
200 mg/kg bw (Weber & Arcelin 2004). Calcifediol is neither a skin nor an eye irritant (Remus
& Eurlings 2016a, 2016b). Calcifediol can penetrate through the skin (see section C.5.2 of this
notice)
Calcifediol showed no potential for mutagenicity in an Ames Test (Woehrle & Sokolowski
2013) and in an MLA Test (Remus & Verspeek-Rip 2016c). Neither in vitro nor in vivo
chromosome damage was noted: No structural chromosomal aberrations were noted in vitro in
human lymphocytes (Weber & Schulz 2005), nor micronuclei in the bone marrow of rats
(Remus & Verbaan 2016e, see section C.5.4.3 of this Notice).
Repeated dose toxicity testing in laboratory rat indicated that 14-day treatment with
calcifediol in corn oil via gavage does not cause increased calcium or phosphorus
concentration or tissue damage in the kidney (calcification) up to 46 nmol calcifediol /d
(approximately equivalent to 170 µg/kg bw/d, Shepard & DeLuca 1980). In a recent OECD 408
compliant 90-day study, calcifediol was administered via dietary admix up to dose levels of
180 µg/kg bw/d. No adverse effects were noted in this study and the NOAEL was 180 µg

NDIN-Hy-D – DSM Nutritional Products 56

calcifediol/kg bw/d. Interestingly, the same vitamin D3 dose level produced significant
toxicity in rats (Thiel et al. 2007). See section C.4.4 of this Notice.
The potential of reprotoxic/developmental effects was investigated using human data on
vitamin D3 because vitamin D3 is the precursor of calcifediol. Indeed, calcifediol is the major
form of vitamin D3 in blood and is used as the marker to determine vitamin D3 status. In
various human studies using vitamin D3 demonstrated that vitamin D3 and consequently also
its metabolite calcifediol do not harm the unborn progeny nor infants when supplemented
with vitamin D3 up to and including the Upper Level of 100 µg/d for adults set forth by IOM
(2011) and the European Food Safety Authority (EFSA 2012). Furthermore, pregnant female
patients suffering from hypoparathyroidism receiving much higher doses delivered healthy
infants. Patients with hypo-parathyroidsm require high vitamin D3 and/or calcium treatment
and therefore these women were treated throughout pregnancy with dose levels exceeding
the Upper Level. See section C.4.5 of this Notice.
Calcifediol was tested in various human studies in healthy adults but also in sub-populations
considered to be susceptible like infants, elderly or non-healthy subjects, Briefly, treatment
in a clinical setting ranged from 5 to 120 µg/d and from single dose up to 4 years of treatment
See section C.7 of this Notice. Based on this data, adverse events or serious adverse events
related to calcifediol in any clinical study were not observed.
Barger-Lux et al., 1998 administered calcifediol without any signs of adversity like
hypercalcemia at dose levels up to 50 µg/d for 4 weeks. In the DSM-owned or sponsored trials
up to 20 µg/d were tested up to 10 and 15 weeks (Cashman et al., 2012, Bischoff-Ferrari et
al., 2012) and up to 6 months (Kunz et al., 2016, Wittwer 2015). In addition, Crilly et al.
(1982) administered 25 µg/d for 6 months.
The efficacy of vitamin D3 and calcifediol has been compared. Efficacy in this context is the
increase of 25-hydroxy vitamin D3 serum/plasma levels from baseline levels upon ingestion of
the either vitamin D3 or calcifediol. Those human studies, directly comparing calcifediol and
vitamin D3 were evaluated See section C.5 in this Notice. The six clinical trials show that per
microgram dose calcifediol is more effective than vitamin D3 to elevate 25(OH)D in serum.
Across the studies, the mean increase of 25-hydroxy vitamin D3 was 1.7  0.5 nmol/L, ranging
from 1.0 to 2.3 nmol/L per microgram vitamin D3, and 5.1  1.6 nmol/L, ranging from 2.4 to
9.0 nmol/L per microgram calcifediol. In other words, calcifediol was on average about 3-
times (range 1.5-5) more effective in raising serum 25-hydroxy vitamin D3, conversely 3 times
more vitamin D3 is needed to achieve the same response as calcifediol.
Based on the presented DSM-proprietary data and published literature on calcifediol and in
consideration that calcifediol is an endogenous substance and a metabolite of vitamin D3 we
propose and ADI of 33 µg/d for adult persons. This is based on the following consideration:
The Upper Level of vitamin D3 for adults is 100 µg/d as set forth by IOM and EFSA. We have
shown based on studies which compare vitamin D3 and calcifediol in their ability to increase
calcifediol plasma/serum level per microgram administered substance. This comparison
revealed calcifediol is 3 times more effective in increasing calcifediol serum concentration as

NDIN-Hy-D – DSM Nutritional Products 57

compared to vitamin D3. As a conservative measure it is proposed to divide the Upper Level
of vitamin D3 by a factor of 3 which results in a proposed ADI of 33 µg/d for adult persons.
Indeed this proposed ADI of 33 µg/d is supported by human clinical studies in adult persons
including elderly and unhealthy subjects. Up to 50 µg/d were safely ingested for up to 4
weeks (Barger-Lux et al., 1998), and 20 µg/d could be safely administered for up to 6 months
(eg. Kunz et al. 2016). See section C.7.2 in this Notice.
In conclusion, an ADI of 33 µg/d is proposed.
This proposed ADI is >3 times the proposed dose, 10 µg/d. We therefore believe that a
sufficiently high margin of safety exists.

NDIN-Hy-D – DSM Nutritional Products 58