VITAMIN D FORTIFICATION OF EGGS ALONE AND IN COMBINATION 
WITH MILK IN WOMEN AGED 44–65 YEARS:  
FORTIFICATION MODEL AND ECONOMIC EVALUATION
Vid VIČIČ1, Ruža PANDEL MIKUŠ1, Saša KUGLER2, Ksenija GERŠAK3,4, Joško OSREDKAR5, Andreja KUKEC6*
1University of Ljubljana, Faculty of Health Sciences, Nursing Department, Zdravstvena pot 5, 1000 Ljubljana, Slovenia
2National Institute of Public Health, Trubarjeva 2, 1000 Ljubljana, Slovenia
3University of Ljubljana, Faculty of Medicine, Division of Obstetrics and 
Gynaecology, Šlajmerjeva 3, 1000 Ljubljana, Slovenia
4University Medical Centre Ljubljana, Clinic of Gynaecology and Obstetrics, Šlajmerjeva 3, 1000 Ljubljana, Slovenia
5University Medical Centre Ljubljana, Institute of Clinical Chemistry and 
Biochemistry, Njegoševa 4, 1000 Ljubljana, Slovenia 
6University of Ljubljana, Faculty of Medicine, Chair of Public Health, Zaloška cesta 4, 1000 Ljubljana, Slovenia
Received: Jan 19, 2022
Accepted: Nov 17, 2022
Original scientific article
*Corresponding author: Tel. + 386 1 543 75 44; E-mail: andreja.kukec@mf.uni-lj.si
10.2478/sjph-2023-0005 Zdr Varst. 2023;62(1):30-38
30
OBOGATITEV JAJC Z VITAMINOM D V KOMBINACIJI Z MLEKOM  
PRI ŽENSKAH, STARIH MED 44 IN 65 LET:  
MODEL OBOGATITVE IN EKONOMSKO VREDNOTENJE
© National Institute of Public Health, Slovenia. 
Vičič V, Pandel Mikuš R, Kugler S, Geršak K, Osredkar J, Kukec A. Vitamin D fortification of eggs alone and in combination with milk in women aged 44–65 years:  
fortification model and economic evaluation. Zdr Varst. 2023;62(1):30-38. doi: 10.2478/sjph-2023-0005.
ABSTRACT
Keywords: 
vitamin D, 25(OH)
D3, pre-menopausal 
women, post-
menopausal women, 
fortification, 
economic evaluation 
IZVLEČEK
Ključne besede: 
vitamin D, 25(OH)
D3, ženske v 
predmenopavzi, 
ženske po menopavzi, 
obogatitev živil, 
ekonomsko 
vrednotenje
Introduction: For almost nine decades, the fortification of foods with vitamin D has been proven effective in 
preventing rickets. This study aims to build and economically evaluate a fortification model based on egg 
biofortification and milk (including yoghurt) fortification. 
Methods: A cross-sectional study was carried out between 1. March and 31. May 2021. Three hundred and nineteen 
healthy women from the Central Slovenian region aged between 44 and 65 were recruited for the study, with 
176 participants included in the final analysis. For the fortification model calculations, the vitamin D contents of 
unenriched milk (including yoghurt) and eggs were replaced by enriched foods containing vitamin D. The economic 
evaluation was done using available drug and food supplement prices. Fortification costs were calculated using 
vitamin D prices provided by suppliers.
Results: Mean vitamin D intake from food was 2.19±1.34 µg/d. With fortification Model 1 (enriched eggs), it would 
be: 6.49±4.45 µg/d, and with Model 2 (enriched eggs and milk): 10.53±6.49 µg/d. Without fortification, none of the 
participants would reach a daily vitamin D intake >10 µg. With fortification Model 1 (egg fortification), 15.3% would 
reach >10 µg and with Model 2 (egg and milk fortification) 46.2% would reach >10 µg. The economic comparison of 
the annual cost of 10 µg vitamin D/d/person was EUR 6.17 for prescription drugs, EUR 6.37 for food supplements, 
EUR 0.09 for direct milk fortification and EUR 0.12 for egg biofortification with vitamin D.
Conclusions: Egg and milk (including yoghurt) fortification could cost-effectively increase vitamin D intake in the 
Slovenian population of women between 44 and 65 by almost five-fold, and could significantly lower the prevalence 
of vitamin D deficiency. Additional research and changes to legislation are needed before this can be introduced.
Uvod: Obogatitev živil z vitaminom D se je kot učinkovita pri preventivi rahitisa izkazala že pred skoraj 90 leti. 
Namen študije je zgraditi obogatitveni model, temelječ na bioobogatitvi kokošjih jajc preko krme, samostojno in 
v kombinaciji z mlekom (vključno z jogurtom). Strošek smo ekonomsko ovrednotili v primerjavi z individualnim 
dodajanjem vitamina D z zdravili ali prehranskimi dopolnili. 
Metode: V obdobju med 1. marcem in 31. majem 2021 smo izvedli presečno študijo, v katero je bilo vključenih 319 
žensk, starih med 44 in 65 let. Ob upoštevanju izločitvenih dejavnikov smo v končno analizo vključili 176 udeleženk. 
Za izračun obogatitvenega modela smo vsebnost vitamina D v neobogatenem mleku (vključno z jogurtom) in jajcih 
nadomestili z vsebnostjo v obogatenih živilih. Ekonomsko vrednotenje smo opravili na podlagi razpoložljivih cen 
zdravil, prehranskih dopolnil in cen surovin.
Rezultati: Povprečni vnos vitamina D s hrano je bil 2,19 ± 1,34 µg/d. Pri modelu obogatitve 1 bi bil vnos: 6,49 ± 4,45 
µg/d in pri modelu 2: 10,53 ± 6,49 µg/d. Brez obogatitve nobena udeleženka ne dosega dnevnega vnosa vitamina D 
> 10 µg. Vnos > 10 µg/d bi z modelom obogatitve 1 (obogatitev jajc) in modelom 2 (obogatitve jajc in mleka) doseglo 
15,3 % in 46,2 % udeleženk. Primerjava letnih stroškov dodajanja 20 µg vitamina D/osebo je bila 6,17 € za zdravila 
na recept, 6,37 € za prehransko dopolnilo, 0,09 € za neposredno obogatitev hrane in 0,12 € za obogatitev jajc z 
vitaminom D. ≥ 1 jajce/teden uživa 88,6 % udeleženk.
Zaključki: Obogatitev jajc in mleka (vključno z jogurtom) lahko stroškovno učinkovito poveča vnos vitamina 
D v slovenski populaciji žensk, starih med 44 in 65 let, za skoraj petkrat in lahko bistveno zmanjša prevalenco 
pomanjkanja vitamina D. Dodajanje vitamina D preko zdravil in prehranskih dopolnil je učinkovito na individualni 
ravni, v nasprotju z obogatitvijo živil pa ne doseže najranljivejših populacijskih skupin. Pred implementacijo bodo 
potrebne dodatne raziskave in prilagoditev zakonodaje.
1 INTRODUCTION
The main vitamin D source for humans is the synthesis 
of cholecalciferol (D3) from 7-dehydrocholesterol in skin 
exposed to UV rays. To a lesser extent, it is obtained 
from fatty fish and eggs in the form of cholecalciferol 
(D3) and from UV-exposed yeast and fungi in the 
form of ergosterol (D2). Animal products also contain 
25-hydroxycholecalciferol (25(OH)D3) (1). 
The interest in vitamin D has increased due to the 
association of vitamin D deficiency with increased risk 
of SARS-CoV-2 infection and disease severity (2). Vitamin 
D deficiency is not a new disease, nor is its prevention. 
Herodotus first described possible vitamin D deficiency 
and its aetiology in a lack of sun exposure in 525 BC 
(3). Beginning in the 17th century, industrialisation and 
increased coal use resulted in air pollution. Particles 
suspended in the air decreased sun exposure and 
contributed to the widespread development of rickets. In 
Germany, Schmorl (4), as cited in Hernigou et al. (3), noted 
that 96% of the children that died in 1906 had signs of 
rickets. In the 19th century, the first associations between 
sun exposure and rickets were made, followed by the 
discovery that cod liver oil cured and prevented rickets. 
In 1904 Buchholz reported that UV irradiation of human 
skin cured rickets (5). Steenbock discovered that milk and 
yeast could be fortified with vitamin D by irradiation with 
UV lamps (6). Hess and Windaus first demonstrated that 
cholecalciferol and ergocalciferol could be synthesised 
with cholesterol and ergosterol (7). Within 20 years, a 
wide variety of foods were fortified, from milk, margarine 
and breakfast cereals to bread and even beer. This led 
to the rapid eradication of rickets in developed nations 
(5). However, some errors in fortification and subsequent 
hypervitaminosis effectively led to the prohibition of 
mass vitamin D fortification in most countries in the 
1950s (8). Prior to EC Regulation 1925/2006 (9), voluntary 
fortification in many European countries was severely 
restricted or tightly controlled (10).
Only a few countries currently maintain mandatory 
fortification of milk (US, Canada: 1–2 µg/100 mL) and fat 
spreads (Canada: 15–30 µg/100 g) (11). There is mandatory 
fortification of bread in Jordan (12) and the voluntary 
fortification of milk (2 µg/100 mL) and fat spreads (20 
µg/100 g) in Finland (13). Instead, rickets prevention relies 
on rickets prophylaxis by vitamin D supplementation in 
infants (14). The fortification programme in Finland has 
proved to be particularly successful, as 71% of men and 55% 
of women reached a dietary vitamin D intake ≥10 μg/d (13). 
Various fortification possibilities have been suggested 
(15, 16): milk (13), flour (17), UV-irradiated yeast for 
the preparation of bread (18), UV-irradiated mushrooms 
(19), and the enrichment of eggs through feed (20). The 
appropriate fortification media should be consumed 
10.2478/sjph-2023-0005 Zdr Varst. 2023;62(1):30-38
31
regularly by most of the population, be affordable, and 
lead to only a minimal increase in production costs (16).
Hen eggs can be readily enriched with vitamin D (20–26), 
and cholecalciferol (D3) has been shown to be more 
effective than ergocalciferol (D2) (25). To achieve even 
higher vitamin D efficacy in eggs, the 25(OH)D3 form can 
be used in addition to cholecalciferol (22). This is an issue 
because most studies have only analysed cholecalciferol 
(20–26). Food composition data frequently undervalues 
vitamin D content, as it is commonly based only on 
cholecalciferol (D3) determination (1, 27). The reported 
efficacy of dietary 25(OH)D3 to increase serum 25(OH)
D is ~5-fold greater than that of cholecalciferol (24). 
Considering its higher potency, the contribution of 25(OH)
D3 intake from eggs may be even greater than that of 
cholecalciferol (D3) (1, 24).
Supplemental vitamin D and 25(OH)D3 for animal nutrition 
are commercially available (28). Some legal barriers to the 
biofortification of animal feed remain, as the maximum 
levels of added vitamin D in animal feed place a limit on 
higher levels of biofortification (29). 
Perimenopausal and postmenopausal women are at high risk 
of vitamin D deficiency and its public health consequences. 
Menopause and age-related changes in vitamin D and 
calcium metabolism increase the risk of vitamin D 
deficiency and secondary hyperparathyroidism (29, 30). 
Milk intake in Slovenia is low; therefore, milk fortification 
alone would not be an effective fortification medium (31). 
Consequently, this article aims to build a fortification 
model based on egg biofortification and the fortification of 
milk (including yoghurt). Additionally, it seeks to evaluate 
the costs compared to individual supplementation.
2 METHODS
2.1 Study design and participants
A cross-sectional study of the Health Interview Survey 
(HIS) type and a Clinical Health Examination Survey (HES) 
was conducted between 1. March and 31. May 2021. Three 
hundred and nineteen healthy women from the Central 
Slovenian region, aged between 44 and 65, were recruited 
for the study. 
Participants were initially recruited by healthcare workers 
during preventive health visits at Kamnik and Vrhnika 
health centres (Figure 1). Since recruitment was hindered 
by Covid-19 preventive measures, the snowball sampling 
method was successfully employed (32). This meant that 
after the participants were informed of their vitamin D 
status, either by post or email, they were asked to invite 
suitable peers to the study. They were also given a link 
to an online contact form through which new participants 
could join. 
10.2478/sjph-2023-0005 Zdr Varst. 2023;62(1):30-38
32
2.2 Inclusion and exclusion criteria
All willing women between the ages of 44 and 65 were 
included in the study. For the final analysis, the exclusion 
criteria, summarised according to (33) and supplemented, 
were: 1) confirmed osteoporosis; 2) current oestrogen 
therapy (prior oestrogen therapy was not an exclusion 
criterion if >3 months have elapsed since the last dose); 3) 
treatment with corticosteroids in the previous 12 months; 
4) treatment of hyperthyroidism or hypothyroidism in the 
previous 12 months; 5) any new or unregulated chronic 
disease (e.g. unregulated diabetes); 6) malignant diseases 
at any time in life; 7) hysterectomy was not an exclusion 
criterion; 8) health conditions associated with vitamin D 
deficiency (malabsorption, chronic inflammatory bowel 
disease, gastrointestinal resection, liver disease, acute 
gallbladder disease, chronic kidney disease grade 3 to 
5); 9) institutionalisation and poor physical mobility; 10) 
excessive vitamin D intake (serum 25(OH)D3 >250 nmol/L). 
2.3 Data collection 
After obtaining signed informed consent forms (ICF), a 
telephone survey was performed by trained registered 
nurses (RN) or nutritionists (MNutr.) using a purpose-made 
questionnaire. The questionnaire was tested in a pilot 
study carried out in 2019 (34). It contained a modified 
food frequency questionnaire (FFQ), body weight, height, 
health status, use of food supplements with vitamin D, 
food intake, menstruation/menopause, sun exposure 
status, and socio-economic and socio-demographic status. 
Blood samples were collected during regular working 
hours at the participating health centres and Ljubljana 
University Medical Centre between 1. March and 31. May 
2021. All samples were transported to a central laboratory 
at Ljubljana University Medical Centre, where they were 
analysed the same day or stored at -80ºC until analysis. 
Measurements were performed at the Clinical Institute of 
Clinical Chemistry and Biochemistry (Ljubljana University 
Medical Centre). In all participants, S-25(OH)D was 
measured by an Architect 25-OH D vitamin kit (Abbott 
Diagnostics, Lake Forest, USA).
2.4 Egg and milk fortification and economic analysis 
model 
There are many studies on the fortification of eggs with 
vitamin D; the best to date, containing data for both 
cholecalciferol (D3) and 25(OH)D3, is by Browning et al. 
(20). The models are made to be safe, cost-effective, 
simple and feasible. 
The input data for the model is: 1) assessment of vitamin 
D status by 25(OH)D3 determination; 2) assessment of food 
intake that will be used for vitamin D intake calculation 
and for the calculation of fortification; 3) food composition 
data; 4) method for achieving the biofortification of eggs 
(20); 5) vitamin D ingredient prices. 
Vitamin D status was assessed by serum 25(OH)D3 
determination. The data for food sources of vitamin D 
was assessed using an FFQ. Fish composition data was 
obtained from the Open Platform for Clinical Nutrition 
(OPEN) database. A mean of 10.01µg/100 g was used for 
fatty fish and a mean of 1.49 µg/100 g was used for lean 
fish (35); for eggs, 2.4 µg/1 egg (63.8 g) was used (20). 
In the fortification model, egg composition data from 
Browning et al. (20) was used. Eggs were produced using 
feed containing 250 µg/kg of cholecalciferol and 69 µg/kg 
of 25(OH)D3. The mean egg weight was 63.8 g and the feed 
conversion-to-egg ratio was 2.27±0.693) (kg feed/kg egg). 
For the production of one enriched egg, 144.82 g of feed 
was used. This amount contained 36.21 µg cholecalciferol 
and 9.99 µg of 25(OH)D3. Conversion from feed to eggs was 
14.32% for cholecalciferol and 13.54% for 25(OH)D3. There 
were no detrimental effects on animal health or production 
parameters (20). For the economic analysis, the prices of 
vitamin D3 and feed grade 25(OH)D3 were provided by 
DSM Nutritional Products. The milk and yoghurt can be 
fortified by direct addition during manufacture (16). Milk 
and yoghurt do not differ significantly in nutrient content 
and method of vitamin D addition.
We built three models: a no-fortification model, Model 
1 (only egg fortification used) and Model 2 (combination 
of egg fortification and milk and yoghurt fortification). 
Fortification model input data is presented in Table 1. For 
the fortification model calculations, vitamin D contents 
for unenriched milk, yoghurt and eggs were substituted 
with enriched food vitamin D content. All participants 
Figure 1. Flow chart of the study in healthy women from the 
Central Slovenian region aged between 44 and 65, 
conducted between 1. March and 31. May 2021.
10.2478/sjph-2023-0005 Zdr Varst. 2023;62(1):30-38
33
were included in the building of both fortification models 
even if they did not consume milk, yoghurt and/or eggs.
No fortification
Model 1
Model 2
Unenriched egg
Enriched eggs**
Enriched eggs**
Enriched milk and yoghurt
2.4 (80 IU)
11.98 (479 IU)
11.98 (479 IU)
3.00 µg (120 IU)/100 mL
0.27
1.35
1.35
1.07
5.18
5.18
3 µg/100 mL
Cholecalciferol 
µg/1 egg 
25(OH)D3
µg/1 egg
Vitamin D efficacy*
µg (IU)/1 egg
Table 1. Fortification model input data.
*Vitamin D efficacy to increase 25(OH)D levels includes cholecalciferol and 25(OH)D3 (20, 24)
**Source of vitamin D efficacy data: (20). 
The mean egg weight used was 63.8 g.
1µg vitamin D=40 IU
2.5 Statistical analysis
The variables observed in the analysis were: 1) serum 
25(OH)D3 vitamin D (dependent variable); 2) taking 
(frequency and quantity) of dietary supplements/
medicines with vitamin D; 3) survey questionnaire (eating 
habits – frequency and quantity, vitamin D content, sun 
exposure); and 4) body mass index (BMI).
Serum 25(OH)D3 levels were stratified using Endocrine 
Society cut-off values: Deficiency: <50 nmol/L, 
Insufficiency: 50–75 nmol/L and target concentration for 
optimal vitamin D effects 75–125 nmol/L (36, 37). 
The data has a normal distribution. The differences 
between users and non-users of supplements were 
examined using a two-sample t-test. The statistical 
analysis was conducted using MS Excel and SPSS, version 
27 (IBM, 2020).
2.6 Ethical approval
The study protocol was approved by the Slovenian 
National Medical Ethics Committee, identification no KME 
0120-68/2019/9 (approval letter ID 0120-68/2019/9, date 
of approval: 22. March 2019). The study was performed 
in compliance with the requirements of local authorities. 
All subjects signed a written informed consent form (ICF) 
before participating in the study. 
3 RESULTS
The population characteristics, vitamin D status, 
supplement use and food intake are presented in Table 2.
10.2478/sjph-2023-0005 Zdr Varst. 2023;62(1):30-38
34
Age
BMI
LABORATORY ANALYSIS
25(OH)D 
 
Education
 
MILK AND EGG INTAKE
Milk and yoghurt intake (mL/d)
Milk consumers (≥200 mL /week)
Egg intake (pcs/week
Egg consumers (≥1 egg/week)
VITAMIN D INTAKE AND SUPPLEMENTATION 
Mean food intake (µg/d)  
Supplement use (≥5 µg vitamin D /d) 
Mean supplemental intake (µg/d)
Mean intake of all sources (µg/d)
FORTIFICATION MODEL
Mean vitamin D intake all sources (µg/d)
54.25±4.83
25.47±4.18
54.63%
31.48%
8.33%
4.63%
0.93%
76.24±27.06
3.70%
6.48%
44.44%
45.37%
29.63% 
70.37%
154.26±171.29
79.63%
3.19±2.52
86.1%
2.24±1.41
100%
35.4±25.26
37.64±25.17
53.14±5.17
26.14±4.63
47.06%
33.82%
14.71%
4.41%
0.00%
50.82±19.55
16.18%
30.88%
41.18%
11.76%
36.76%
63.24%
103.33±137.03
73.53%
3.14±2.34
92.7%
2.11±1.22
0.00%
0±0.08
2.48±3.33
0.150
0.322
0.000
0.0400
0.895
0.532
0.000
0.000
53.83±4.99
25.74±4.36
51.70%
32.39%
10.80%
4.55%
0.57%
66.42±27.35
8.52%
15.91%
43.18%
32.39%
32.39% 
67.61%
 
134.58±160.46
72.16%
3.17±2.44
88.6%
2.19±1.34
61.36% 
21.72±26.24
24.06±26.19
year
kg/m²
18.5–24.9
25.0–29.9
30.0–34.9
35.0–39.9
over 40.0
nmol/L
<30
30–50 
50–75 
>75
Primary and 
secondary school
Higher education
Fortified  
eggs
(Model 1)
29.49±
26.56
Fortified  
eggs
(Model 1)
43.33±
25.33
Fortified  
eggs
(Model 1)
7.49±
4.22
Fortified eggs 
and milk
(Model 2)
33.52±
27.24
Fortified eggs 
and milk
(Model 2)
47.96±
25.40
Fortified eggs 
and milk
(Model 2)
10.59±
6.13
Category/
Unit
Variables All participants 
n=176
Users of 
supplements
n=108
Non-users of 
supplements
N=68
p– value
Table 2. Population characteristics, vitamin D status, supplementation and food intake in healthy women from the Central Slovenian 
region aged between 44 and 65, carried out between 1. March and 31. May 2021 (n=176).
All values are presented as Mean ±SD or %. T-test analysis was performed to compare variables among users and non-users of 
supplements. Values are presented as mean ±SD, 𝑝<0.05 was considered statistically significant (𝑝 values of significant variables are in
bold print).
Figure 2 shows the difference in serum 25(OH)
D3 concentrations between users and non-users of 
supplements. The 5 µg vitamin D cut-off was chosen 
because it is the lowest level found in multivitamin 
supplements.
3.1 Fortification model
Mean vitamin D intake from food without fortification 
was 2.19±1.34 µg/d. With fortification Model 1, it would 
be6.49±4.45 µg/d and with Model 2 it would be 10.53±6.49 
µg/d. Without fortification, none (0%) of the participants 
would reach a daily vitamin D intake >10 µg. With fortification 
Model 1 (egg fortification), 15.3% would reach >10 µg and 
with Model 2 (egg and milk fortification) 46.2% would reach 
>10 µg. Vitamin D supplementation was not included.
Figure 3 shows vitamin D intake without fortification, 
with fortified eggs, and fortified eggs in combination with 
fortified milk (including yoghurt). 
10.2478/sjph-2023-0005 Zdr Varst. 2023;62(1):30-38
35
Figure 2. The differences of serum 25(OH)D3 concentrations 
between users and non-users of supplements in 
healthy women from the Central Slovenian region 
aged between 44 and 65 were assessed between 1 
March and 31 May 2021 (n=176).
Figure 3.
Figure 4.
Vitamin D intake from food, without fortification 
with projection using fortified eggs and fortified eggs 
and milk (including yoghurt) in healthy women from 
the Central Slovenian region aged between 44 and 
65, was assessed in the period between 1 March and 
31 May 2021 (n=176). 
Comparison of the annual cost of 10 µg of vitamin 
D daily for Slovenians >1 year of age (n=2,050,916)
(38). (Key: MEUR – millions of Euros).
*25(OH)D3 analysis, physician visits and dispensing costs are not 
included. The cheapest drug was used.
**Cheapest food supplement per µg, cost with VAT.
***No additional cost is predicted. 
3.2 Economic analysis
Figure 4 presents the annual cost of fortification compared 
with vitamin D-containing prescription drugs and food 
supplements. The annual cost of 10 µg vitamin D/d/person 
is shown in Table 3. 
4 DISCUSSION
The results of our study indicate that egg biofortification 
in combination with milk and yoghurt fortification could 
lead to a 4.8-fold increase in vitamin D intake. In Slovenia, 
vitamin D drug prescriptions amounted to EUR 6,029,647 
in 2020 (39). Considering the low cost of fortification, 
even a slight decrease in dosage from fortified food would 
bring substantial savings.
Vitamin D intake and status in Slovenia were assessed in 
2017/2018 by Hribar et al. (40–42). The supplementation 
rate in our study was increased due to the Covid-19 
pandemic. Consequently, we report higher serum 25(OH)
D3. However, a study of a population of people aged 
between 18 and 74 that looked at the intake of the specific 
foods addressed in this study produced very similar 
findings. The intake of eggs in adults and older adults was 
between three and four eggs/week. The daily intake of 
milk and dairy products in was 169 g in older adults and 
174 g in adults (31, 40, 41). Consequently, their milk-based 
fortification model was ineffective because milk intake in 
the Slovenian population is low (41). 
The impact of Model 2 (eggs and milk) compared to milk 
and fat spread fortification in Finland would be lower 
but still substantial: fortification Model 2 would result in 
a mean vitamin D intake of 10.53±6.49 µg/d and 46.2% 
of women reaching vitamin D intake >10 µg/d, compared 
with the Finnish intake of 12 μg/d (95% CI: 11, 12 μg/d) and 
58% of women reaching vitamin D intake >10 µg/d (13).
Similar economic evaluations of food fortification have 
been performed in Germany. A nationwide fortification 
programme with 20 µg of vitamin D and 200 mg of calcium 
per day would prevent 36,705 fractures in women aged 65 
and older and save EUR 315 million in health expenditure 
in this part of the population alone. The yearly cost of 
a fortification programme for Germany would be EUR 
41 million. The cost would be EUR 0.11 per person for 
cholecalciferol (D3) and EUR 0.22 per person for calcium 
(43). In our study, the calculated costs were higher, as a 
small volume of ingredient prices were used in the case 
of egg biofortification; the higher cost reflects a <15% 
carryover of dietary vitamin D from hen to egg (15).
4.1 Effectiveness and safety considerations
Food supplements are an effective strategy for preventing 
vitamin D deficiency on an individual level. However, there 
are significant limitations. Supplements have a higher risk of 
excessive intake than food fortification (16, 44). Typically, 
they do not reach people at the highest risk of deficiency, as 
supplement intake is positively associated with a healthier 
lifestyle and higher socioeconomic status (16). 
Achieving 25(OH)D3 levels >50 nmol/L in the entire 
population by fortification is unrealistic and unsafe, 
as it would mean a very high median intake. The food 
fortification goal set out in the World Health Organization 
(WHO) guidelines is ‘to provide most (97.5%) of individuals 
in the population group(s) at greatest risk of deficiency 
with an adequate intake of specific micronutrients, 
without causing a risk of excessive intakes in this or 
other groups. Inadequate intake is thus defined as intake 
below Estimated Average Requirements (EAR) and not the 
recommended intake (RI) value (45). Therefore, based on 
the WHO guidelines (45), the target intake should be at 
least EAR (46), which is 10 µg (16). The RI of vitamin D 
to reach serum 25(OH)D3 levels >50 nmol/L for people 
over one year of age is set at 20 µg (800 IU) by DACH 
(45, 46,47), at 15 µg (600 IU) by the European Food Safety 
Agency (EFSA) and the Institute of Medicine (IOM) (46,47), 
and at 25 µg (1000 IU) by the Endocrine Society (36, 37,47).
It is important to note that supplementation with 10 
µg of vitamin D/day is not enough to achieve vitamin D 
sufficiency (49). As in the Finnish case, food fortification 
effectively prevented 25(OH)D levels <30 nmol/L, but 
13.7% of non-users of supplements still did not achieve 
levels >50 nmol/L. Importantly, it did not cause a decline 
in vitamin D supplementation, as supplementation with 
vitamin D increased almost fourfold (13). 
The EFSA report concluded that a long-term daily dose of 
250 µg (10,000 IU) is considered to reflect a no observed 
adverse effect level (NOAEL). Using a safety factor, the 
upper tolerable intake level (UL) was established at 100 µg 
(4,000 IU) per day (48). In our fortification models, none 
of the participants would exceed >30 µg/day even with 
the maximum modelled fortification.
The effect of the fortification can be difficult to predict as 
serum 25(OH)D3 levels do not increase proportionally; in 
populations with lower baseline concentrations of serum 
25(OH)D3, there is a higher increase than in populations 
with higher baseline levels. In Finland, mean serum levels 
of 65 nmol/L 25(OH)D3 were achieved by systematic 
vitamin D food fortification and with guidelines suggesting 
supplementation. This was sufficient to decrease the 
prevalence of 25(OH)D3 concentrations <30 nmol/L below 
1% (15). 
 
4.2 Strengths and limitations of the study
Our study is the first to implement fortification modelling 
to calculate the effects of egg biofortification on vitamin 
D intake. Previous studies have been concerned only with 
egg fortification (19–25). The main limitation of this study 
is that it is based on data about egg vitamin D content 
from a single study (20), as other studies lacked 25(OH)D3 
data (20–26). Before implementation, additional studies 
on the transfer of vitamin D from feed to eggs that would 
include 25(OH)D3 would need to be conducted. 
10.2478/sjph-2023-0005 Zdr Varst. 2023;62(1):30-38
36
Supplementation with physician-prescribed vitamin D drugs*
Vitamin D food supplements**
Egg biofortification with vitamin D***
Direct food (milk) fortification with vitamin D3***
0.016911
0.017440 
0.000342 
0.000240 
6.17
6.37
0.12
0.09
Variables Cost per 10 µg  
(EUR)
Annual costs of 10 µg 
vitamin D/d/person (EUR)
Table 3. Annual costs of 10 µg vitamin D/d/person. 
*Serum 25(OH)D3 analysis, physician visits and dispensing costs are not included.
**Cheapest food supplement per µg, cost with VAT.
***No additional cost is predicted. 
Additionally, before biofortified eggs can be put on the EU 
market, feed legislation will need to be changed to allow 
higher vitamin D levels (29), as current legislation allows 
for vitamin D content in eggs to a maximum of 4.54±1.38 
μg of vitamin D/egg (16). Currently, such eggs would be 
illegal in the EU. Since changes were made in 2006 to 
vitamin D legislation concerning the addition of vitamins 
and minerals in 2006 (9), feed legislation could also see 
changes in the future, especially if research provides the 
basis for doing so.
Enrolment in the study was offered to all women coming 
for preventive medical examinations; however, those who 
chose to enrol in this voluntary study had significantly 
higher education and lower BMI than the general 
population (50). Additionally, socio-economic factors, 
such as language barrier, access to transport and flexibility 
of working hours, influenced withdrawal from the study.
These fortification models are simple and could be readily 
applied to any country with significant egg intake. In 
countries with very high egg intake, such as Mexico (>350 
eggs/capita/year) or Japan (>325 eggs/capita/year), egg 
fortification would be viable even with lower (51) legal 
levels of fortification (16).  
The models could also be applied to already-collected 
data from representative populations older than one 
year to confirm suitability for the entire population (31). 
Additionally, simultaneous fortification of eggs with several 
other nutrients, e.g. iodine, would also be possible (52). 
5 CONCLUSION
Due to the Covid-19 pandemic, almost two thirds of our 
subjects took vitamin D supplements. However, less than 
one third of subjects had optimal 25(OH)D3 levels, and 
25(OH)D3 levels were particularly low in those who did not 
use supplements. Vitamin D biofortification of hens’ eggs, 
especially in combination with milk fortification, could be 
a simple, safe and cost-effective way of increasing vitamin 
D intake in the population. 
CONFLICTS OF INTEREST
No conflict of interest.
FUNDING 
The study was financed by: Department of Public Health 
and Department of Gynecology and Obstetrics, Faculty 
of Medicine, University of Ljubljana, and the Division of 
Gynaecology and Obstetrics, University Medical Centre 
Ljubljana research program ARRS P3-0124, Metabolic and 
congenital factors of reproductive health, childbirth II. 
ETHICAL APPROVAL 
The study protocol was approved by the Slovenian 
National Medical Ethics Committee, identification no KME 
0120-68/2019/9 (approval letter ID 0120-68/2019/9, date 
of approval: 22. March 2019). The study was performed 
in compliance with the requirements of local authorities. 
All subjects signed a written informed consent form (ICF) 
before participating in the study.
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