SCIENTIFIC OPINIONADOPTED: 28 November 2019
doi: 10.2903/j.efsa.2020.5948

Safety of vitamin D2 mushroom powder as a novel foodpursuant to Regulation (EU) 2015/2283
EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA),
Dominique Turck, Jacqueline Castenmiller, Stefaan de Henauw, Karen-Ildico Hirsch-Ernst,
John Kearney, Alexandre Maciuk, Inge Mangelsdorf, Harry J McArdle, Androniki Naska,
Carmen Pelaez, Kristina Pentieva, Alfonso Siani, Frank Thies, Sophia Tsabouri, Marco Vinceti,
Francesco Cubadda, Karl-Heinz Engel, Thomas Frenzel, Marina Heinonen,€user-Berthold, Morten Poulsen, Yolanda Sanz,
Rosangela Marchelli, Monika Neuha
n-Torres, Hans Steinkellner andJosef Rudolf Schlatter, Henk van Loveren, Ruth Rolda
Helle Katrine Knutsen

Abstract
Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods andFood Allergens (NDA) was asked to deliver a scientiﬁc opinion on vitamin D2 mushroom powder as anovel food (NF) pursuant to Regulation (EU) 2015/2283. The NF is an ingredient produced from
Agaricus bisporus mushrooms that have been exposed to ultraviolet (UV) light to induce theconversion of provitamin D2 (ergosterol) to vitamin D2 (ergocalciferol). The NF contains concentrationsof vitamin D provided by vitamin D2 in the ranges of 1,000–1,300 lg/g. The information provided on
the manufacturing process, composition and speciﬁcations of the NF does not raise safety concerns.
The applicant intends to add the NF in a variety of foods and beverages, including food for special
medical purposes and food supplements. The target population is the general population except forfood supplements, for which the target population is individuals above seven months of age. The
Panel concludes that the NF, used as an ingredient, is safe for the general population at the proposed
condition of use in foods and beverages and that the NF used as a food supplement, is safe forindividuals above 1 year. The Panel, however, notes that the UL for infants aged 0–6 months may be
exceeded in high consumers of infant formula (IF) and/or follow-on formula (FoF) that may also behigh consumers of foods fortiﬁed with the NF and for infants aged 7–12 months consuming a daily
vitamin D oral supplementation of 10 lg. However, the Panel considers this scenario unlikely ascomplementary feeding in high consumers of IF and/or FoF may be limited. Furthermore, thecombined consumption of vitamin D via fortiﬁed foods and supplements does not speciﬁcally concern
this NF application. The Panel concludes that the NF is safe under the proposed conditions of use forthe proposed target populations.
© 2020 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalfof European Food Safety Authority.

Keywords: Novel food, ingredient, safety, mushroom powder, UV treatment, vitamin D

Requestor: European Commission following an application by Oakshire Naturals, LP
Question number: EFSA-Q-2018-00601
Correspondence: nda@efsa.europa.eu

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Safety of vitamin D2 mushroom powder

Panel members: Dominique Turck, Jacqueline Castenmiller, Stefaan De Henauw, Karen Ildico HirschErnst, John Kearney, Helle Katrine Knutsen, Alexandre Maciuk, Inge Mangelsdorf, Harry J McArdle,
Androniki Naska, Carmen Pelaez, Kristina Pentieva, Alfonso Siani, Frank Thies, Sophia Tsabouri andMarco Vinceti.
Acknowledgments: The Panel wishes to thank Davide Arcella for the support provided to thisscientiﬁc opinion.
Suggested citation: EFSA NDA Panel (EFSA Panel on Nutrition, Novel Foods and Food Allergens),
Turck D, Castenmiller J, de Henauw S, Hirsch-Ernst K-I, Kearney J, Maciuk A, Mangelsdorf I, McArdle
HJ, Naska A, Pelaez C, Pentieva K, Siani A, Thies F, Tsabouri S, Vinceti M, Cubadda F, Engel K-H,
€user-Berthold M, Poulsen M, Sanz Y, Schlatter JR, vanFrenzel T, Heinonen M, Marchelli R, Neuha
n-Torres R, Steinkellner H and Knutsen HK, 2020. Scientiﬁc Opinion on the safety ofLoveren H, Rolda
vitamin D2 mushroom powder as a novel food pursuant to Regulation (EU) 2015/2283. EFSA Journal
2020;18(1):5948, 23 pp. https://doi.org/10.2903/j.efsa.2020.5948
ISSN: 1831-4732© 2020 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalfof European Food Safety Authority.
This is an open access article under the terms of the Creative Commons Attribution-NoDerivs License,
which permits use and distribution in any medium, provided the original work is properly cited and no
modiﬁcations or adaptations are made.
The EFSA Journal is a publication of the European Food
Safety Authority, an agency of the European Union.

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Table of contents
Abstract................................................................................................................................................... 1
1.
Introduction................................................................................................................................. 4
1.1.
Background and Terms of Reference as provided by the requestor................................................... 4
1.2.
Additional information on existing evaluations and authorisations..................................................... 4
2.
Data and methodologies ............................................................................................................... 4
2.1.
Data............................................................................................................................................ 4
2.2.
Methodologies.............................................................................................................................. 5
3.
Assessment.................................................................................................................................. 5
3.1.
Introduction................................................................................................................................. 5
3.2.
Identity of the NF......................................................................................................................... 5
3.3.
Production process ....................................................................................................................... 5
3.4.
Compositional data....................................................................................................................... 5
3.4.1. Stability ....................................................................................................................................... 8
3.5.
Speciﬁcations ............................................................................................................................... 8
3.6.
History of use of the NF and/or of its source .................................................................................. 9
3.6.1. History of use of the source .......................................................................................................... 9
3.6.2. History of use of the NF................................................................................................................ 9
3.7.
Proposed uses and use levels and anticipated intake....................................................................... 9
3.7.1. Target population ......................................................................................................................... 9
3.7.2. Proposed uses and use levels ........................................................................................................ 9
3.7.3. Anticipated intake of the NF .......................................................................................................... 12
3.7.4. Combined vitamin D intake from the NF and other sources.............................................................. 13
3.7.5. Estimate of exposure to undesirable substances in mushrooms........................................................ 15
3.8.
Absorption, distribution, metabolism and excretion (ADME) ............................................................. 15
3.9.
Nutritional information .................................................................................................................. 16
3.10. Toxicological information............................................................................................................... 16
3.10.1. Genotoxicity................................................................................................................................. 16
3.10.2. Subacute, subchronic and chronic toxicity ...................................................................................... 17
3.10.3. Carcinogenicity............................................................................................................................. 17
3.10.4. Reproductive and developmental toxicity........................................................................................ 17
3.10.5. Human data................................................................................................................................. 17
3.11. Allergenicity ................................................................................................................................. 17
4.
Discussion ................................................................................................................................... 17
5.
Conclusions.................................................................................................................................. 18
Steps taken by EFSA ................................................................................................................................ 18
References............................................................................................................................................... 18
Abbreviations ........................................................................................................................................... 20
Appendix A – Subacute and chronic toxicity animal studies with powder of UV-radiated Agaricus bisporus ...... 22
Appendix B – Human intervention studies with powder of UV-radiated Agaricus bisporus............................... 23

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1.

Introduction

1.1.

Background and Terms of Reference as provided by the requestor

On 17 July 2018, the company Oakshire Naturals, LP submitted a request to the European
Commission in accordance with Article 10 of Regulation (EU) No 2015/22831 to place vitamin D2
mushroom powder on the Union market as a novel food.
The novel food (vitamin D2 mushroom powder) is intended for use in foods, beverages and foodsupplements. The target population is the general population with the exception of food supplements,
which are intended for the general population upwards of 7 months of age.
In accordance with Article 10(3) of Regulation (EU) 2015/2283, the European Commission asks theEuropean Food Safety Authority to provide a scientiﬁc opinion on vitamin D2 mushroom powder as anovel food.

1.2.

Additional information on existing evaluations and authorisations

Controlled ultraviolet (UV) irradiation is used to enhance the concentration of vitamin D2 inmushrooms. Commercially grown UV-treated Agaricus bisporus mushrooms are approved in theEuropean Union (EU) market as a novel food (NF) since 2016 (FSAI, 2017). No safety concerns
associated with the consumption of commercially cultivated mushrooms subjected to controlled UVlight were raised by the competent authorities of EU Member States.
UV irradiation technique to enhance the content of vitamin D has been used in other approved
novel foods evaluated by EFSA: UV-treated baker’s yeast (Saccharomyces cerevisiae) (EFSA NDA
Panel, 2014), UV-treated bread (EFSA NDA Panel, 2015) and UV-treated milk (EFSA NDA Panel,
2016c). All of them are currently under the Commission Implementing Regulation (EU) 2017/2470 of
20 December 2017 establishing the Union list of novel foods.

2.

Data and methodologies

2.1.

Data

The safety assessment of this NF is based on data supplied in the application and information
submitted by the applicant following EFSA requests for supplementary information.
During the assessment, the Panel identiﬁed additional data which were not included in theapplication (Gordon et al., 2008; Gallo et al., 2013; Cashman et al., 2016).
The applicant also provided a comprehensive literature search conducted in several databases to
identify issues of potential safety relevance for vitamin D2 mushroom powder (Annex V of the dossier).
Administrative and scientiﬁc requirements for NF applications referred to in Article 10 of Regulation(EU) 2015/2283 are listed in the Commission Implementing Regulation (EU) 2017/24692.
A common and structured format on the presentation of NF applications is described in the EFSA
guidance on the preparation and presentation of a NF application (EFSA NDA Panel, 2016a). As indicatedin this guidance, it is the duty of the applicant to provide all of the available (proprietary, conﬁdential andpublished) scientiﬁc data, including both data in favour and not in favour to supporting the safety of the
proposed NF.
This NF application includes a request for protection of proprietary data in accordance with Article
26 of Regulation (EU) 2015/2283. Data claimed to be proprietary by the applicant include: Rawmaterials and processing aids (Annex I to the dossier), Certiﬁcates of Analysis and batch data (Annex
II to the dossier) and Stability reports (Annex III to the dossier).

1

Regulation (EU) 2015/2283 of the European Parliament and of the Council on novel foods, amending Regulation (EU) No 1169/2011of the European Parliament and of the Council and repealing Regulation (EC) No 258/97 of the European Parliament and of theCouncil and Commission Regulation (EC) No 1852/2001. OJ L 327, 11.12.2015, p. 1.
2
Commission Implementing Regulation (EU) 2017/2469 of 20 December 2017 laying down administrative and scientiﬁcrequirements for applications referred to in Article 10 of Regulation (EU) 2015/2283 of the European Parliament and of theCouncil on novel foods. OJ L 351, 30.12.2017, pp. 64–71.
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2.2.

Methodologies

The assessment follows the methodology set out in the EFSA guidance on NF applications and theprinciples described in the relevant existing guidance documents from the EFSA Scientiﬁc Committee.
The legal provisions for the assessment are laid down in Article 11 of Regulation (EU) 2015/2283 andin Article 7 of the Commission Implementing Regulation (EU) 2017/2469.
This assessment concerns only risks that might be associated with consumption of the NF under
the proposed conditions of use and is not an assessment of the efﬁcacy of vitamin D2 mushroom
powder with regard to any claimed beneﬁt.

3.

Assessment

3.1.

Introduction

The NF which is the subject of the application is vitamin D2 mushroom powder as a novel food as
deﬁned in article 3 of the NF Regulation 2015/2283. The NF falls under the category (ii) ‘food
consisting of, isolated from or produced from microorganisms, fungi or algae’.
The NF contains concentrations of vitamin D as vitamin D2 in the range of 1,000–1,300 lg/g.
The applicant intends to market the NF as an ingredient in foods and beverages for consumption
by the general population. Vitamin D2 mushroom powder food supplements are intended forindividuals above 7 months of age.

3.2.

Identity of the NF

The NF is a whole mushroom powder containing vitamin D2 (ergocalciferol) induced by UVtreatment.
According to the applicant, this vitamin D2 whole mushroom powder is a slightly brown or tan
granular powder made from homogenised Agaricus bisporus mushrooms that have been exposed to
UV light to induce the conversion of provitamin D2 (ergosterol) to vitamin D2 (ergocalciferol).
The product contains vitamin D2 with registered CAS number 50-14-6 and its IUPAC 5Z,7E,22E)(3S)-9,10-seco-5,7,10(19),22-ergostatetraen-3-ol. The molecular formula of vitamin D2 is C28H44O.
The source of the NF is the fungus Agaricus bisporus as listed in the Index fungorum (http://www.
indexfungorum.org/names/names.asp). Various common synonyms for this mushroom are closed cap,large – ﬂat, common, cremini, button, cultivated, chestnut or portobello mushroom, depending on size,
shape, and colour of the mushroom.

3.3.

Production process

The mushrooms used in the production of vitamin D2 mushroom powder are cultivated Agaricus
bisporus. The cultivation is in accordance with current Good Agricultural Practices (GAP) and all
facilities comply with Food Safety Modernization Act (FSMA) produce rules. The producer has a Hazard
Analysis Critical Control Points (HACCP) plan in place.
As a ﬁrst step, the mushrooms are washed and homogenised in a mixing kettle to produce amushroom puree. The mushroom puree is transferred onto a shaker tray that passes under a UV
lamp. The slurry is then ﬁltered, dried and ground, producing vitamin D2 mushroom powder.
The process conditions of the UV treatment, i.e. energy input and range of wavelength, have been
provided by the applicant, after request for additional information from EFSA.
Based on previous EFSA assessments on UV-treated novel foods (EFSA NDA Panel, 2014, 2015,
2016c) that are currently authorised, the Panel considers the speciﬁed parameters, including the rangeof wavelength for the UV treatment, as being similar.
The Panel considers that the production process is sufﬁciently described and does not raise safetyconcerns.

3.4.

Compositional data

The applicant provided product analysis of six independent batches of the NF (Table 1). The
applicant provided the certiﬁcate of analysis for each batch.

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Table 1:

Batch-to-batch analysis of six batches of the NF

Parameter

Batch number and date of manufacture

Vitamin D2(lg/g)
Moisture (%)

Method of analysis

Batch #1

Batch #2

Batch #3

Batch #4

Batch #5

Batch #6

1,276.9

1,129.2

1,066.6

1,257.3

1,172.1

1,169.5

4.39

3.24

5.07

4.77

3.39

4.87

AOAC 964.22

6.12

4.87

4.80

6.40

6.28

5.71

AOAC 945.46

Ash (%)HPLCAOAC: Association of Ofﬁcial Analytical Chemists; HPLC: high-performance liquid chromatography.

The analytical method used by the applicant for vitamin D2 is validated in-house as well as bysample exchange with two reference laboratories. Vitamin D2 is quantiﬁed using high-performanceliquid chromatography (HPLC) with UV detection and [3H]-vitamin D3 as the internal standard as
described in Phillips et al. (2011).
Another form of vitamin D described as vitamin D4 (22-dihydroergocalciferol) has been observedand conﬁrmed by HPLC in edible mushrooms in a compositional analysis by Phillips et al. (2012). To
account for the separation of vitamin D4 from the D3 internal standard, modiﬁed HPLC conditions as
described by Phillips et al. (2012) are applied by the applicant.
Upon an EFSA request, the applicant provided the chromatograms of 6 reported batches of the NF
showing the peaks for the different forms of vitamin D (vitamin D2, vitamin D3 internal standard andvitamin D4). The analyses indicated that the concentrations of vitamin D4 in the NF are at
approximately 1/10th the concentration of vitamin D2.
The physical parameters particle size and appearance indicated that the six analysed batches of the
NF are a slightly brown or tan, granular powder that passes through an 80-mesh sieve.
Upon request of EFSA, the applicant provided full compositional information, including nutritional
composition, on 5 batches.
The applicant also provided analyses of chemical and microbiological contaminants of 6
independent batches of the NF (Tables 2 and 3). All investigated batches met the speciﬁcations (seeSection 3.5) regarding microbiological and chemical contaminants.
The chemical contaminants within the investigated batches of the NF, where applicable, were below
the limits speciﬁed in Commission Regulation (EC) No 1881/2006 of 19 December 2006 setting
maximum levels (MLs) for certain contaminants in foodstuffs.3 For contaminants with maximum levels
in mushrooms (i.e. the source of the NF), speciﬁc MLs from the legislation were used (i.e. lead).
The results for arsenic, lead, mercury, and cadmium have been converted by the applicant toequivalent wet weight values based on the moisture content of vitamin D2 mushroom powder (10%)
and of Agaricus bisporus mushrooms (approximately 90% (OECD, 2007)).
Table 2:Analysis of chemical contaminants of 6 batches of the NF

Potential chemical
contaminants

Method ofanalysis

Batch number and date of manufacture
Batch #1 Batch #2 Batch #3 Batch #4 Batch #5 Batch #6

Arsenic (mg/kg)
Lead (mg/kg)

0.011*
0.006*

0.011*
0.006*

0.011*
0.006*

0.011*
0.006*

0.011*
0.006*

0.011*
0.006*

Mercury (mg/kg)
Cadmium (mg/kg)

0.004*
0.003*

0.004*
0.003*

0.004*
0.003*

0.004*
0.003*

0.004*
0.003*

0.004*
0.003*

Aﬂatoxins (sum of
B1, B2, G1 and G2)(lg/kg)

< 0.7

< 0.7

< 0.7

< 0.7

< 0.7

< 0.7

USP 730

AOAC 994.08

AOAC: Association of Ofﬁcial Analytical Chemists; USP: United States Pharmacopeia.
*: Equivalent wet weight values calculated based on a 10% moisture content of vitamin D2 mushroom powder and a moisture
content of approximately 90% (OECD, 2007)] for Agaricus bisporus mushrooms Calculation = reported value 9 (1/9).

3

Commission Regulation (EC) No 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants infoodstuffs. OJ L 364, 20.12.2006, p. 5–24. Available online: https://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:
02006R1881-20180319&from=EN [Consolidated]

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The applicant states that screenings for the presence of pesticides in batches of the NF are
conducted using internationally accepted methods (EN 15662/Canadian Food Inspection Agency (CFIA)
PMR-006).
The applicant conducted a multi-residue pesticide screen on a representative batch of vitamin D2
mushroom powder, and the full results were provided in the certiﬁcates of analysis in Annex II of theapplication. None of the pesticides were detected (detection limit of < 0.005 mg/kg for all compounds,
with the exception of 2-(thiocyanomethylthio) benzothiazole (< 0.006 mg/kg), folpet (< 0.012 mg/kg)
and fenamiphos sulfone (< 0.013 mg/kg)). The Panel notes that those levels are below the EU
maximum residue levels for pesticides.4
Microbiological analyses for vitamin D2 mushroom powder are presented in Table 3.
Table 3:

Microbiological analysis of six batches of the NF

Microbiologicalcontaminants

Batch #1 Batch #2
Standard plate
count (CFU/g)
Yeast (CFU/g)
Mould (CFU/g)
Salmonella spp.
Staphylococcus
aureus (CFU/g)
Escherichia coli
(CFU/g)Coliforms (CFU/g)
Enterobacteriaceae(CFU/g)Listeria
monocytogenes

Method ofanalysis

Batch number and date of manufacture
Batch #3

Batch #4

Batch #5

Batch #6

100

< 100

600

< 100

< 100

< 100

FDA (BAM) CH. 3

< 10

< 10

< 10

< 10

< 10

10

FDA (BAM) CH. 18

20
Conforms

20
Conforms

60
Conforms

FDA (BAM) CH. 18
FDA (BAM) CH. 5

40
30
50
Conforms Conforms Conforms
< 10

< 10

< 10

< 10

< 10

< 10

FDA (BAM) CH. 12

< 10

<3

<3

<3

<3

<3

FDA (BAM) CH. 4

< 10
< 10

<3
< 10

<3
< 10

<3
< 10

<3
< 10

<3
< 10

FDA (BAM) CH. 4
FDA (BAM) CH. 29

Conforms

Conforms

Conforms

Conforms Conforms Conforms

AOAC 2004.06

AOAC: Association of Ofﬁcial Analytical Chemists; CFU: colony forming units; NT: not tested; FDA (BAM): United States Food andDrug Administration (Bacteriological Analytical Manual).

The conversion of ergosterol into vitamin D2 with UV exposure is accompanied by photochemical
isomerisations resulting in photoisomers such as lumisterol and tachysterol (Havinga et al., 1960). Bothlumisterol and tachysterol are biologically inactive and known to be formed in the course of theUV-induced conversion of epidermal 7-dehydrocholesterol into vitamin D3 (Holick et al., 1981).
The applicant, therefore, performed analyses to investigate the potential formation of these sterols
under the conditions of the UV treatment of the mushroom powder. The results of HPLC showed that,apart from the intended vitamin D2, both tachysterol and lumisterol were detected. The applicant
provided information on three representative batches of the NF (UV-treated batches) and three
representative batches of the NF not exposed to UV treatment (untreated batches). The parameters
analysed were vitamin D2, ergosterol and lumisterol at a limit of detection of 0.5 lg/g and tachysterolat a limit of detection of 1 lg/g.
In the UV-treated batches, the mean vitamin D2 content was 1,500 lg/g and the mean ergosterol
content was 8,747 lg/g. The untreated batches reported a mean vitamin D2 content of 0.72 lg/g anda mean ergosterol content of 9,223 lg/g. The mean contents of the photoisomers in UV-treatedbatches were lumisterol at concentrations of 206 lg/g of NF and tachysterol at 111 lg/g of NF.
The applicant indicated a content of vitamin D from the NF of 2.25 lg/100 g in food products otherthan beverages that would result in intakes of lumisterol and tachysterol of 0.0005 lg/100 g and
0.0003 lg/100 g, respectively.
The NDA Panel noted that the tachysterol concentrations are lower than those reported in previous
EFSA Scientiﬁc Opinions on UV-treated novel foods (EFSA NDA Panel, 2014, 2015, 2016c). The exposure
to these compounds from the NF can therefore be considered not to be of toxicological relevance.

4

Regulation (EC) No 396/2005 of the European Parliament and of the Council of February 2005 on maximum residue levels of
pesticides in or on food and feed of plant and animal origin and amending Council Directive 91/414/EEC. OJ L 70, 16.3.2005, p. 1.

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The Panel considers that the information provided on the composition of the NF is sufﬁcient anddoes not raise safety concerns.

3.4.1.

Stability

The applicant performed stability tests with ﬁve batches of the NF (stability of the bulk powder).
The tests were carried out at representative storage conditions (room temperature in sealed
polypropylene bags). The batches were analysed for vitamin D2 content. The results demonstrated
that the vitamin D2 content of the mushroom powder is stable for at least 3 years (intended shelf-lifeof 2 years).
The applicant also provided stability studies of vitamin D2 mushroom powder as an ingredient in foods.
The NF was used in the production process of granola bars. Granola bars with and without the NF werestored at room temperature for up to 3 months. The batches were analysed for vitamin D2 content. A
minor reduction of vitamin D2 was identiﬁed over time. The applicant noted that vitamin D is a liposoluble
vitamin and degradation over time due to oxidation is reported in the literature (EVM, 2003). The granola
bar samples with the added NF showed no difference in taste in comparison with the controls.
The applicant also tested the stability of the NF as an ingredient in soya drink. Results showedvariability (increase and decrease in vitamin D2 concentrations) and were therefore considered as notsuitable for the risk assessment.
The Panel considers that vitamin D2 from the NF is not expected to behave differently from vitamin
D2 from other production process.
The Panel considers that the data provided are sufﬁcient with respect to the stability of the NF.

3.5.

Speciﬁcations

The applicant describes the NF ingredient, i.e. vitamin D2 mushroom powder, as a slightly brown or tan
granular powder made from homogenised Agaricus bisporus mushrooms that have been exposed to UV
light.
The speciﬁcations for vitamin D2 mushroom powder as proposed by the applicant are reported inTable 4. These speciﬁcations include physical and chemical parameters as well as chemical andbiological contaminants.
Upon request of EFSA, the applicant provided a maximum speciﬁcation limit for vitamin D2 andclariﬁed the non-inclusion of the photoisomers tachysterol and lumisterol in the speciﬁcations (basedon the very low concentrations in the ﬁnal product and low exposure through the NF, see Section 3.4).
Table 4:

Speciﬁcations for vitamin D2 mushroom powder
Speciﬁcation limit

MethodVitamin D2
Moisture

1,000–1,300 lg/g(a)≤ 10.0%HPLCAOAC 964.22

Ash≤ 13.5%AOAC 945.46

100% through 80 mesh sieve
Slightly brown or tan, granular powder––

Standard plate count

5,000 CFU/g

FDA (BAM) CH. 3

Yeast
Mould

100 CFU/g
100 CFU/g

FDA (BAM) CH. 18
FDA (BAM) CH. 18

Salmonella
Staphylococcus aureus

Absent in 25 g
< 10 CFU/g

FDA (BAM) CH. 5
FDA (BAM) CH. 12

Escherichia coli
Coliform< 10 CFU/g< 10 CFU/g

FDA (BAM) CH. 4
FDA (BAM) CH. 4

Enterobacteriaceae
Listeria monocytogenes< 10 CFU/g
Absent in 25 g

FDA (BAM) CH. 29
AOAC 2004.06

Chemical parameters

Physical parameters
Particle size
Appearance
Microbiological parameters

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Speciﬁcation limit

Method< 4 lg/kg

AOAC 991.08

Arsenic≤ 0.3 mg/kg

USP 730

Lead (as Pb)
Mercury≤ 0.5 mg/kg≤ 0.1 mg/kg

USP 730
USP 730

Cadmium< 0.5 mg/kg

USP 730

Arsenic≤ 0.3 mg/kg

USP 730

Mycotoxins
Aﬂatoxins (sum of B1+B2+G1+G2)
Heavy metals

AOAC: Association of Ofﬁcial Analytical Chemists; CFU: colony forming units; FDA (BAM): United States Food and Drug
Administration (Bacteriological Analytical Manual); HPLC : high-performance liquid chromatography; USP: United States
Pharmacopeia; –: no information available.
(a): Converted from International Units (IU) using the conversion factor of 0.025 lg = 1 IU stated in the EFSA Technical Reporton Dietary Reference Values for nutrients (EFSA, 2017).

The Panel considers that the information provided on the speciﬁcations of the NF is sufﬁcient anddoes not raise safety concerns.

3.6.

History of use of the NF and/or of its source

3.6.1.

History of use of the source

The source of the NF is the mushroom Agaricus bisporus. The applicant listed several publications
describing the history of the consumption, cultivation and production for human consumption of thesemushrooms within and outside the EU (FAO, 2004; OECD, 2007; FSAI, 2017).
In addition, according to the applicant, UV-treated Agaricus bisporus mushrooms have a history ofuse in the EU (as they have been approved as a novel food ingredient since 2016) and in several nonEU countries, including the United States, Canada and Australia (FSAI, 2017).

3.6.2.

History of use of the NF

The NF, i.e. the powder from UV-treated Agaricus bisporus mushrooms, has no history of use in the EU.

3.7.

Proposed uses and use levels and anticipated intake

3.7.1.

Target population

The target population for the consumption of the NF added to foods and beverages is the general
population.
The target population for the consumption of the NF added to foods for special medical purposes(FSMPs) as deﬁned in Regulation (EU) No 609/20135 is individuals above 1 year of age.
The target population proposed by the applicant for the consumption of the NF added to foodsupplements is individuals above 7 months of age.

3.7.2.

Proposed uses and use levels

The applicant intends to use the NF as an ingredient in a variety of foods and beverages as
indicated in Tables 5 and 6, in foods for special medical purposes as deﬁned in Regulation (EU) No
609/2013 (excluding those intended for infants) and in food supplements. The use level of the NF infood or beverages is determined by the applicant based on the vitamin D2 content per gram of NF(1,000–1,300 lg vitamin D2/gram NF). The proposed levels of vitamin D2 from the NF are 2.25 lg/100
g for products other than beverages (excluding food supplements) and 1.125 lg/100 mL for beverages(i.e. using the minimum speciﬁcation for vitamin D content in the NF of 1,000 lg vitamin D2/gram NF).
5

Regulation (EU) No 609/2013 of the European Parliament and of the Council of 12 June 2013 on food intended for infants andyoung children, food for special medical purposes, and total diet replacement for weight control and repealing Council Directive
92/52/EEC, Commission Directives 96/8/EC, 1999/21/EC, 2006/125/EC and 2006/141/EC, Directive 2009/39/EC of theEuropean Parliament and of the Council and Commission Regulations (EC) No 41/2009 and (EC) No 953/2009 Text with EEA
relevance, OJ L 181, 29.6.2013, p. 35–56.

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Safety of vitamin D2 mushroom powder

The maximum proposed level of vitamin D2 as consumed using the maximum speciﬁcation for vitamin Dcontent in the NF (i.e. 1,300 lg vitamin D2/gram NF) would result in an intake of 2.93 lg of vitamin D2/
100 g in foods and 1.46 lg of vitamin D2/100 mL for beverages.
The applicant also intends to market the NF as an ingredient in food supplement. In their initial
proposal, the applicant proposed to market the NF as food supplement at the maximum daily intake ofvitamin D2 of 15 lg for the general population excluding infants and children. Upon question fromEFSA, the applicant modiﬁed the use levels and proposed 15 lg/day for individuals above 1 year and
10 lg/day in food supplements for infants from 7 to 11 months. The applicant based those use levels
on the adequate intakes (AIs) for vitamin D speciﬁed by EFSA (EFSA, 2017). According to theapplicant, the NF used in food supplement would typically be taken as an alternative supplement
replacing other vitamin D2-containing supplements on the market.
The applicant also intends to market the NF as an ingredient in FSMPs (excluding those intended for
infants). For the adult population, the maximum intended use level in FSMPs is 15 lg vitamin D2/day.
Table 5:

Proposed uses and use levels for the NF in the EU
Proposed food category name

Proposed maximum use
level of the NF (mg/100 g
or 100 mL), as consumed(a)Breakfast cereals

Breakfast cereals

2.25

Bakery products
Processed cereal foodYeast-leavened bread and pastries
Grain products and pastas

2.25
2.25

Fruit juice and fruit/vegetableblend beverages
Dairy products

Fruit and vegetable juices (incl. powders and 1.125
concentrates)
Milk and dairy products (excl. ﬂuid milks)
2.25/1.125 (beverages)

Cheese and cheese products

Cheese (excl. cottage cheese, ricotta cheese, 2.25
and hard-grating cheeses)
Meal replacement bars and beverages, not2.25/1.125 (beverages)
intended for weight control

Food groups

Meal replacement
Dairy analogues
Meat imitates

Dairy Imitates
Meat imitates

2.25/1.125 (beverages)
2.25

Soups and broths

Soups (RTE and dry mixtures)

2.25

Vegetable snacks

Extruded vegetable snacks

2.25

RTE: ready to eat.
(a): Proposed maximum use level of the NF per 100 g or 100 mL of each food category, considering the minimum speciﬁcationfor vitamin D content in the NF of 1,000 µg vitamin D2/gram of NF.

Table 6:

Proposed food categories based on the FoodEx2 classiﬁcation system and use levels for
the NF in the EU
FoodEx2
level

FoodEx2 name

Proposed maximum use
level of the NF (mg/100 g
or 100 mL), as
consumed(a)

A00CV

2

Breakfast cereals

2.25

A0BY0
A00BK

3
3

Leavened bread and similar
Yeast leavened pastry

2.25
2.25

A000K
A007D

2
3

Cereals and cereal primary derivatives
Pasta and similar products

2.25
2.25

A040M
A0BX9

3
2

Pastas and rice (or other cereal) – based dishes
Fruit/vegetable juices and nectars

2.25
1.125

A03BM
A02LR

2
2

Concentrated or dehydrated fruit/vegetables juices
Milk and dairy products

7.875(b)
2.25

A02MZ
A02NA

3
3

Fermented milk or cream
Sour cream products

2.25
2.25

FoodEx2
code

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Safety of vitamin D2 mushroom powder

Proposed maximum use
level of the NF (mg/100 g
or 100 mL), as
consumed(a)FoodEx2
code

FoodEx2
level

A0C69
A02NE

4
4

Fermented milk products
Yoghurt

2.25
2.25

A02NQ
A02NR

4
4

Yoghurt drinks
Probiotic milk-like drinks

1.125
1.125

A0C69
A02NT

4
5

Fermented milk products
Traditional sour milk products

2.25
2.25

A02NV
A02NY

5
5

Keﬁr
Traditional Nordic fermented milks

1.125
1.125

A02PC
A02PD

5
3

Flavoured traditional sour milk products
Milk and dairy powders and concentrates

2.25
2.25

A02PE
A02PH

3
4

Milk and dairy concentrate
Milk and dairy powders

2.25
22.5(c)

A02PJ
A02PM

4
4

Milk powder
Cream powder22.5(c)92.25(d)

A02PN
A02MP

4
4

Whey powder
Flavoured milks22.5(c)
1.125

A02MK
A0EZB

3
3

Cream and cream products
Whey

2.25
2.25

A02MV
A02PT

3
2

Buttermilk
Dairy dessert and similar

2.25
2.25

A02QE
A02QF

3
4

CheeseFresh uncured cheese

2.25
2.25

A02QH
A02QJ

4
4

Mascarpone
Mozzarella

2.25
2.25

A02QK
AS04NV

4
4

Quark
Miscellaneous fresh uncured cheeses

2.25
2.25

A0CRN
A02RA

4
4

Cheese curd
Brined cheese (feta-type and similar)

2.25
2.25

A02RB
A02RG

4
4

Soft brined cheese (feta-type)
Ripened cheese

2.25
2.25

A02RH
A031A

4
3

Soft – ripened cheese
Processed cheese and spreads

2.25
2.25

A03RS
A03RV

4
4

Food for weight reduction(e)Single meal replacement for weight reduction

2.25(f)
2.25

A03TH
A03TQ

4
4

Milk imitates
Dairy imitates other than milks

1.125
2.25

A0BXC
A03TE

4
2

Dairy imitates
Meat imitates

2.25
2.25

A0B9J
A041L

3
3

Soups, dry mixture, uncooked
Soups, ready-to-eat

20.25(g)
2.25

A0EQV

4

Vegetable snacks, puffs/curls-type extruded snack

2.25

A011L

5

Potato crisps or sticks

2.25

FoodEx2 name

NF: novel food.
(a): The use level of vitamin D2 mushroom powder is determined based on maximum 1,000 lg vitamin D2/gram of mushroom
powder.
(b): Adjusted for being present in concentrated or dehydrated form; reconstitution factor of 7 (based on available products on
the market).
(c): Adjusted for being present in powder form; reconstitution factor of 10 (based on available products on the market).

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Safety of vitamin D2 mushroom powder(d): Adjusted for being present in powder form – assumed to be used as coffee whitener; reconstitution factor of 41 (based on
available products on the market).
(e): While the food use name includes the term ‘for weight reduction’, these are representative of use in the general populationand not intended to be used for special dietary purposes.
(f): Unable to distinguish between food products and beverages. Therefore, the higher use level (i.e., 2.25 for food products)
was applied in this category.
(g): Adjusted for being present in powder form; reconstitution factor of 9 (based on available products on the market).

3.7.3.

Anticipated intake of the NF

Anticipated intake of the NF from the proposed food uses, as an ingredient in foods andbeverages
The applicant provided estimates of the anticipated daily intake of the NF for each food category
indicated in Table 6, based on the summary statistics from the EFSA Comprehensive European Food
Consumption Database.6
EFSA performed an additional intake assessment of the NF based on the individual consumption
data from the EFSA Comprehensive Food Consumption Database (EFSA, 2011). For this estimation,
EFSA considered the food categories and the maximum use levels proposed by the applicant in Table 6.
The ranges of the estimated daily intake of the NF (i.e. lowest and highest means and 95th
percentiles among surveys covered by the EFSA Database), from foods to which the NF is added, arepresented in Table 7 (on a mg/kg body weight (bw) per day basis). The estimated daily intake of the
NF for each population group from each EU dietary survey is available in the excel ﬁle annexed to thisscientiﬁc opinion (under supporting information).
Table 7:

Anticipated daily intake of the NF on a mg/kg bw per day basis: lowest and highestmeans and 95th percentiles anticipated daily intake of the NF among the EU surveys in the
EFSA Comprehensive European Food Consumption Database (calculated by EFSA)
Estimated intake of the NF as food/beverage ingredient(mg/kg bw per day)

Population group

Number of
EU surveys

Range of means(minimum andmaximum) acrossEU dietary surveys

Range of 95th percentile(minimum and maximum) acrossEU dietary surveys

Infants (≤ 11 months)

110.06–0.52

0.29–1.87

Young children7(12–35 months)
Children (3–9 years)

14

0.34–0.67

0.65–1.28

19

0.26–0.50

0.49–0.97

Adolescents (10–17
years)
Adults (18–64 years)

18

0.13–0.22

0.26–0.45

19

0.08–0.15

0.15–0.31

Pregnant women
Lactating women2
2

0.10–0.12
0.12–0.14

0.18–0.21
0.22–0.24

Elderly (65–74 years)

18

0.07–0.14

0.13–0.32

Very elderly (≥ 75
years)

14

0.07–0.14

0.13–0.27

bw: body weight; NF: novel food.

As stated in Section 3.5 on Speciﬁcations, the NF contains 1,000–1,300 lg vitamin D2/g NF.
Therefore, based on estimated daily intakes of the NF and using a maximum content of 1,300 lg
vitamin D2/g NF, estimated daily intakes of vitamin D2 calculated both in absolute values (lg/day) andon a per body weight basis (lg/kg bw per day) are reported in Table 8.

67

https://www.efsa.europa.eu/en/food-consumption/comprehensive-database
Referred as toddlers in the EFSA food consumption comprehensive database (EFSA, 2011).

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Safety of vitamin D2 mushroom powder

Table 8:

Anticipated P95 of daily intake of the vitamin D2 from the NF as ingredient in foods andbeverages considering a vitamin D2 concentration in the NF of 1,300 lg/g (calculated by
EFSA)
Anticipated P95 daily intake of vitamin D2 from the NF as
ingredient in foods and beverages(lg/kg bw per day)(lg/day)(a)Infants (≤ 11 months)

2.43

12.2

Young children (12– 35 months)
Other children (3–9 years)

1.66
1.26

19.9
28.9

Adolescents (10–17 years)
Adults (18–64 years)

0.59
0.40

25.3
28.1

Pregnant women
Lactating women

0.27
0.31

19.2
21.9

Elderly (65–74 years)

0.42

29.2

Very elderly (≥ 75 years)

0.36

25.0

Population classbw: body weight; NF: novel food.
(a): Based on default body weights as given for the different age groups as described in (EFSA Scientiﬁc Committee, 2012).

The Panel notes that the intake estimates for vitamin D2 from the foods added with the NF arebased on conservative assumptions, which are that the NF always contains the upper speciﬁcation limitof vitamin D (i.e. 1,300 lg/g NF) and that all proposed food items consumed by an individual actuallycontain the NF at the maximum proposed use levels.

3.7.4.

Combined vitamin D intake from the NF and other sources

The potential combined intake of vitamin D from the NF (vitamin D2) and other sources (vitamin D2
or D3) is estimated by summing the contribution to vitamin D intake from the NF as estimated by EFSA
(Table 8) and the high vitamin D intakes from other food sources as reported by the EFSA NDA
Panel in 2012 based on a literature review (EFSA NDA Panel, 2012).
In this Opinion from 2012, the highest 95th percentile (P95) dietary intake across surveys in adults
was 16 lg vitamin D/day. The P95 exposure from the background diet alone was not available for all
children in the EFSA opinion from 2012, and as a substitute, the highest mean intakes across thecovered surveys for each age category of children were used. The highest mean intakes were 5.6 lg/
day in younger children (1–5 years), 2.7 lg/day in older children (4–13 years) and 4.0 lg/day inadolescents (11–18 years). The P95 intakes from the sum of food and supplements were however
available for children (up to 15 lg/day) and adolescents (up to 8 lg/day) in this previous opinion of
the EFSA NDA Panel (2012) and were also used in the present calculations, but without it to the intake
of vitamin D2 from the NF as food supplement.
Table 9 provides an overview of the exposure to vitamin D from different sources separately andcombined, and a comparison of the total estimated intake with the tolerable upper intake levels (ULs)
established for young children, children, adolescents and adults.

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Safety of vitamin D2 mushroom powder

Table 9:Estimated daily intakes of vitamin D (lg/day) from the different sources and combined
intake of vitamin D compared with ULs per population group > 1 year

Population
group1) Intake ofvitamin D from
4) Combined
2) Highest P95
foods in theintake ofbackground diet estimated intake
3) Intake of vitamin D fromUL
of vitamin D2(P95 intake forvitamin D2
1+2+3(lg/day) EFSA
adults andfrom the NF as
from the NF as (background
NDA Panelhighest mean
an ingredient in
diet, NF as
food(2012)
intakes forfoods andsupplements ingredient, NF
children andbeverage (fromas supplement
adolescents)
Table 8)
if applicable)
EFSA NDA
Panel (2012)

Young children(12–35months)5.6(a)
15(b)

19.9
19.9

15
–

40.5
34.9(c)50

Other children(3–9 years)
Adolescents(10–17 years)

2.7(a)
15(b)
4(a)
8(b)

28.9
28.9
25.3
25.3

15
–
15
–

46.6
43.9(c)44.3
33.3(c)50

Adults(18–64 years)
Pregnant
women

16

28.1

15

59.1

100

16

19.2

15

50.2

100

Lactating
women
Elderly(65–74 years)

16

21.9

15

52.9

100

16

29.2

15

60.2

100

Very elderly(≥ 75 years)

16

25.0

15

56.0

100

100

UL: tolerable upper intake level; NF: novel food.
(a): Maximum mean/median intake of vitamin D from foods only. Data collected from different surveys/studies (EFSA NDA Panel,2012).
(b): Combined vitamin D intake from foods and supplements; vitamin D intake from high consumers (90th or 95th percentile
depending on surveys) in infants, children and adolescents (EFSA NDA Panel, 2012).
(c): Dietary intake of vitamin D included in foods and food supplements (EFSA NDA Panel, 2012). In order to avoid
overestimation of vitamin D intake, the maximum intake of vitamin D from the total diet (combined intake, column 5) does
not include the contribution of the vitamin D from the NF used as ingredient in food supplements.

The Panel notes that estimates for combined intake of vitamin D2 from the NF (added to the foods,
beverages and food supplements) plus estimated intake of vitamin D from the background diet resultin overall vitamin D intakes of 44, 59, 60 and 56 lg/day for adolescents, adults, elderly and very
elderly, respectively.
For pregnant and lactating women, such combined intake estimates result in values of 50 and 53
lg/day, respectively.
Those intake estimates are below the UL of 100 lg/day (EFSA NDA Panel, 2012) for each of these
population groups.
In children, estimating the combined intake of vitamin D2 from the NF (19.9 and 28.9 lg/day foryoung children and other children, respectively) plus intake of vitamin D from other dietary sources(including 15 lg/day from the NF when used in food supplements) results in vitamin D intakes of 40
and 47 lg/day for young children and other children, respectively. The Panel notes that thoseestimated combined intakes are below the upper level (UL) established by EFSA for children aged 1–10
years (50 lg/day) (EFSA NDA Panel, 2012).
For infants aged from 4 to 12 months, data on vitamin D intake were estimated by EFSA using
composition data from the EFSA nutrient composition database and individual consumption data fromnational surveys from 6 European countries (EFSA NDA Panel, 2018). In addition to the vitamin D
intake provided by infant formula (IF) or follow-on formula (FoF), the vitamin D intake fromwww.efsa.europa.eu/efsajournal

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Safety of vitamin D2 mushroom powder

complementary feeding was considered, including foods naturally containing vitamin D and foods
fortiﬁed with vitamin D, but intake of vitamin D via supplements was not considered.
For this age group, P95 intakes for vitamin D ranged across the surveys from 13.2 to 16.9 lg/day
in formula consumers8 not consuming (voluntarily) fortiﬁed foods. For non-formula consumers thatwere also not consuming (voluntarily) fortiﬁed foods, the P95 vitamin D intake from the diet ranged
between 0.7 and 2.8 lg/day (EFSA NDA Panel, 2018).
For formula consumers8 consuming also fortiﬁed foods, the P95 vitamin D intake ranged from 15.2
to 22.2 lg/day. For non-formula consumers, the P95 intake from diet including fortiﬁed foods ranged
from 1.6 to 10 lg/day (based on scenario 6 from Annex B of EFSA NDA Panel, 2018).
For infants, the estimated P95 intake of vitamin D2 from the NF as an ingredient in foods is 12.2
lg/day (see Table 8). The addition of this amount to the highest P95 vitamin D intake of formula
consumers not consuming fortiﬁed foods (16.9 lg/day) results in a combined intake of 29.1 lg/day(for comparison, the highest P95 intake of vitamin D in formula consumers consuming also fortiﬁed
foods was 22.2 lg/day, according to EFSA, 2018). This estimated combined intake of 29.1 lg/day canbe considered an overestimation as highest formula consumers can be assumed not to be also highest
consumers of fortiﬁed foods including foods with the added NF. The Panel notes that this estimatedcombined intake of 29.1 lg/day is below the UL of 35 lg/day for infants aged 6 to less than 12
months established by EFSA (EFSA NDA Panel, 2018).
The addition of 10 lg/day of vitamin D2 from the NF used as an ingredient in supplement (which
the applicant intends to market for infants aged from 7 to 12 months) to the (most likely
overestimated) intake of 29.1 lg/day would result in an intake of 39.1 lg/day and thus would exceed
the UL.
When adding 10 lg/day to the previously estimated intake of 22.2 lg/day (as calculated by EFSANDA Panel, 2018), the combined estimated intake of vitamin D from formula, fortiﬁed foods andsupplements containing the NF amounts to 32.2 lg vitamin D/day and thus would be below the UL for
vitamin D of 35 lg/day established by EFSA in 2018.

3.7.5.

Estimate of exposure to undesirable substances in mushrooms

The undesirable toxicological substances in mushrooms have been described by the Consensus
document on compositional considerations for new varieties of the cultivated mushrooms Agaricus
bisporus (OECD, 2007). Agaritine (b-N-[c-L-(+)-glutamyl]-4-hydroxymethylphenyl-hydrazine) is anatural occurring phenylhydrazine derivative found in mushrooms. Agaritine ranges were reported by
OECD between 80 and 1,730 mg/kg fresh weight of mushrooms (OECD, 2007).
As explained in Section 3.3 Production process, the NF is produced from the fresh cultivatedAgaricus bisporus mushrooms. Based on the NF production yield from fresh mushroom and on thehighest estimated intakes provided by the applicant, and considering that a food portion size ofmushrooms is approximately 70 g (FSA, 2002), the estimated maximum additional intake due to the
NF would correspond to less than 1.3% of a single serving of mushrooms.
As reported in Section 3.4 Compositional data, the UV processing inducing the conversion ofergosterol to vitamin D2 is accompanied by isomerisations resulting in tachysterol and lumisterol. The
applicant indicated a maximum use level of the NF of 2.25 lg/100 g for food products other thanbeverages that would result in intakes of lumisterol and tachysterol of 0.0005 lg/100 g and 0.0003
lg/100 g, respectively. The Panel also noted that the tachysterol concentrations are lower than thosereported in previous EFSA Scientiﬁc Opinions on UV-treated novel foods, which did not give rise tosafety concerns (EFSA NDA Panel 2014, 2015, 2016c).
The Panel considers that there is no concern with respect to the exposure to undesirable
substances from mushrooms (i.e. agaritine) and from the NF production process (lumisterol andtachysterol) from the consumption of NF at the proposed uses.

3.8.

Absorption, distribution, metabolism and excretion (ADME)

The applicant has not provided speciﬁc ADME studies for the NF, but has submitted publicly
available animal and human studies that suggest that vitamin D2 from powder from UV-irradiatedmushrooms is bioavailable, as dose-related increase in serum concentrations of 25(OH)D2 are observed
upon its oral consumption. Some of these studies have been carried out with material similar to the NF
8Containing maximum regulated vitamin D content (2.5 µg/100 kcal for IF; 3.0 µg/100 kcal for FoF), in accordance with
Commission Delegated Regulation (EU)2019/828 amending Delegated Regulation (EU) 2016/127.

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Safety of vitamin D2 mushroom powder(i.e. powder from UV-radiated Agaricus bisporus mushrooms). These studies (Bennett et al., 2013;
Calvo et al., 2013; Koyyalamudi et al., 2009; Stephensen et al., 2012; Shanley et al., 2014; Stepien
et al., 2013) are presented in Appendices A and B.
Results from a meta-analysis on randomised clinical trials (RCTs) with UV-exposed and vitamin D2enriched mushrooms suggest an effect of their consumption on serum total 25(OH)D concentration
when the mean baseline vitamin D status of the participants is inadequate [serum 25(OH)D < 50
nmol/L], but no effect when vitamin D status is ‘high’ [mean baseline 25(OH)D: 81.5 nmol/L]], due toa reduction in serum 25(OH)D3 that accompanies the increase in serum 25(OH)D2 (Cashman et al.,2016).
Studies comparing the effects of vitamin D2 and D3 supplementation on serum 25(OH)D
concentrations in a small number of infants, showed no statistically signiﬁcant differences between theeffects of the two vitamin forms (Gordon et al., 2008; Gallo et al., 2013).

3.9.

Nutritional information

The applicant refers to a publication by Simon et al. (2011) where the vitamin content of Agaricus
bisporus mushrooms exposed to UV light was investigated. In this study, the nutritional content of themushrooms (including macronutrients, fatty acids, amino acids, water-soluble vitamins) remained
unchanged by the UV treatment, with the exception of the intended increase in the vitamin D2
content.
The Panel notes that the intakes of vitamin D2 in high consumers of foods and beverages fortiﬁed
with the NF are above the AIs for all age groups set by the NDA Panel of EFSA (EFSA NDA Panel,2016b). However, together with the intake of vitamin D from background diet and possible intake of
the NF supplement (see Section 3.7.4), the total daily vitamin D intakes do not exceed the ULs set forchildren aged 1–10 years (50 lg/day), adolescents and adults (100 lg/day) (EFSA NDA Panel, 2012).
For infants, the Panel notes that, based on the intake assessment for vitamin D in infants (EFSA
NDA Panel, 2018), combined highest P95 intakes of vitamin D from background diet, including IF
and/or FoF and fortiﬁed foods and foods in which the NF would be added (see Section 3.7.4), do
not exceed the ULs set for infants aged 0–6 months at 25 lg/day and for infants 6–12 months at
35 lg/day. With regard to infants aged ≥ 7 to 12 months, the Panel notes that the additional intake of 10
lg/day of vitamin D2 from the NF used as an ingredient in food supplements, in high consumers ofvitamin D (from background diet, including IF and/or FoF and fortiﬁed foods), would result in a total
vitamin D intake of 32 lg/day, close to, but not exceeding the UL of 35 lg/day.
Given the assumption that infants who are high consumers of IF and/or FoF may also be highconsumers of foods fortiﬁed with the NF, then some infants aged 0–6 months may be at risk of
exceeding the UL for vitamin D (25 lg/day). For infants aged 7–12 months, using the (most likely
overestimated) intake of 29.1 lg vitamin D/day, the additional intake of 10 lg/day could result inexceeding the UL of 35 lg/day. However, the Panel considers this scenario unlikely as complementary
feeding in high consumers of IF and/or FoF may be limited.
Considering that daily oral supplementation of 10 lg vitamin D is generally recommended for all
infants during the ﬁrst year of life starting from birth onwards (ESPGHAN Committee on Nutrition,
Braegger et al., 2013 cited in EFSA NDA Panel, 2016b), there is a potential risk of approaching or
exceeding the UL for vitamin D in infants. However, this is a general issue related to the combined
consumption of vitamin D via fortiﬁed foods and supplements and does not speciﬁcally concern the NF
of this application.
The Panel considers that consumption of the NF is not nutritionally disadvantageous.

3.10.

Toxicological information

No speciﬁc toxicity studies on the NF have been submitted by the applicant. For previous scientiﬁc
opinions on similar NFs (on UV-treated baker’s yeast, bread or milk), no toxicological studies wereconsidered necessary (EFSA NDA Panel, 2014, 2015, 2016c).

3.10.1.

Genotoxicity

No speciﬁc studies on genotoxicity of the NF were provided. The Panel notes that given the source,
nature, and the intended use of the NF, genotoxicity studies are not required.

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Safety of vitamin D2 mushroom powder

3.10.2.

Subacute, subchronic and chronic toxicity

No speciﬁc studies with the NF have been carried out, but several publicly available animal studies
with material similar to the NF have been submitted by the applicant. The studies in which powder
from UV-treated Agaricus bisporus was tested are summarised in Appendix A (subacute and chronic
toxicity studies).

3.10.3.

Carcinogenicity

No studies on carcinogenicity were provided. The Panel notes that given the source, nature, andthe intended use of the NF carcinogenicity studies are not required.

3.10.4.

Reproductive and developmental toxicity

No studies on reproductive and developmental toxicity were provided. The Panel notes that given
the source, nature, and the intended use of the NF reproductive and developmental toxicity studies
are not required.

3.10.5.

Human data

No speciﬁc studies on the NF were provided, but the applicant provided several publicly available
human intervention studies where material similar to the NF has been tested. The studies in which
powder from UV-treated Agaricus bisporus was investigated are summarised in Appendix B.
The studies presented in Annex B show that consumption of powder from UV-treated mushrooms
from 4 to 6 weeks, signiﬁcantly increases serum concentrations of 25(OH)D2 and total 25(OH)D in
humans, while no other changes in blood parameters or adverse effects (see Appendix B for individual
parameters) were observed. The doses of vitamin D2 (contained in the mushroom powders) given ranged
between 15 and 17.1 lg/day. The Panel notes that the estimated ‘worst case’ vitamin D2 intakes inhumans, combining intake of vitamin D2 from the NF together with other dietary sources (seeSection 3.7.4 ‘Combined vitamin D intake from the NF and other sources’ of this opinion), were higher(ranging from 40.5 to 60.2 lg/day in the different age groups) than these doses. Therefore, the humanstudies presented are of limited value for the assessment of potential effects on human health of the NF.

3.11.

Allergenicity

The applicant refers to the conclusion by the Food Safety Authority of Ireland (FSAI) in 2017
regarding UV-treated mushrooms approved as novel food, i.e. that ‘allergenicity or other foodhypersensitivities associated with Agaricus bisporus are rare and there is no reason to believe that theadditional UV treatment will alter that risk’ (FSAI, 2017). The Panel concurs with this view.

4.

Discussion

The NF is produced from Agaricus bisporus mushrooms that have been exposed to UV light toinduce the conversion of provitamin D2 (ergosterol) to vitamin D2 (ergocalciferol). The NF contains
levels of vitamin D provided by vitamin D2 in the range of 1,000–1,300 lg/g.
The information provided on the manufacturing process, composition and speciﬁcations of the NF
does not raise safety concerns.
The applicant intends to add the NF in a variety of foods and beverages, including food for special
medical purposes (excluding those intended for infants) and food supplements. The target populationis the general population except for food supplements, for which the target population is individuals
above seven months of age.
The conservative highest vitamin D estimates for combined intake of vitamin D2 from the NF together
with intake from other dietary sources were below the upper levels for vitamin D as established previously
by the NDA Panel for ‘Young children’ and ‘Other children’, ‘Adolescents’, ‘Adults’, ‘Pregnant women’,‘Lactating women’, ‘Elderly’ and ‘Very elderly’ (NDA Panel, 2012) and ‘Infants’ (NDA Panel, 2018).
The Panel notes that vitamin D intake in high consumers of IF and or FoF with the maximum
regulated content of vitamin D consuming also fortiﬁed foods as calculated by EFSA (NDA Panel, 2018)
does not result in exceeding the ULs set for infants aged 0–6 months or ≥ 7–12 months.
Given the assumption that infants aged 0–6 months who are high consumers of IF and/or FoF may
also be high consumers of foods fortiﬁed with the NF, some infants may be at risk of exceeding the UL for
vitamin D (25 lg/day). For infants aged 7–12 months, using the (most likely overestimated) intake ofwww.efsa.europa.eu/efsajournal

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EFSA Journal 2020;18(1):5948

Safety of vitamin D2 mushroom powder29.1 lg vitamin D/day, the additional intake of 10 lg/day could result in exceeding the UL of 35 lg/day.
However, the Panel considers this scenario unlikely as complementary feeding in high consumers of IF
and/or FoF may be limited.
Considering that daily oral supplementation of 10 lg vitamin D is generally recommended for all
infants during the ﬁrst year of life starting from birth onwards (ESPGHAN Committee on Nutrition,
Braegger et al., 2013 cited in EFSA NDA Panel, 2016b), there is a potential risk of approaching or
exceeding the UL for vitamin D in infants. However, this is a general issue related to the combined
consumption of vitamin D via fortiﬁed foods and supplements and does not speciﬁcally concern the NF
of this application.

5.

Conclusions

The Panel concludes that the NF, vitamin D2 mushroom powder, is safe under the proposed
conditions of use. The target population is the general population, except for food supplements forwhich the target population is individuals above 7 months of age.
The Panel concludes that the NF, used as an ingredient, is safe for the general population at the
proposed condition of use in foods and beverages.
The Panel concludes that the NF is safe for the adult population at intake levels up to 15 lg vitamin
D2/day used in foods for special medical purposes.
The Panel concludes that the NF, used as a food supplement, is safe for individuals above 1 year at
a level up to 15 lg vitamin D2/day.
The Panel, however, notes that the UL for infants may be exceeded in high consumers of IF and/or
FoF that may also be high consumers of foods fortiﬁed with the NF, and infants aged 7–12 months
consuming the food supplement containing 10 lg of vitamin D. However, the Panel considers this
scenario unlikely as complementary feeding in high consumers of IF and/or FoF may be limited.
Considering that daily oral supplementation of 10 lg vitamin D is generally recommended for all infants
during the ﬁrst year of life starting from birth onwards (ESPGHAN Committee on Nutrition, Braegger et al.,2013 cited in EFSA NDA Panel, 2016b), there is a potential risk of approaching or exceeding the UL for
vitamin D in infants. However, this is a general issue related to the combined consumption of vitamin D via
fortiﬁed foods and supplements and does not speciﬁcally concern the NF of this application.
The Panel could not have reached the conclusions on the safety of the NF under the proposed
conditions of use without the following data claimed as proprietary by the applicant:•••Annex I: Raw materials and processing aids
Annex II : Certiﬁcates of Analysis and batch data
Annex III: Stability reports.

Steps taken by EFSA
1) Letter from the European Commission to the European Food Safety Authority with therequest for a scientiﬁc opinion on the safety of vitamin D2 mushroom powder as a NF. Ref.
Ares (2018)5350698, dated 18 October 2018.
2) On 18 October 2018, EFSA received a valid application from the European Commission on
vitamin D2 mushroom powder as NF, which was submitted by Oakshire Naturals, LP, and thescientiﬁc evaluation procedure started.
3) On 30 January, 4 June and 28 October 2019, EFSA requested the applicant to provide
additional information to accompany the application and the scientiﬁc evaluation was
suspended.
4) On 4 April, 21 August, 12 and 26 November 2019, additional information was provided by
the applicant and the scientiﬁc evaluation was restarted.
5) During its meeting on 28 November 2019, the NDA Panel, having evaluated the data,
adopted a scientiﬁc opinion on the safety of vitamin D2 mushroom powder as a novel foodpursuant to Regulation (EU) 2015/2283.

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Abbreviations
24,25(OH)2D325(OH)D225(OH)D3
ADME
AOAC
AI
AST
BMI
BP
bw
CAS
CPK
FoF
FSAI
FSMA
GAP
HACCP
HDL
HPLC
hsCRP
IF
IL-6
iPTH
IU
LDH
LDL
MB
ML
ND
NDA
NEFA
NF

24,25-hydroxycholecalciferol
25-hydroxyvitamin D2
25-hydroxyvitamin D3
absorption, distribution, metabolism and excretion
Association of Ofﬁcial Analytical Chemists
adequate intake
aspartate aminotransferase
body mass index
blood pressurebody weight
Chemicals Abstracts Service
creatine phosphokinase
follow-on formula
Food safety Authority of Ireland
Food Safety Modernization Act
Good Agricultural Practices
Hazard Analysis Critical Control Points
high-density lipoprotein
high-performance liquid chromatography
high-sensitivity C-reactive protein
Infant formulainterleukin 6
intact parathyroid hormone
International Unitslactate dehydrogenase
low-density lipoprotein
myoglobinmaximum levels
not detected
Nutrition, Novel Foods and Food Allergens
non-esteriﬁed fatty acids
novel foodwww.efsa.europa.eu/efsajournal

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Safety of vitamin D2 mushroom powder

NFI
OECD
PAI-1
PTH
RCT
TAG
TNF-a
UL
USP
UV

novel food ingredient
Organisation for Economic Cooperation and Development
plasminogen activator inhibitor-1
parathyroid hormone
randomised clinical trial
triacylglycerol
tumour necrosis factor-atolerable upper intake level
United States Pharmacopeia
ultraviolet

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Safety of vitamin D2 mushroom powder

Appendix A – Subacute and chronic toxicity animal studies with powder of UV-radiated Agaricus bisporus
Route ofSpecies (strain), sex;
administration/ Test item and dose groups
number of animals
duration
Mouse (transgenic
APPswe/PS1dE9 andwild-type); M; 10–13
per group

Oral (diet);
7 months

Rat (SD); F; 6 pergroup

Oral (diet);
10 weeksRat (SD); M; 5 pergroup

Oral (gavage);
3 weeks

Parameters evaluatedWild-type groups: control (basal diet); 5% UV-Cirradiated Agaricus bisporus mushroom powder (54IU vitamin D2/kg or 1.35 lg vitamin D2/kg feed)
Transgenic groups: control (basal diet); 5% UV-Cirradiated Agaricus bisporus mushroom powder(54IU vitamin D2/kg or 1.35 lg vitamin D2/kg feed).
These feed concentrations correspond to a dose ofaround 0.18 lg vitamin D2/kg bw per day(a)Clinical signs, bw, Morris
water maze, Barnes maze,
Y-maze, liver function
biomarkers (plasma total
protein, albumin, globulin),cholesterol, calcium, 25(OH)D2, 25(OH)D3, b-amyloid
load (Ab40, Ab42) in plasma
and brain,
immunohistochemistry(brain)Control (basal diet); control (vitamin D-deﬁcient
Bw, femur density, plasmadiet); Control (non-irradiated Agaricus bisporus
25(OH)D2 andmushroom powder); 2.5 or 5% UV-B-irradiatedhistopathology (kidney, liver,
Agaricus bisporus mushroom powder corresponding and spleen)
to 300 or 600 IU or 7.5 or 15 lg vitamin D2/day.
These correspond to doses of around 37.5 or 75 lg
vitamin D2/kg bw per day(a)Control (non-irradiated Agaricus bisporus
bw, food intake and plasma
mushrooms) 50, 100 or 200 mg/kg bw per day UV- 25(OH)D2
C-irradiated Agaricus bisporus mushroom powder.
At a concentration of 17.6 lg vitamin D2/g (asreported in the paper), this corresponds to doses of
0.88, 1.76 and 3.52 lg vitamin D2/kg bw per day

Results

Reference

No reported clinical signs or
Bennettdifferences in bw gain
et al.
between groups. No(2013)
differences in liver function
biomarkers or cholesterol
between groups. ↑ 25(OH)D2
and 25(OH)D3, ↑learning andmemory and ↓ b-amyloid
plaque load in brain of treatedanimals
No reported clinical signs or
Calvo et al.
differences in bw between(2013)
groups. ↑ 25(OH)D2 in treatedanimals
No test item-related
histopathological ﬁndings
Koyyalamudi
No reported clinical signs oreffects on bw or food intake.↑ et al.
(2009)
25(OH)D2 in animals given
vitamin D2 mushroom powder(all doses)

25(OH)D2: 25-hydroxyvitamin D2; 25(OH)D3: 25-hydroxyvitamin D3; bw: body weight; F: females; IU: International Units; M: males; SD: Sprague–Dawley; UV: ultraviolet.
(a): Because doses were not reported, concentrations in feed were converted to doses following EFSA guidance (EFSA Scientiﬁc Committee, 2012).

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Safety of vitamin D2 mushroom powder

Appendix B – Human intervention studies with powder of UV-radiated Agaricus bisporus
Study design

Duration

Tested material

DosageDouble-blinded,
randomised, placebocontrolled trial; 38
healthy adults (9–10
per group)

6 weeks

UV-B-irradiatedAgaricus bisporus
mushroom powder(10–25 lg/serving)

Placebo control: untreated mushrooms
Stephensen
Serum 25(OH)D2, 25(OH)D3; No reported
providing 34 IU (or 0.85 lg) vitamin D2/day; 24,25(OH)2D3
et al. (2012)
adverse effects. ↑25(OH)D2 and ↓ 25
Test group 1: UV-B-irradiated mushroom(OH)D3 in Test
powder (352 IU (8.8 lg) vitamin D2/day);Test Group 2: UV-B-irradiated mushroomgroups 1, 2 andpowder (684 IU (17.1 lg) vitamin D2/day);supplement group
Supplement Group: puriﬁed ergocalciferol +untreated mushrooms (total of 1,128 IU(28.2 lg) vitamin D2/day)Parameters investigatedResults

Reference

Double-blinded,
randomised, placebocontrolled trial; 33
vitamin D insufﬁcienthighschool athletes(16–17 per group)

6 weeks

UV-B-irradiatedAgaricus bisporus
mushroom powderPlacebo control: Untreated mushroom
Serum total 25(OH)D, 25
powder (ND vitamin D2); Test Group: UV-B- (OH)D2, 25(OH)D3, LDH,
irradiated mushroom powder (600 IU (15 lg) AST, CPK, MB
vitamin D2/day)

No reported
adverse effects. ↑25(OH)D2, ↓ 25
(OH)D3 and ↑ total
25(OH)D in testgroup

Shanely et al.
(2014)

No effects on the
blood parametersinvestigatedDouble-blinded,
randomised, placebocontrolled trial 90
healthy adults (22 pergroup)

4 weeks

UV-B-irradiatedAgaricus bisporus
mushroom powder

Study 1: Placebo control: Untreatedmushroom powder (ND vitamin D2)
Test Group: UV-irradiated mushroom powder(600 IU (15 lg) vitamin D2/day)Study 2: Placebo control: placebo capsule(ND vitamin D3); Supplement Group: vitamin
D3 capsule (600 IU (15 lg) vitamin D3/day)Bw, height, BMI, BP (systolic No reported
adverse effects. ↑and diastolic)25(OH)D2 in test
Serum total 25(OH)D, 25(OH)D2, 25(OH)D3, glucose, group and ↑ 25
(OH)D3 in vitamininsulin, HR, HOMA-IR, CD3 supplement
peptide, TAG, cholesterol(total, HDL, and LDL), NEFA, group
PTH, calcium, adiponectin,
ferritin, leptin, resistin, sCRP, No effects on the
TNF-a, IL-6, PAI-1
blood parametersinvestigated

Stepien et al.
(2013)

24,25(OH)2D3: 24,25-hydroxycholecalciferol; 25(OH)D2: 25-hydroxyvitamin D2; 25(OH)D3: 25-hydroxyvitamin D3; AST: aspartate aminotransferase; BMI: body mass index; BP: blood pressure; bw:
body weight; CPK: creatine phosphokinase; HDL: high-density lipoprotein; hsCRP: high-sensitivity C-reactive protein; IL-6: interleukin 6; iPTH: intact parathyroid hormone; IU: International Units;
LDH: lactate dehydrogenase; LDL: low-density lipoprotein; MB: myoglobin; ND: not detected; NEFA: non-esteriﬁed fatty acids; PAI-1: plasminogen activator inhibitor-1; PTH: parathyroid hormone;
TAG: triacylglycerol; TNF-a: tumour necrosis factor-a; UV: ultraviolet.

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