Position papers

ACNFP Advice on the safety of UV-treated A. Bisphorus Mushroom Powder with increased vitamin D2 as a novel food.

Committee Advice Document

Last updated: 23 January 2025

Summary

An application was submitted to the Food Standards Agency (FSA) and Food Standards Scotland (FSS) in April 2022 from Monterey Mushrooms, LLC (“the applicant”) for the authorisation for UV-treated Agaricus bisphorus Mushroom Powder with increased vitamin D2 as a novel food.

The novel food is an ingredient produced by the exposure of A. bisporus mushrooms to ultraviolet light which catalyses the conversion of endogenous ergosterol within the mushrooms to vitamin D2. The mushrooms are then dehydrated and ground into a powder.

This new application is seeking to use the novel food as an ingredient for various food products, beverages and food supplements with levels of vitamin D2 in the range of 125 - 463 µg/g.

To support the FSA and FSS in their evaluation of the application, the Advisory Committee on Novel Foods and Processes (ACNFP) were asked to review the safety dossier and supplementary information provided by the applicant. The Committee did not consider any potential health benefits or claims arising from consuming the food, as the focus of the novel food assessment is to ensure the food is safe, and not putting consumers at a nutritional disadvantage.

The Committee advised that the applicant had provided sufficient information to assure that the novel food, UV-treated A. bisphorus Mushroom Powder with increased vitamin D2, was safe under the proposed conditions of use. The anticipated intake level and the proposed use in food supplements was considered not to be nutritionally disadvantageous.

1. Introduction

1. The ACNFP assessed the food safety risks of UV-treated A. bisphorus Mushroom Powder with increased vitamin D2 and its production under the proposed uses, in line with Article 7 of regulation (EU) 2017/2469, retained in UK law. The regulatory framework and the retained technical guidance (published by EFSA for full novel food applications) formed the basis and structure for the assessment (EFSA NDA Panel, 2016).

2. In April 2022, Monterey Mushrooms, LLC (“the applicant”) submitted a full novel food application for the authorisation of UV-treated A. bisphorus Mushroom Powder with increased vitamin D2. The novel food is produced by the exposure of Agaricus bisporus mushrooms to ultraviolet light which catalyses the conversion of endogenous ergosterol within the mushrooms to vitamin D2. The novel food is intended to be used as an ingredient in various food products, beverages and food supplements. The target population is the general population excluding those under 7 months of age for food supplements.

3. Following the review by the ACNFP in September 2023, further information was requested concerning the production process, composition, specification, stability and allergenicity of vitamin D2 mushroom powder. The final advice from the Committee was agreed in October 2024, allowing the FSA and FSS to complete the risk assessment.

4. This Committee Advice Document (CAD) outlines the conclusions of the ACNFP on the safety of UV-treated A. bisphorus Mushroom Powder with increased vitamin D2 as a novel food.

2. Assessment

2.1 Identity of Novel Food

5. The novel food is a mushroom powder containing vitamin D2 which is produced by subjecting fungi of the species Agaricus bisporus, commonly known as cultivated mushrooms, to ultraviolet irradiation. Confirmation that Agaricus bisporus mushrooms are used in the production process was carried by high-performance thin-layer chromatography (HPTLC).

6. Vitamin D2 is characterised by the following information:

IUPAC name (3β,5Z,7E,22E)-9,10-secoergosta-5,7,10(19),22-tetraen-3-ol

CAS number 50-14-6

Molecular formula C28H44O

Structural formula

2.2 Production Process

7. The production process for UV-treated A. bisphorus Mushroom Powder with increased vitamin D2 involves one raw material; Agaricus bisporus mushrooms, which are grown and harvested in the USA under controlled environmental conditions in compliance with Regulation (EC) No. 852/2004 and within the principles of HACCP.

8. The mushrooms are diced to increase the surface area for exposure to the UV light units which are adjusted according to the targeted level of vitamin D2. The diced mushrooms are then dehydrated, ground and packaged in a temperature-controlled environment to control moisture levels in the product.

9. Prior to packaging, microbial and vitamin D2 content analysis is performed to ensure safety as well as determine that the amount of vitamin D2 is within the food specification level of 125-46 µg/g.

10. The ACNFP raised some questions on the details of the production process. It was noted that the variability of the vitamin D2 content resulted from the variation of the effectiveness of the ultraviolet irradiation part of the process due to factors including exposure time, lamp properties, as well as variation in the raw material. The conditions are managed to ensure the vitamin D2 level in the final product falls within the target range. It was noted that the product may be blended again after final testing if the vitamin D levels do not meet client-specific requirements.

11. Further queries were raised on how it was ensured that the final product was homogeneous and that samples of the novel food tested for safety parameters such as moisture and Vitamin D2 content reflected the composition of the batch tested. Details of the blending and sampling processes were provided. The applicant undertook a blender validation study involving 3 batches of vitamin D2 mushroom powder, to demonstrate the efficiency of the cone blender for creating homogeneous blends with targeted levels of vitamin D2 according to the specification. Information was also provided on how contamination was minimised during these stages of production and controls in the packaging stage to manage moisture levels.

12. The production process has characterised the potential hazards and it was concluded that the corresponding control measures are appropriate and did not raise safety concerns.

2.3 Compositional Information

13. To verify the compositional analysis provided was accurate and reliable, certification was provided to demonstrate that the laboratories were accredited to perform these analytical studies. Where in-house analysis was utilised, full methodology and supporting validation documentation were provided.

14. Results from five independent batches of UV-treated A. bisphorus Mushroom Powder with increased vitamin D2 demonstrated that the novel food is produced consistently (Table 1). Vitamin D2 levels were assayed using high-performance liquid chromatography (HPLC). The analytical method is similar to that described by Phillips et al. (2011, 2012).

Table 1. Compositional analysis of the novel food.

Parameter	Specification	Method of Analysis	Batch No. 1	Batch No. 2	Batch No. 3	Batch No. 4	Batch No. 5
Vitamin D2 (μg/g)	125 to 462.5*	HPLC	151.5	169.4	167.0	159.3	182.6
Moisture	≤7.0	O'haus Scale	<5	<5	<5	<5	<5

Physical Parameters

Parameter	Specification	Method of Analysis	Batch No. 1	Batch No. 2	Batch No. 3	Batch No. 4	Batch No. 5
Mesh size	90% through 80 mesh sieve	Sieving	Conforms	Conforms	Conforms	Conforms	Conforms
Appearance	Light to dark brown milled powder	NA	Conforms	Conforms	Conforms	Conforms	Conforms
Aroma	Earthy	NA	Conforms	Conforms	Conforms	Conforms	Conforms
Taste	Mushroom savoury, umami flavour	NA	Conforms	Conforms	Conforms	Conforms	Conforms

Microbiological

Parameter	Specification	Method of Analysis	Batch No. 1	Batch No. 2	Batch No. 3	Batch No. 4	Batch No.5
Aerobic plate count (CFU/g)	<5,000	AOAC 966.23	<10	<10	<10	<10	<10
Yeast (CFU/g)	<100	FDA-BAM, 7th ed.	30	30	30	30	30
Mould (CFU/g)	<100	FDA-BAM, 7th ed.	Negative	Negative	Negative	Negative	Negative
Salmonella spp.	Negative in 25 g	AOAC 2004.03	Negative	NT	Negative	Negative	Negative
S. aureus	Negative in 10 g	AOAC 975.55
E. coli	Negative in 10 g	Current USP/NF, 62	Negative	Negative^a	Negative	Negative	Negative
Coliforms (MPN/g)	<100	AOAC 966.24	43	23	23	93	9.1

Listeria			Batch No. 7	Batch No. 8	Batch No. 9	Batch No. 10	Batch No.11
Listeria spp. (CFU/g)	Negative in 25 g	AOAC 2004.06	Negative	Negative	Negative	Negative	Negative

HPLC = high-performance liquid chromatography; NA = not applicable. *Converted from 5,000 to 18,500 International Units (IU) using the conversion factor of 0.025 μg = 1 IU stated in the EFSA Technical Report on Dietary Reference Values for nutrients (EFSA, 2017).

AOAC = Association of Official Analytical Chemists; CFU = colony-forming units; MPN = most probable number; NT = not tested; FDA-BAM = United States Food and Drug Administration Bacteriological Analytical Manual.

^a3 tube MPN method (Method Reference: AOAC 966.24). No growth of the target organism at the limit of detection, 3 MPN/g

15. It was noted that one batch was analysed using a different method of analysis, MPN-most probable number technique, which estimates the concentration of a microbe in a sample. No microbial growth was seen below the higher limit of detection for this test of 3MPN/g hence, all samples were considered to be within specification. Laboratory analysis showed that microbial contamination was appropriately managed and that controls were working effectively.

16. A Certificate of Analysis for testing of a wide range of pesticides showed that no residues were detected in the novel food.

17. Heavy metal levels were analysed and compared to established EU limits (Table 2). All analyses were below EU limits.

Table 2. Heavy metals analysis for 6 batches of the novel food.

Parameter	EU Limits^a(wet weight)	Monterey Specification	Method of Analysis	Batch No. 1	Batch No. 2	Batch No. 3	Batch No. 4	Batch No. 5	Batch No.6
Arsenic (mg/kg)	0.3	≤0.3	USP 730	0.23	0.19	0.28	0.29	0.20	0.22
Cadmium (mg/kg)	0.2	≤0.5	USP 730	0.067	0.061	0.066	0.063	0.050	0.035
Lead (mg/kg)	0.3	≤0.5	USP 730	0.02	0.02	0.02	0.02	0.04	0.01
Mercury (mg/kg)	0.1	≤0.1	USP 730	0.027	0.021	0.042	0.035	0.027	0.025

EU = European Union; USP = United States Pharmacopeia.

^a Maximum level according to Commission Regulation (EC) No. 1881/2006 (taken from the closest relevant categories)

18. Three batches of the novel food were analysed for the presence of biologically inactive photoisomers (lumisterol and tachysterol) which are formed during the conversion of ergosterol to vitamin D2. These photoisomers are also formed endogenously in humans during cutaneous production of vitamin D following exposure to UV-B radiation from sunlight. The results demonstrated that only negligible amounts are detected in the final ingredient (Table 3), and the amounts are lower than those in other UV-treated foods such as baker’s yeast and bread, which gave rise to no safety concerns in previous EFSA Scientific Opinions (EFSA NDA Panel, 2014 & 2015).

Table 3. Concentration of Lumisterol and Tachysterol in the novel food.

Parameter	Method of Analysis	Batch No. 12	Batch No. 13	Batch No. 14
Lumisterol (mg/g)	HPLC	0.05	0.03	0.08
Tachysterol (mg/g)	HPLC	0.004	0.006	0.01

19. The presence of aflatoxins was measured in 6 independent batches of UV- treated A. bisphorus Mushroom Powder with increased vitamin D2. The maximum levels permitted in foods were taken from the closest relevant category (cereals and all products derived from cereals, including processed cereal products) detailed in assimilated Commission Regulation (EC) No 1881/2006, as amended, for aflatoxin B1 and the total sum of B1, B2, G1 and G2. Results demonstrated that the levels to be low and within established limits (Table 4).

Table 4. Aflatoxin analysis for 6 Batches of the novel food.

Parameter	Method of Analysis	EU Limit^a(μg/kg)	Batch No. 15	Batch No. 16	Batch No. 17	Batch No. 18	Batch No. 19	Batch No. 20
Aflatoxins (sum of B1, B2, G1 and G2) μg/kg (w/w)	AOAC 991.31 (Mod.)^b	4	<0.7	<0.7	<0.7	<0.7	<0.7	<0.7
Aflatoxin B1 μg/kg (w/w)	AOAC 991.31 (Mod.)^b	4	<0.6	<0.6	<0.6	<0.6	<0.6	<0.6

EU = European Union.

^a Maximum levels according to assimilated Commission Regulation (EC) No. 1881/2006.

^b The modification applies to the initial steps in the assessment of the performance of the immuno affinity columns used for the isolation and purification of the aflatoxin compounds. Whereas the AOAC method uses a manual fluorescence procedure, the modified method uses a fortified sample (spiked sample) of 5 ng of each aflatoxin type (20 ng total) carried through the entire procedure including the full HPLC steps.

20. The data presented indicated the novel food can be produced consistently. No additional hazards to be explored further or included in the specification were identified.

2.4 Stability

21. Stability tests were carried out on four batches for 3 years and on 1 batch for 4 years to test the stability of the vitamin D2 content under representative storage conditions (17^oC-35^oC and 40%-63% humidity). The results showed the vitamin D2 levels remained within the specified range and supported the proposed 3-year shelf life. Microbial analysis was carried out on the samples in the 3-year study at the start and end of the storage period, without any concerns identified. The results of both tests suggests that that the product is stable for up to 3 years under the recommended storage conditions.

22. The stability of vitamin D2 content was also tested within some foods that the novel food would be an added ingredient (fruit juice drink and cereal bar) under typical commercial storage conditions for the products. In addition to the analysis of vitamin D2 content, sensory parameters were tested. No significant degradation of vitamin D2 was observed throughout the test period of 14 days (the standard shelf life) for the juice and 3 months for the cereal bar. Sensory results did not indicate notable taste changes for the products.

23. The ACNFP concluded that the data provided did not raise safety concerns for the stability of the novel food within the period of 36 months and storage conditions proposed.

2.5 Specification

24. The specification parameters for the novel food were provided by the applicant (Table 5).

Table 5: Specification for UV-treated A. bisphorus Mushroom Powder with increased vitamin D2.

General Description - UV-treated A. Bisphorus Mushroom Powder with increased vitamin D2 is a light to dark brown milled powder, produced by exposing sliced/diced Agaricus bisporus mushrooms to ultraviolet light followed by homogenisation to form a powder.

Parameter	Specification	Method
Vitamin D2	125 to 462.5 μg/g*	HPLC-DAD
Moisture	≤7%	O'haus Scale
Water activity	<0.5%	AOAC 978.18
Ash	≤13.5%	AOAC 945.46

Physical Parameter

Parameter	Specification	Method
Appearance	Light to dark brown milled powder	Organoleptic
Aroma	Earthy	Organoleptic
Taste	Mushroom savoury, umami flavour	Organoleptic

Proximate Analysis

Parameter	Specification	Method
Total Fat	≤4.5%	AOAC 996.06
Total Carbohydrate	≤60%	Calculation
Protein	≤40%	AOAC 992.23

Microbiological Parameters

Parameter	Specification	Method
Standard plate count	<5,000 CFU/g	AOAC 966.23
Total yeast and mould count	<100 CFU/g	FDA-BAM, 7th ed.
Salmonella	Negative in 25 g	AOAC 2004.03
Staphylococcus aureus	Negative in 10 g	AOAC 975.55
Escherichia coli	Negative in 10 g	Current USP/NF, 62
Coliform	<100 MPN/ g	AOAC 966.24
Listeria spp.	Negative in 25 g	AOAC 2004.06
Aflatoxins (sum of B1, B2, G1 and G2)	<4 μg/kg	AOAC 991.31 (Mod.)
Aflatoxin B1	<2 μg/kg	AOAC 991.31 (Mod.)

Heavy Metals

Parameter	Specification	Method
Arsenic	≤0.3 mg/kg	USP 730
Lead (as Pb)	≤0.5 mg/kg	USP 730
Mercury	≤0.1 mg/kg	USP 730
Cadmium	≤0.5 mg/kg	USP 730

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; MPN = most probable number; USP = United States Pharmacopeia.

*Converted from International Units (IU) using the conversion factor of 0.025 μg = 1 IU stated in the European Food Safety Authority Technical Report on Dietary Reference Values for nutrients (EFSA, 2017).

25. The ACNFP concluded the information provided is sufficient for the specification of UV-treated A. bisphorus Mushroom Powder with increased vitamin D2, and appropriately characterises the novel food.

2.6 History of Use

26. It was cited that wild edible fungi, including macrofungi such as mushrooms, have been collected and consumed for thousands of years (FAO, 2004). Cultivated and wild grown A. bisporus mushrooms (the source of the novel food) have been consumed within and outside of the UK for a long time (FSAI, 2017).

27. The novel food, UV-treated A. bisphorus Mushroom Powder with increased vitamin D2 has no history of use in the UK. As the UV irradiation process is considered a novel process and the parameters for its operation will be slightly different between operators each is subject to a separate review and authorisation.

28. UV-treated A. bisporus mushrooms have also been consumed in the UK with their approval as a novel food ingredient since 2016 (FSAI, 2027). A similar ingredient produced by a different manufacturer using a different production process, vitamin D2 mushroom powder produced by homogenisation of mushrooms before exposure to UV light was authorised as a novel food in 2020 (EUR-Lex, 2020). Another form of vitamin D2 mushroom powder was considered safe under its intended uses (EFSA, 2021) and is authorised for use in the EU. The novel food that is subject to this application, uses a different process whereby mushrooms are first exposed to UV light before being dried and ground into a powder.

29. The history of use does not indicate any further areas for concern.

2.7 Proposed Use and Anticipated Intake

30. UV-treated A. bisphorus Mushroom Powder with increased vitamin D2 used in foods and beverages is intended to be consumed by the general population. The vitamin D2 mushroom powder is to be used in food supplements as defined in GB food supplements legislation and is intended for the general population of 7 months of age or above.

31. A summary of the foods proposed to use the novel food as an ingredient and the maximum use levels for the novel ingredient in the form of a powder is provided in Table 6. These include breakfast cereals, dairy analogues, fruits and vegetable juices and non-alcoholic beverages.

Table 6. Proposed Food Uses and Use Levels for UV treated A. bisphorus Mushroom Powder with increased vitamin D2.

Conventional Food Categories:

Food Categories [for inclusion in the Union list of novel foods]	Specified Food Categories^a	Expected level of Vitamin D2 from proposed use, as consumed (μg/100 g or 100 mL)	Novel Food Use Level (mg/100 g or 100 mL)
Breakfast cereals (RTE)	Breakfast cereals (RTE)	2.25	4.9 to 18.0
Cereal and nutrition bars	Cereal bars	2.25	4.9 to 18.0
Cereal and nutrition bars	Energy bars	2.25	4.9 to 18.0
Cereal and nutrition bars	Protein bars	2.25	4.9 to 18.0
Chocolate confectionary	Superfood chocolate bars^b	2.25	4.9 to 18.0
Dairy analogues	Milk imitates	1.125	2.4 to 9.0
Dairy analogues	Cream imitates, including non-dairy coffee creamer	2.25	4.9 to 18.0
Dairy analogues	Non-dairy yogurt	2.25	4.9 to 18.0
Dairy analogues	Non-dairy cheese	2.25	4.9 to 18.0
Dairy analogues	Non-dairy ice cream	2.25	4.9 to 18.0
Fruit juices	Fruit juices and nectars (including powders and concentrates)	1.125	2.4 to 9.0
Vegetable juices	Vegetable juices (including powders and concentrates)	1.125	2.4 to 9.0
Hot breakfast cereals (dry and RTE)	Hot breakfast cereals (instant and regular)	2.25	4.9 to 18.0
Non-alcoholic beverages	Energy Drinks	1.125	2.4 to 9.0
Non-alcoholic beverages	Sports and Isotonic Drinks	1.125	2.4 to 9.0
Non-alcoholic beverages	Fortified water beverages	1.125	2.4 to 9.0
Non-alcoholic beverages	Fruit smoothies (RTD)	1.125	2.4 to 9.0
Non-alcoholic beverages	Fruit juice-based drinks	1.125	2.4 to 9.0
Non-alcoholic beverages	Protein drinks	2.25	4.9 to 18.0
Non-alcoholic beverages	Nutritional drinks	2.25	4.9 to 18.0
Soups and broths	Soups (RTE and dry mixtures)	2.25	4.9 to 18.0

Specialised Food Categories:

Food Categories [for inclusion in the Union list of novel foods]	Expected level of Vitamin D2 from proposed use, as consumed (μg/100 g or 100 mL)	Novel Food Use Level (mg/100 g or 100 mL)
Food supplements as defined in Directive 2002/46/ECc, excluding food supplements for infants and young children	15 μg/day	32 to 120
Food supplements (infants 7–11 months)	10 μg/day	22 to 80
Foods for special medical purposes as defined in Regulation (EU) No 609/2013^d,e(excluding those intended for infants)	15 μg/day	32 to 120
Total diet replacement for weight control as defined in Regulation (EU) No 609/2013^d,e	5 μg/meal	10.8 to 40

EU = European Union; RTD = ready-to-drink; RTE = ready-to-eat; UK = United Kingdom.

^aSurrogates were included for this food use (see Appendix C).

^b All batches of UV treated A. Bisphorus Mushroom Powder with increased vitamin D2 are produced with a vitamin D2 content within the proposed specifications 125 to 462.5 μg/g (see Table 2.c.1-1 for further details). Thus, use levels of UV treated A. Bisphorus Mushroom Powder with increased vitamin D2 are dependent on the vitamin D2 content of the selected batch/batches used. In order to produce a conservative estimate, the highest use level (g powder/100 g food) was utilised.

^c EUR-Lex, 2002. Directive 2002/46/EC of the European Parliament and of the Council of 10 June 2002 on the approximation of the laws of the Member States relating to food supplements. Available online: https://www.legislation.gov.uk/eudr/2002/46/contents.

^d EUR-Lex, 2013. Regulation (EU) No 609/2013 of the European Parliament and of the Council of 12 June 2013 on food intended for infants and young 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 the European Parliament and of the Council and Commission Regulations (EC) No 41/2009 and (EC) No 953/2009. Available online: https://www.legislation.gov.uk/eur/2013/609/contents.

^e EUR-Lex, 2016. Commission Delegated Regulation (EU) 2016/128 of 25 September 2015 supplementing Regulation (EU) No 609/2013 of the European Parliament and of the Council as regards the specific compositional and information requirements for food for special medical purposes. Available online: https://www.legislation.gov.uk/eur/2016/128/contents.

32. The proposed maximum use levels of the novel food provide the following maximum additional intake of vitamin D2: for food products 2.25 μg/100g (15% of the adequate intake for vitamin D identified by EFSA), 1.125 μg/100ml (7.5% of the adequate intake identified by EFSA), for beverages, 15 μg/day for food supplements for age +1yr. 10μg/day for food supplements for infants 7-11 months, which is intended to provide the full adequate intake for vitamin D for the relevant age groups. This is based on EFSAs recent defined adequate intake (15μg/day for adults including children above the age of 1 and 10 μg/day for ages 7-11 months) as a replacement for nutrient reference value (EFSA, 2016b).

33. Estimated intakes of vitamin D2 calculated based on the proposed use levels as presented in Table 7 were provided. The estimated total intake of novel food (mg/person/day) from all proposed food uses were calculated using the lowest dose of vitamin D in the powder i.e., 125μg vitamin D2/g powder, giving a maximum dose of 18μg powder per 100g.

34. Also provided was a summary of the estimated daily per kilogram body weight intake of the novel food from all proposed food categories in the UK by population group (Table 7). This was calculated with consumption data from the UK National Diet and Nutrition Survey (NDNS).

Table 7. Summary of the Estimated Daily Intake of Vitamin D2 from the novel food from All Proposed Food Categories in the UK by Population Group (NDNS Data, Years 7 to 9).

Population Group	Age Group (Years)	n	Total Population Intake (μg/person/day) Mean	Total Population Intake (μg/person/day) 95th Percentile
Toddlers	1.5 to 3	309	0.252	0.691
Children	4 to 10	740	0.175	0.482
Female Teenagers	11 to 18	373	0.066	0.171
Male Teenagers	11 to 18	363	0.081	0.232
Female Adults	19 to 64	833	0.047	0.159
Male Adults	19 to 64	618	0.048	0.173
Elderly	≥65	430	0.034	0.107
Total Population	≥1.5	3,666	0.065	0.235

bw = body weight; n = sample size; NDNS = National Diet and Nutrition Survey; UK = United Kingdom.

35. The maximum intended use level in Foods for Special Medical Purposes (FSMPs) is 15μg vitamin D2/day, excluding those intended for infants. This is with the assumption that the powder would be the only source of vitamin D in the diet as many Foods for Special Medical Purposes replaces significant components in the diet. This is also the same maximum amount intended for total diet replacement for weight control and meal replacements for weight control.

36. The applicant considered the combined intake from the novel food and other sources. Results from the summary of the estimated daily intake of vitamin D from the total diet (Table 8) is used to calculate the cumulative exposure of vitamin D from the total diet. It is concluded that based on these results, 29μg/day for infants up to and including 11 months of age, and 25 and 26.3μg/day for toddlers and other children, respectively, do not exceed the tolerable upper intake level (UL) of 35μg/day established by EFSA for infants aged 6 to 12 months of age and that of 50μg/day for children aged 1 to 9 years. Similarly, the cumulative intakes of vitamin D from the total diet of 19.9, 30.5 and 23.3μg/day among adolescents, adults, and elderly subjects, respectively, are well below the UL of 100μg/day for each of these population groups.

Table 8. Estimated Daily Intake of Vitamin D from the Total Diet.

Population Group	Intake of Vitamin D from Foods (ug/day)^a 95^th percentile	Intakes of Vitamin D2 from the novel food (μg/day) 95^th percentile	Intake of Vitamin D2 from proposed use in Food Supplements (μg/day)	Intake of Vitamin D from the Total Diet (μg/day)^b 95^th percentile	UL (μg/day)
Infants (≤11 months)	19^c	10.0^d	10.0 (infants 7 to 11 months)	29^e	25 (0 to 6 months)^f 35 (6 to 12 months)^f
Toddlers (1 to 3 years)	15^c	10.0^d	15.0	25^e	50^g
Other children (3 to 9 years)	15^c	11.3	15.0	26.3^e	50^g
Adolescents (10 to 17 years)	8^c	9.8 to 11.9	15.0	17.8 to 19.9^e	100^g
Adults (18 to 64 years)	16	10.7 to 14.5	15.0	26.7 to 30.5	100^g
Elderly (65 to 74 years)	16	7.3	15.0	23.3	100^g

NDNS = National Diet and Nutrition Survey; UL = tolerable upper intake level.

^a EFSA NDA Panel (2016b): https://www.efsa.europa.eu/en/efsajournal/pub/4547.

^b Intake of vitamin D from the total diet = (intake of vitamin D from foods) + (intake of vitamin D2 from UV treated A. Bisphorus Mushroom Powder with increased vitamin D2 + (intake of vitamin D2 from food supplements).

^c Intake from foods and food supplements.

^d No data on intakes for infants in the NDNS; as such, the estimated values for toddlers were applied to the cumulative calculation for both infants and toddlers.

^e As the intake of vitamin D from foods also includes the intake from food supplements for this population group, the cumulative intake of vitamin D2 from the total diet does not include the intake of vitamin D2 from food supplements (to avoid duplication).

^f EFSA NDA Panel (2018): https://www.efsa.europa.eu/en/efsajournal/pub/5365.

^g EFSA NDA Panel (2012): https://www.efsa.europa.eu/en/efsajournal/pub/2813.

37. The ACNFP noted the data on the likely impact of vitamin D exposure and the vitamin D2 vitamin, in particular from consumption of the novel food. The additional consumption is predicted not to lead to increases above safe levels.

2.8 Absorption, Distribution, Metabolism and Excretion (ADME)

38. The applicant undertook 2 studies using UV-Irradiated mushroom products supplied by Monterey. The first was a human study performed in a randomised, double-blinded, placebo-controlled 6-week study involving 38 healthy adults (14 males and 24 females) allocated 1 of 4 groups. The groups consisted of; a placebo control group receiving nonexposed mushroom powder, a group receiving UV-B-exposed mushroom powder providing 8.8µg/day, a group receiving 17.1 μg/day of vitamin D2, plus the placebo control dose and 1 supplement control group receiving vitamin D2 supplements providing 28.2 μg/day vitamin D2. All groups consumed similar meals containing 1 serving (87.9 g) of cooked white button mushrooms. The study measured serum 25(OH)D2, 25(OH)D3, 24,25- hydroxycholecalciferol [24,25(OH)2D3] and total 25(OH)D from blood samples taken at baseline, after 3 weeks and after 6 weeks.

39. The authors of the study concluded that that vitamin D2 from UV-exposed mushrooms is absorbed and metabolized to 25(OH)D2. They measured a small but significant decrease in serum 25(OH)D3. The authors hypothesise that this is a result of the study being undertaken in a sunny climate and so exposure to vitamin D from sunlight was a confounder in interpreting their results due to wider regulation of vitamin D levels by the body (Stephensen et al., 2012).

40. The second human ADME study involved a 12-week randomised study carried out during winter to minimise influence of environmental UV-B exposure, where 25 healthy adults consumed either UV-exposed mushroom extract (supplied by Monterey), vitamin D2 supplement or vitamin D3 supplement (all providing 50μg vitamin D2/day) in capsules once daily for 12 weeks. The study measured serum 25(OH)D, 25(OH)D2 and 25(OH)D3 from blood samples taken weekly throughout the study, to determine the bioavailability of the supplements. The study concluded that the mushroom extract was as effective at increasing and maintaining total serum 25(OH)D levels as supplemental vitamin D2 (Keegan et al., 2013).

2.9 Nutritional information

41. The nutritional analysis of the novel food was provided. It consists of approximately 50% carbohydrates, 33% protein, 20% dietary fibre, 9% ash, 3% fat, and less than 5% moisture. It also contains minerals and vitamins.

42. The nutritional content of mushrooms exposed to UV light is unchanged, with the exception of the intended increase in vitamin D2 content (Simon et al., 2011). Vitamin D2 from UV-irradiated mushrooms has been shown to be bioavailable in several human studies using 25(OH)D as an indicator (Outila et al., 1999; Keegan et al., 2013; Stepien et al., 2013; Mehrotra et al., 2014; Shanely et al., 2014), and that increases in 25(OH)D have also been observed in animal studies (Jasinghe et al., 2005; Koyyalamudi et al., 2009; Bennett et al., 2013; Calvo et al., 2013).

43. The ADME studies outlined bioavailability, and it was noted that while serum levels increased in some studies this was not necessarily related to changes in vitamin D status. This might be because of a difference in starting vitamin D status or exposure to sunlight. This was explored further in a study undertaken in the winter months.

44. Based on this information, the consumption of the novel food as a source of vitamin D2 is not expected to be nutritionally disadvantageous for consumers under the proposed conditions of use.

2.10 Toxicological information

2.10.1 Sub-chronic toxicology and Genotoxicity

45. No genotoxicity and sub-chronic toxicology studies were provided by the applicant on the basis that the impact of the novel process is on the vitamin D2 content of mushrooms. There are not expected to be specific concerns as a result of the conversion of the endogenous precursor to vitamin D2.

46. It was noted there are a number of authorised foods that are UV-treated; UV-treated baker’s yeast, UV-treated bread and UV-treated milk. They also highlighted studies with ingredients similar to the novel food i.e. vitamin D2 mushroom powder produced by homogenisation of mushrooms before exposure to UV light (EFSA NDA Panel, 2020). No specific toxicological issues beyond vitamin D exposure were highlighted.

2.10.2 Human studies

47. The applicant provided a review of six human studies (Keegan et al., 2013, Mehrotra et al.,2014; Stephensen et al., 2012; Urbain et al., 2011, Stepien et al., 2013, Shanely et al., 2014), with the conclusion that daily consumption of UV-B-exposed A. bisporus mushrooms providing up to 65μg (2,600 IU) vitamin D2/day for 16 weeks. There were not any associated reported adverse effects. The studies indicated Vitamin D2 from UV-B-exposed A. bisporus mushroom products was bioavailable, as evidenced by increased serum 25(OH)D2 (the direct metabolite of vitamin D2) concentrations.

Toxicology conclusions

48. The ACNFP concluded that there were no additional toxicological issues to be considered in addition to vitamin D exposure in assessing the safety of the novel food.

2.11 Allergenicity

49. UV-treated A. Bisphorus Mushroom Powder with increased vitamin D2 is expected to have the same allergenic risk as that associated with consumption of A. Bisporus mushrooms, as the ultraviolet treatment did not alter the composition of the mushrooms apart from the content of vitamin D2.

50. The novel food is intended to be added as an ingredient to foods that do not normally contain mushrooms. Mushroom allergic consumers would not expect to find the novel food, and mushrooms more generally, in the range of products in which the applicant intends to add them. Risk managers will need to consider how the product will be labelled to support those with mushroom allergy avoiding the novel food.

3. Discussion

51. The novel food is UV-treated Agaricus bisphorus Mushroom Powder with increased vitamin D2, produced by the exposure of Agaricus bisporus mushrooms to ultraviolet light which catalyses the conversion of endogenous ergosterol within the mushrooms to vitamin D2. The mushrooms are then dehydrated and ground into a powder.

52. The target population is the general population. UV-treated A. bisphorus Mushroom Powder with increased vitamin D2 in food supplements is not intended to be consumed by those under the age of 7 months.

53. The novel food contains vitamin D2 content ranging between 125.0 to 463 μg/g and is intended to be used as ingredient to achieve a maximum dose of vitamin D2 of 2.25 μg/100 g for food products, and 1.125 μg/100 mL for beverages. In food supplements the proposed maximum dose of vitamin D2 is 15 μg/day in food supplements for the general population older than 1 year of age and at 10 μg/day in food supplements for infants from 7 to 11 months.

54. The proposed maximum vitamin D2 levels meet the Acceptable Intake level set by EFSA for these population groups. These use levels ensure that the required amount to be deemed as a source of vitamin D [as stipulated in Regulation (EC) No 1924/200619] would be met.

55. Combined intake of vitamin D2 from the novel food with that of the other dietary sources was considered to not exceed the upper limits for vitamin D previously established by the NDA Panel for infants (EFSA NDA Panel, 2018), and for children, adolescents and adults (EFSA NDA Panel, 2012).

4. Conclusions

56. The ACNFP have undertaken the assessment of U- treated mushrooms from Monterey Mushrooms Inc. and concluded that the novel food is safe under the proposed conditions of use and does not pose a safety risk to human health. The proposed uses are not considered nutritionally disadvantageous.

These conclusions based on the information in the novel food dossier submitted by the applicant plus the supplementary information and could not have been reached without the following data claimed as proprietary by the applicant:

annexes to the dossier which relate to the identity of the novel food, the production process, composition, stability, and the intakes assessment report.

57. With thanks to the members of the ACNFP during the course of the assessment were; Dr Camilla Alexander White, Dr Anton Alldrick, Ms Alison Austin, Dr Mark Berry, Professor George Bassel, Dr Christine Bosch, Professor Dimitris Charalampopoulos, Dr Meera Cush, Dr Cathrina Edwards, Professor Susan Fairweather-Tait, Dr Sophie Foley, Professor Paul Fraser, Dr Hamid Ghoddusi, Dr Andy Greenfield, Professor Wendy Harwood, Professor Huw D. Jones, Dr Kimon-Andreas Karatzas, Dr Ray Kemp, Dr Elizabeth Lund, Professor Harry J. McArdle, Dr Lynn McIntyre, Rebecca McKenzie, Professor Clare Mills, Dr Antonio Peña-Fernández, Dr Isabel Skypala, Dr Lesley Stanley, Professor Hans Verhagen, Dr Maureen Wakefield, and Professor Bruce Whitelaw.

5. References

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Calvo MS, Babu US, Garthoff LH, Woods TO, Dreher M, Hill G and Nagaraja S, 2013. Vitamin D2 from light-exposed edible mushrooms is safe, bioavailable and effectively supports bone growth in rats. Osteoporosis International, 24, 197-207. https://doi.org/10.1007/s00198-012-1934-9

EFSA NDA Panel (EFSA Panel on Dietetic Products, Nutrition and Allergies), 2016b. Scientific opinion on dietary reference values for vitamin D (Question no EFSA-Q-2011-01230, adopted: 29 June 2016). EFSA Journal, 14(10), 4547 [145 pp]. DOI:10.2903/j.efsa.2016.4547. Available online: https://www.efsa.europa.eu/en/efsajournal/pub/4547.

EFSA NDA Panel (EFSA Panel on Nutrition, Novel Foods and Food Allergens), 2020. Scientific Opinion on the safety of vitamin D2 mushroom powder as a novel food pursuant to Regulation (EU) 2015/2283 (Question no: 2018-00601, adopted: 28 November 2019, published: 21 January 2020). EFSA Journal, 18(1), 5948 [23 pp.] DOI:10.2903/j.efsa.2020.5948. https://www.efsa.europa.eu/en/efsajournal/pub/5948.

EFSA NDA Panel (EFSA Panel on Nutrition and Novel Foods), 2016. Guidance on the Preparation and Presentation of an Application for Authorisation of a Novel Food in the Context of Regulation (EU) 2015/2283. EFSA Journal 2016, 14(11): 4594. https://doi.org/10.2903/j.efsa.2016.4594.

EFSA NDA Panel (EFSA Panel on Nutrition, Novel Foods, Food Allergens), Turck D, Castenmiller J, De Henauw S,Hirsch-Ernst KI, Kearney J, Maciuk A, Mangelsdorf I, McArdle HJ, Naska A, Pelaez C, Pentieva K, Siani A, ThiesF, Tsabouri S, Vinceti M, Cubadda F, Frenzel T, Heinonen M, Marchelli R, Neuh€auser-Berthold M, Poulsen M,Maradona MP, Schlatter JR, van Loveren H, Gelbmann W and Knutsen HK, 2021b. Scientific Opinion on thesafety of extended uses of UV-treated baker’s yeast as a Novel Food pursuant to Regulation (EU) 2015/2283.EFSA Journal 2021;19(6):6602, 13 pp. https://doi.org/10.2903/j.efsa.2021.6602

EFSA NDA Panel (EFSA Panel on Nutrition, Novel Foods and Food Allergens), Turck D, Castenmiller J, De HenauwS, Hirsch-Ernst KI, Kearney J, Maciuk A, Mangelsdorf I, McArdle HJ, Naska A, Pelaez C, Pentieva K, Siani A,Thies F, Tsabouri S, Vinceti M, Cubadda F, Frenzel T, Heinonen M, Marchelli R, Neuhauser-Berthold M, PoulsenM, Prieto Maradona M, Schlatter JR, van Loveren H, Roldan-Torres R and Knutsen HK, 2021a. Safety of VitaminD2 mushroom powder (Agaricus bisporus) as a Novel food pursuant to Regulation (EU) 2015/2283. EFSAJournal 2021;19(4):6516, 19 pp. https://doi.org/10.2903/j.efsa.2021.6516

EUR-Lex, 2020. Commission Implementing Regulation (EU) 2020/1163 of 6 August 2020 authorising the placing on the market of vitamin D2 mushroom powder as a novel food under Regulation (EU) 2015/2283 of the European Parliament and of the Council and amending Commission Implementing Regulation (EU) 2017/2470. Available online: https://www.legislation.gov.uk/eur/2020/1163/contents

FAO (Food and Agriculture Organization of the United Nations), 2004. Wild Edible Fungi: A Global Overview ofTheir Use and Importance to People in NON-WOOD FOREST PRODUCTS 17, by Eric Boa. Available online: https://www.fao.org/3/y5489e/y5489e.pdf

FSAI (Food Safety Authority of Ireland), 2017. Safety assessment: UV-treated mushrooms (Agaricus bisporus) with increased vitamin D content. Available online: https://www.fsai.ie/uploadedFiles/Science_and_Health/Novel_Foods/Applications/2017%2520UVtreated%2520mushrooms%2520(Agaricus%2520bisporus)%2520with%2520increased%2520vitamin%2520D%2520content.pdf

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Koyyalamudi SR, Jeong SC, Song CH, Cho KY and Pang G, 2009. Vitamin D2 formation and bioavailability from Agaricus bisporus button mushrooms treated with ultraviolet irradiation. Journal of Agricultural and Food Chemistry, 57, 3351-3355. https://doi.org/10.1021/jf803908q

Mehrotra A, Calvo MS, Beelman RB, Levy E, Siuty J, Kalaras MD and Uribarri J, 2014. Bioavailability of vitamin D2 from enriched mushrooms in prediabetic adults: a randomized controlled trial. European Journal of Clinical Nutrition, 68, 1154-1160 [plus supplementary data]. https://doi.org/10.1038/ejcn.2014.157

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Phillips KM, Ruggio DM, Horst RL, Minor B, Simon RR, Feeney MJ, Byrdwell WC and Haytowitz DB, 2011. Vitamin D and sterol composition of 10 types of mushrooms from retail suppliers in the United States. Journal of Agricultural and Food Chemistry, 59, 7841-7853. https://doi.org/10.1021/jf104246z

Phillips KM, Horst RL, Koszewski NJ and Simon RR, 2012. Vitamin D4 in mushrooms. PLoS ONE, 7, e40702 [10pp]. https://doi.org/10.1371/journal.pone.0040702.

Shanely RA, Nieman DC, Knab AM, Gillitt ND, Meaney MP, Jin F, Sha W and Cialdella-Kam L, 2014. Influence of vitamin D mushroom powder supplementation on exercise-induced muscle damage in vitamin D insufficient high school athletes. Journal of Sports Sciences, 32, 670-679. https://doi.org/10.1080/02640414.2013.847279

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Abbreviations

ACNFP	Advisory Committee on Novel Foods and Processes
ADME	Absorption, Distribution, Metabolism and Excretion
AOCS	Association of Official Analytical Chemists
BW	body weight
CAS	Chemical Abstracts Service
CFU	Colony Forming Unit
cGMP	Current Good Manufacturing Practice
EC	European Commission
EFSA	European Food Safety Agency
EU	European Union
FAO	Food and Agriculture Organisation
FDA	Food and Drug Administration
FSA	Food Standards Agency
FSAI	Food Safety Authority Ireland
FSS	Food Standards Scotland
GLP	Good Laboratory Practice
HACCP	Hazards Analysis and Critical Control Points
HPLC	High Performance Liquid Chromatography
HPTLC	High Performance Thin-Layer Chromatography
ISO	International Organisation for Standardization
IU	International Unit
IUPAC	International Union of Pure and Applied Chemistry
LOD	Limit of detection
LOQ	Limit of quantification
ND	not determined
NDA	Dietetic products, Nutrition and Allergies
NLT	not less than
NMT	not more than
NMR	Nuclear Magnetic Resonance
NOAEL	No Observable Adverse Effect Level
OECD	Organisation for Economic Co-operation and Development
USP	United States Pharmacopoeia
UL	Tolerable Upper Intake Level
UV	Ultra-Violet
25(OH)D	25-hydroxy vitamin D

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