Summary of Application for Authorisation as Novel Food acc ... · Enteron Science GmbH ENTEROBAL...

Enteron Science GmbH ENTEROBAL Date: June 4, 2018

Summary of Application for Authorisation as Novel Food

acc. to Art. 35 of Regulation (EU) No. 2015/2283

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ENTEROBAL






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CONTENTS

1. SUMMARY BY THE APPLICANT ........................................................................................................... 3

1.1 INTRODUCTION ........................................................................................................................................ 3 1.2 IDENTITY OF THE NOVEL FOOD ................................................................................................................ 4 1.3 PRODUCTION PROCESS OF THE TRICHURIS SUIS EGGS .............................................................................. 7 1.4 COMPOSITIONAL DATA .......................................................................................................................... 13 1.5 SPECIFICATION OF THE NOVEL FOOD ...................................................................................................... 14 1.6 HISTORY OF USE OF THE NOVEL FOOD AND/OR ITS SOURCE ................................................................... 16 1.7 PROPOSED USES AND USE LEVELS AND ANTICIPATED INTAKE ................................................................ 17 1.8 ABSORPTION, DISTRIBUTION, METABOLISM AND EXCRETION (ADME) ................................................ 18 1.9 NUTRITIONAL INFORMATION ................................................................................................................. 18 1.10 TOXICOLOGICAL INFORMATION ............................................................................................................. 19 1.11 ALLERGENICITY (HERE ANTI-ALLERGENICITY) .................................................................................... 21

FIGURES

Figure 1. Example of the pooling procedure of food substance starting material bulk suspension ........ 8 Figure 2. Microscopy of Trichuris suis eggs, top: embryonated, bottom: non-embryonated ............. 9 Figure 3. Container with the novel food bulk suspension (approximately 1 million eggs). ................... 10 Figure 4. Comprehensive flow chart of manufacture of novel food ENTEROBAL ................................... 11 Figure 5. Example of Manufacture (adjustment by mixing) of finished novel food product for sale. .... 12

TABLES

Table 1. Composition of ENTEROBAL Product for Sale ............................................................................ 3 Table 2. Comparison of Trichuris suis ova from Master Egg Bank MEB-32 with wild-type Trichuris

suis and differentiation between Trichuris suis and other Trichuris species ............... 6 Table 3. Intra-individual and intra-species (inter-individual) variability of the ITS1, 5.8S and ITS2

sequences of TSO (Cutillas et al. 2007) ....................................................................... 6 Table 4. Results of nutritional analysis .................................................................................................. 13




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1. Summary by the Applicant

This is to summarise this current application for authorisation of ENTEROBAL as novel food

acc. to Regulation (EU) No. 2015/2283 on Novel Food and is submitted by the following

company:

Enteron Science GmbH

Bordesholmer Str. 10

22143 Hamburg, Germany

Tel.: +49 (40) 94777-160

E-Mail: [email protected]

Enteron Science GmbH is developing Trichuris suis ova (TSO= eggs of the porcine

whipworm) for food (as is) purposes and for use as food ingredient.

1.1 INTRODUCTION

The novel food ENTEROBAL is defined as the viable embryonated eggs of the whipworm

Trichuris suis. Its bulk form is a suspension with 9 to 19 million eggs per millilitre. Following

adjustments, the offered units (15 ml) contain concentrations of 250, 2’500 and/or 7’500

active Trichuris suis eggs per bottle.

The eggs are suspended in phosphate buffer pH 3.0, and the suspension medium contains

potassium sorbate as antimicrobial preservative in a concentration of 0.5 mg/ml (equivalent to

0.05%).

The product is intended to be offered as a food supplement.

Table 1. Composition of ENTEROBAL Product for Sale

One bottle of 15 ml (corr. 15.06 g) 1 TSO 250 / 2’500 / 7’500 contains

Ingredient TSO 250 TSO 2’500 TSO 7’500 Function

Trichuris suis ova 2 250 TSOactive 2’500 TSOactive 7’500 TSOactive Novel food

Sodium dihydrogen

phosphate dihydrate

0.2895 g 0.2895 g 0.2895 g Buffer

Potassium sorbate 0.0075 g 0.0075 g 0.0075 g Preservative

Purified water 14.7630 g 14.7630 g 14.7630 g solvent

Total mass 15.060 g 15.060 g 15.060 g 1 Density of the solution: 1.004 g/ml 2 Influence of TSO on volume / mass is negligible and was neglected in the composition

mailto:[email protected]




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At first sight, the apparent lack of a significant nutritional value of ENTEROBAL may give rise

to the question, for what purpose the product is meant. The following idea may be helpful to

understand the background of this complex application.

The product should be seen as comparable to probiotic foods which range from biological

supplements through yogurt, kefir and even kosher pickles A probiotic product is a food

preparation containing viable microorganisms, frequently Bifidobacterium longum,

Lactococcus lactis, Lactobacillus spp., Enterococcus faecium and Streptococcus and many

others. Yogurt is a typical probiotic food. When consumed orally in sufficient amounts,

probiotic foods are thought to exert a beneficial influence on the health of consumers.

While ENTEROBAL contains viable eggs of the porcine whipworm Trichuris suis it does not

contain microorganisms intentionally. Beneficial properties of ENTEROBAL have not been

proven so far in double blind human clinical trials. A clinical development program has been

stopped due to lack of medicinal efficacy. The majority of safety data generated to date

support this food supplement program.

Without any aspiration for completeness, we point to the publication Walk et al. (2010)

dealing with positive influences of another helminth (Heligmosomoides polygyrus) on animal

gut microflora and to the approved patents of Tufts University Prof. Joel Weinstock.

At this time, beneficial properties are neither justified nor claimed. This may be the aim of a

later and separate step using another application procedure according to art. 14 (1) lit. a) of

regulation (EU) No. 1924/2006.

1.2 IDENTITY OF THE NOVEL FOOD

The porcine whipworm Trichuris suis is a common intestinal nematode with a high

prevalence in the pig, its natural host.

The systematic classification of the nematode is:

Phylum: ASCHELMINTHES

Class: NEMATODA

Subclass: Adenophorea (Aphasmidia)

Order: Enoplida Chitwood, 1933

Superfamily: Trichurioides

Trichuris




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Structure elucidation by morphological and biometrical determinations

Embryonated eggs of Trichuris suis can be clearly identified by microscope due to their

distinct morphological characteristics (see Figure 2 below). The microscopic image allows

for a structure identification of embyronated Trichuris suis eggs by their morphology, shape

and size dimensions:

Morphology:

Lemon-shaped

Two clear, transparent protruding plugs

Thick egg shell.

Size dimensions:

Lengths: 50 to 70 μm

Width: 20 to 30 μm.

Viability as a potency parameter is linked to morphology due to the fact that this parameter

can only be measured in embryonated, i.e. morphologically developed eggs. Non-

embryonated eggs, identifiable by their granular brownish and unsegmented contents, are not

viable. The factors “structure” and “biological function” of Trichuris suis eggs are mutually

dependent.

The analytical characterisation of the source organism Trichuris suis is based on molecular

identification techniques (RT-PCR). This is part of the analytical release specification.

The selected sequence region ITS1 and ITS2 is a segment of the ribosomal DNA and specific

for Trichuris suis, with no significant homology to other Trichuris species like T. trichiura, T.

leporis, T. vulpis, T. ovis, T. muris or Ascaris suum, Oesophagostomum dentatum and

Hyostrongylus rubidus.




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Table 2. Comparison of Trichuris suis ova from Master Egg Bank MEB-32 with wild-type

Trichuris suis and differentiation between Trichuris suis and other Trichuris species

ITS-1 5.8S ITS-2 Reference

TSO (MEB-32) versus TSO

(wild-type) a)

99.85% 100.00% 99.25% Development

study

TSO (wild-type) versus

T. trichiura

(from non-human primate)

86.00% 96.00% 81.00% Cutillas et al

2007

TSO (wild-type) versus T. vulpis 62.00% 96.80% 58.70% Cutillas et al

2007


T. arvicolae

56.97% 96.80% 57.60% Cutillas et al

2007

TSO (wild-type) versus T. ovis 48.04% 96.80% 59.10% Cutillas et al

2007

TSO (wild-type) versus T. leporis 48.46% 96.80% 59.00% Cutillas et al

2007

TSO (wild-type) versus T. muris 58.60% 96.80% 59.50% Cutillas et al

2007


T. srkjabini

49.98% 96.80% n.a. Cutillas et al

2007 a) TSO isolated from conventional domestic pigs is considered as wild-type.

Table 3. Intra-individual and intra-species (inter-individual) variability of the ITS1, 5.8S and

ITS2 sequences of TSO (Cutillas et al. 2007)

ITS-1 5.8S ITS-2

Intra-individual variability (host: domestic pig) 0.9 % 1.3 % n.a.

Intra-individual variability (host: wild boar) 0.61 % 0.63 % 0.19 %

Intra-species variability (host: domestic pig) 0.0 % n.a. n.a.

Intra-species variability (host: wild boar) 0.45 % n.a. 0.0 %

Structure elucidation by molecular identification

The molecular identification of Trichuris suis allows for a reliable differentiation of Trichuris

species on the genetic level (Cutillas et al. 2007; Cutillas et al. 2009).

Based upon the ITS1-5.8S-ITS2 region of the ribosomal DNA segment, a molecular

identification of Trichuris suis and a differentiation to other Trichuris species and common

porcine parasites such as Ascaris suum, Oesophagostomum dentatum and Hyostrongylus

rubidus is possible.




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1.3 PRODUCTION PROCESS OF THE Trichuris suis EGGS

The novel food ENTEROBAL is a suspension of viable, purified, embryonated eggs of the

whipworm Trichuris suis. It exists as a bulk suspension with 9 to 19 million eggs per millilitre

and, following adjustment, with concentrations of 250, or 2’500 or 7’500 active Trichuris suis

eggs per bottle (product for sale).

Overview of the manufacturing process:

The established small-scale manufacturing process of the food consists of both in vivo and in

vitro steps which mimic the natural life cycle of the nematode, at least to some degree.

Inoculation of pigs and pooling of faecal fractions (in vivo)

Isolation and purification of the non-embryonated eggs from the faecal pools

Pooling of TSONEE, formation of the novel food starting material

Maturation & embryonation of eggs (in vitro)

Egg bank system

Manufacture of products for sale

Inoculation of pigs and pooling of faecal fractions

The starting point of a food batch is a group of 2 to 5 domestic pigs (female, 80 to 150 days,

approximately. 6 to13 kg body weight) with a defined and documented health status.

Each animal is inoculated with a dose of 2’000 active Trichuris suis eggs. The inoculation

doses are prepared from the Working Egg Bank. After an incubation period of 49 days the

faeces from inoculated domestic pigs are collected and pooled.

The faecal material obtained from the domestic pigs contains non-embryonated eggs

(TSONEE) shed (excreted) by the oviparous females of whipworm living in the small intestine

and/or cecum of the pigs. The faeces are collected until day 57 post inoculation (p.i.), leading

to 9 individual fractions (day 49 to day 57 p.i.).

Three pools are collected from the individual daily faecal fractions (pool 1 from the faeces of

days 49 to 51, pool 2 from the faeces of days 52 to 54 and pool 3 from the faeces of days 55

to 57).

Isolation and purification of the non-embryonated eggs from the faecal pools

The subsequent steps include the isolation and purification of the non-embryonated eggs from

the faecal pools to obtain three individual aqueous egg suspensions.




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Pooling of TSONEE, formation of the novel food starting material bulk suspension

The egg count per suspension is determined and the overall yield is calculated.

Based on the percentage of egg excretion, portions of eggs are taken from each suspension

and pooled to one combined novel food starting material with a total of 8 to 19 million eggs.

An exemplary pooling procedure is illustrated in Figure 1.

Figure 1. Example of the pooling procedure of food substance starting material bulk

suspension

Number of domestic pigs = 5

Collection of faecal material (day 49 p.i. to day 57 p.i.)

Faecal fraction 1 Faecal fraction 2 Faecal fraction 3

Day 49 to 51 p.i. pool Day 52 to 54 p.i. pool Day 55 to 57 p.i. pool

Isolation and purification of TSONEE:

Egg suspension 1 Egg suspension 2 Egg suspension 3

Day 49 to 51 p.i. pool Day 52 to 54 p.i. pool Day 55 to 57 p.i. pool

32.8 million TSONEE 44.0 million TSONEE 51.5 million TSONEE

Total egg excretion: 128.3 million TSONEE = 100 %


25.6 % of total TSONEE 34.3 % of total TSONEE 40.1 % of total TSONEE

Pooling of TSONEE to 1 batch of the novel food starting material:

Batch size: 17 million TSONEE (9-19 million)


25.6 % of 17 million 34.3 % of 17 million 40.1 % of 17 million

= 4.4 million TSONEE = 5.8 million TSONEE = 6.6 million TSONEE

Excess of Excess of Excess of

32.8 -4.4 = 28.4 million 44.0 – 5.8 = 38.2 million 51.5 – 6.8 = 44.7 million

TSONEE used for repeated

pooling according to the

defined procedure



defined procedure



defined procedure

Several (further) batches of the novel food starting material

17 million TSONEE




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The excess eggs that remain in each egg suspension is used to pool additional final bulk

suspensions with the same size and to establish the preliminary egg bank system.

The novel food starting material is free of potential microbial and parasitic contaminants

and has an egg concentration of 40’000 non-embryonated Trichuris suis eggs per millilitre

(total eggs: approximately 8 to 19 million).

Maturation and embryonation of eggs (in vitro)

The eggs are maturated in vitro under controlled and reproducible conditions (25°C, 90 – 110

days, 40’000 eggs/ml of 0.1 N sulphuric acid pH 1) to yield embryonated and viable eggs.

The first 30 days of the embryonation process are also considered the virus inactivation step.

During the maturation step (TSONEE TSOEE), a decisive structural change occurs within the

eggs. Embryonated and non-embryonated eggs can be clearly differentiated by microscopic

observation. While non-embryonated eggs are identifiable by their granular brownish and

unstructured contents, a folded parasite larva (“a baby worm”) is visible in the embryonated

eggs. Figure 2 illustrates a microscopic image of an embryonated and a non-embryonated egg

of Trichuris suis.

Figure 2. Microscopy of Trichuris suis eggs,

top: embryonated, bottom: non-embryonated

Finally, the eggs are transferred into the storage medium (phosphate buffer pH 3.0 with

0.05% potassium sorbate), and after concentration adjustment, the novel food bulk

suspension is obtained.




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Figure 3. Container with the novel food bulk suspension (approximately 1 million eggs).

Egg bank system - General considerations

Inoculation of domestic pigs with a defined dose of Trichuris suis ova (eggs, TSO) is the first

step in the manufacturing process of the novel food batches. Therefore, a system of TSO

suspensions needs to be established and maintained to allow for the continuous supply of

inoculation doses with a defined and reproducible egg quality.

To date, a proven concept of an egg bank system has been developed, which is considered

sufficient for the manufacture of first novel food batches. The available system consists of a

series of suspensions with TSOactive that was classified as Master Egg Bank (MEB) and

Working Egg Banks (WEB). The inoculation doses are prepared from the WEB. The WEB

itself has been derived from the MEB. The egg bank system is currently re-established

regularly in parallel to a production campaign of a novel food batch.

The MEB and the novel food batch are, therefore, derived by the same route of manufacture:

The pools of non-embryonated Trichuris suis eggs (TSONEE) obtained from the 3 faecal

fractions (pool 1 = days 49 to 51; pool 2 = days 52 to 54; pool 3 = days 55 to 57) are used for

the manufacture of both the novel food and the MEB. In addition, pool 4 = faecal fractions of

days 58 to 60 and pool 5 = days 61 to 63 are currently also included in the egg bank process.




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Figure 4. Comprehensive flow chart of manufacture of novel food ENTEROBAL

Prerequisites 2-5 domestic pigs (female, 80 to 150 days)

animal food

Working Egg Bank

In vivo phase 1. Manufacture of inoculation doses (2’000 TSOactive/10 ml) from

the Working Egg Bank

2. Inoculation of domestic pigs

3. Collection of faecal material from day 49 to 58 post inoculation

(p.i.)

Fraction 1: Fraction 2 : Fraction 3

Days 49 to 51 p.i. Days 52 to 54 p.i. Days 55 to 57 p.i.

Faecal mass/animal: Faecal mass/animal: Faecal mass/animal:

Approx. 600 g Approx. 600 g Approx. 600 g

Manufacture of the

starting material

bulk suspension

4. Isolation of non-em-

bryonated eggs

Isolation of non-em-

bryonated eggs

Isolation of non-em-

bryonated eggs

5. Purification of non-

embryonated eggs

Purification of non-

embryonated eggs

Purification of non-

embryonated eggs

6. Determination of

egg count

Determination of

egg count

Determination of

egg count

7. Pooling of eggs considering the actual egg excretion rate in the

relevant fractions 1 to 3 ()

Note: The final pool only contains a portion of all eggs isolated

per fraction

8. Suspending of eggs in 0.1 N sulphur acid pH 1 and concentration

adjustment to 40’000 Trichuris suis eggs/ml (total: 9 to 19 m)

Manufacture of the

novel food substance

bulk suspension

9. Replacement of the suspension medium

10. Embryonation of the eggs of the starting material bulk in 0.1 N

sulphuric acid pH 1 (25 °C, 90 days)

11. Inactivation of potential viruses within the first 30 days of the

embryonation steps (see 10).

12. Change of suspension medium to phosphate buffer pH 3 with 0.05

% potassium sorbate and concentration adjustment to e.g. 5’000

embryonated and active Trichuris suis eggs/ml (total 8 to 19

million)




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Manufacture of product batches TSO 250, TSO 2’500, TSO 7’500 for sale

The manufacturing process of one batch of approximately 1’000 bottles consists of:

Manufacture of 10 (2 x 5) litres of suspension medium (phosphate buffer pH 3 with

0.05% potassium sorbate)

Manufacture of 5 litres of TSO bulk suspension (175 TSOactive/ml or 525 TSOactive/ml or

1’050 TSOactive/ml, resp.)

Filling of suitable volumes of suspension medium and respective TSO bulk suspension

into 30 ml bottles; depending on the respective volumes, the 3 concentrations 250,

2,500, 5’000 and 7’500 egg/bottle are obtained

Closing of the vial by screw caps.

Emplacing a safety seal over the top of the bottle to prevent tampering.

Figure 5. Example of Manufacture (adjustment by mixing) of finished novel food product for

sale.




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1.4 COMPOSITIONAL DATA

The novel food ENTEROBAL contains live embryonated eggs of the porcine whipworm

Trichuris suis in different concentrations as a preserved (E 202) and phosphate buffered

suspension at pH 3.0.

Such eggs are built up by the same constituent classes as found frequently in similar non-

vertebrate eggs, i.e. protein, fat, carbohydrate, water and also micronutrients. The shell of the

eggs is built of chitin. Eggs and larvae can be regarded like eggs or meat and are digested like

eggs or meat, i.e. can be cleaved (hydrolysed) and resorbed.

ENTEROBAL is not intended to provide significant amounts either of macro- or micronutrients

or energy. The caloric and macronutrient content of ENTEROBAL is minimal. Analytical

information about the biochemical / food-chemical composition was obtained:

Table 4. Results of nutritional analysis

Analyte Results

Protein < 0.10 g/100ml

fat 0.1 g/100 ml

Dry matter 1.69 g/100 ml

water 99.11 g/100 ml

Ash (minerals) 0.87 g/100 ml

fibres < 0.02 g/100 ml

Carbohydrates 0.7 g

Energy value 4 kJ / 16 kcal per 100 ml

D-Glucose < 0.10 g/100 g

D-Fructose < 0.10 g/100 g

Sucrose < 0.10 g/100 g

Maltose < 0.10 g/100 g

Lactose < 0.10 g/100 g

Total sugars < 0.10 g/100 g

Salt 0.927 g/100 g

sodium 370.6 mg/100 g




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1.5 SPECIFICATION OF THE NOVEL FOOD

Below, we reprint the current release specification for ENTEROBAL:

Test parameter (Method) Specifications

Appearance of solution

(Visual examination)

Clear solution, not more intensively coloured than reference

solution BY5. A slight sediment might be visible on standing

which disappears completely on shaking.

Odour of solution

(Organoleptic examination)

Odourless to slightly fruity-like

Identity of Trichuris species,

embryonated (Microscopic

determination of morphological

characteristics)

Positive for Trichuris species, embryonated (lemon-shaped

eggs with a thick shell and transparent protruding polar caps

containing a folded larva)

Identity of Trichuris suis species,

embryonated (RT-PCR of the

Trichuris suis ITS2 sequence)

Positive amplification of the Trichuris suis ITS2 sequence

Identity of potassium sorbate

(HPLC)

Positive for potassium sorbate

Purity – Product-related

impurities: TSOinactive

(embryonated and non-

embryonated) (Microscopic

determination; see motility index)

< 20% (in relation to TSOtotal)

Microbiological quality

(Ph.Eur. 2.6.12/13, 5.1.4.-1.)

TAMC ≤ 104 CFU/ml

TYMC ≤ 102 CFU/ml

E.coli Absent (1 ml)

Enterobacteria and certain other

gram (-) bacteria ≤ 102 CFU/ml

Salmonella Absent (10 ml)

Staphylococcus aureus Absent (1 ml)

Assay TSOtotal

• TSO 250 (Microscopic

counting)

• TSO 2,500 (Automatic

counting)

• TSO 7,500 (Automatic

counting)

Assay TSOtotal

120 – 495 TSOtotal/bottle (8 – 33 TSOtotal/ml) corresponding to

120 to 390 TSOactive/bottle1

1‘875 – 3’915 TSOtotal/bottle (125 – 261 TSOtotal/ml)

corresponding to 1’875 – 3’135 TSOactive/bottle2

5’625 – 11’715 TSOtotal/bottle (375 – 781 TSOtotal/ml)

corresponding to 5’625 – 9’375 TSOactive/bottle3

(continued)




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Test parameter (Method) Specifications

Assay potassium sorbate (HPLC) ≥ 0.40 mg/ml equivalent to ≥ 80% of the labelled amount

Potency in vitro (TSOactive)

• Motility index (Microscopic

determination)

• ATP assay (Luciferase reaction)

≥ 80 % of the eggs contain motile/active larva

≥ 25 nM of ATP is induced by 1,260 (1,120 to 1,400) eggs

(after thermal stimulation)

Potency in vivo (TSOviable)

• Pig infectivity test4

(Establishment rate in pigs)

The infectivity rate in at least 1 out of 5 analysed pigs is

≥ 20% with reference to TSOtotal or ≥ 25% with reference to

TSOactive (as measured in the corresponding drug substance

batch at time of manufacture)

Uniformity of content of single-

dose preparations

• TSO 250 (Microscopic counting)

• TSO 2,500 and TSO 7,500

(Automatic counting)

Level 1: 10 units tested

9 out of 10 values within average content

± 15% and 1 out of 10 value > ±15% but

< ± 25% of average content

Level 2: If max. 3 out of 10 values > ±15% but < ± 25%

of average content test another 20 units

27 out of 30 single values within average

content ± 15% and 3 out of 30 single values

> ±15% but < ± 25% of average content

Container closure integrity

(Visual examination)

No leakage perceptible after 24h storage in a refrigerator in

inverted position (n=3) 1 Adjusted to: TSOactive = 17/ml ± 50% → Calculated range of TSOtotal/ml = 17*0.50 to 17*1.25*1.5 (considering

a motility index of ≥80% at release) 2 Adjusted to: TSOactive = 167/ml ± 25% → Calculated range of TSOtotal/ml = 167*0.75 to 167*1.25*1.25

(considering a motility index of ≥80% at release)

3 Adjusted to: TSOactive = 500/ml ± 25% → Calculated range of TSOtotal/ml = 500*0.75 to 500*1.25*1.25

(considering a motility index of ≥80% at release) 4 Results adopted from the respective drug substance batch at time of manufacture




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1.6 HISTORY OF USE OF THE NOVEL FOOD AND/OR ITS SOURCE

History unintended exposure

Humans have cohabitated with pigs for centuries. In such surroundings, specificly farm

workers are constantly exposed to a certain background patterns of viable mature eggs.

Trichuris suis eggs form part of this background helminth patterns where humans were and

are exposed to them. A complete lack of reported diseases by Trichuris suis suggests they are

remarkably safe and may reside in humans for a short time without the host’s knowledge.

Even farmers who raise pigs do not appear to get sick from Trichuris suis exposure.

Intended previous human exposure to the novel food

Early data on the tolerability of Trichuris suis eggs in humans were obtained after an

inoculation of a 23-year old healthy individual and of a human female. Eggs were detected in

stool samples not before day 40 p.i. No symptoms of distress and no diarrhoea were

experienced (Beer, 1971; Beer, 1976). Heitman et al. (2007) published a similar case report of

an 8-year old boy. After 10 doses of Trichuris suis eggs, no adverse events were reported.

Sandborn et al. (2013) evaluated in a dose-escalation (500 to 7’500 viable embryonated

TSO), randomised, double-blind, placebo-controlled study the safety of a single dose of oral

TSO suspension in 11 Crohn’s disease patients. GI disorders were reported by both verum-

treated and placebo-treated patients. No dose-dependent relationship was observed.

Clinical trial reports, intended exposure

During a preceding clinical development phase, experiences with intended exposure to the

novel food ENTEROBAL have been collected:

Table 5. Subjects exposed to Trichuris suis eggs in clinical trials and reports

Study placebo verum

(CD)

verum

(UC)

verum

(non-IBD)

exposed

total

Summers et al. 2003 --- 4 3 --- 7

Summers et al. 2005b 24 --- 30 -- 54

Elliot et al. 2005 15 --- 17 - 17 *

Summers et al. 2005b,

discussion --- approx. 100 --- approx. 100

Summers et al. 2005c --- 29 --- --- 29

Bager et al. 2010 50 - - 50 50

Flemming et al. 2009 5 5

total 89 approx. 183 55 262 * this publication reports – among other things - the following cross over phase of the study described by

Summers et al (2005b): Selected subjects who were given placebo for the first 12 weeks were switched to verum

for a second 12-week interval, and vice versa. The blind was maintained.

read: subjects with CD = Crohn’s disease, UC = ulcerative colitis, IBD = inflammatory bowel diseases




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The outcomes of these human studies regarding safety are compiled below in the section 1.10

TOXICOLOGICAL INFORMATION.

Registration in Thailand, intended exposure

The product has been approved, manufactured and sold in Thailand, formally as an “herbal

product.” Overall, 15,000 individuals of all ages have consumed the eggs of Trichuris suis.

Based on this experience, we conclude that Trichuris suis eggs are safe to consumers.

1.7 PROPOSED USES AND USE LEVELS AND ANTICIPATED INTAKE

Proposed uses and use levels

Intended targeted population will be the general population.

Children are not exempted from the target population. However, ENTEROBAL mainly

addresses to adults.

The applicant intends to start marketing of ENTEROBAL as a food supplement for the general

population. As can be depicted from the term “food supplement” or from its legal definition,

the purpose of such product is to supplement the normal diet and not to replace other foods.

The applicant want to keep open the possibility of a later marketing as an ingredient to be

mixed with other foods.

It is possible that consumers suffering from chronic bowel conditions, such as IBD, could

hear about the beneficial properties of ENTEROBAL and decide to become consumers.

Consumers with IBD belong to the particular population group as explicitly addressed by

recommendation 97/618/EC and so requiring separate consideration: Available human

clinical data demonstrates that the intake of ENTEROBAL is well tolerated by such consumers

and there is no specific concern.

Anticipated intake of the novel food

It is anticipated that ENTEROBAL will be used as a food supplement according to the proposed

marketed presentation.

The consumption recommendation is “take one bottle daily”, corresponding to 250, 2’500 or

7’500 whipworm eggs per day.




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It is not expected that that the general consumption pattern of the consumer will show any

remarkable change and human nutritional status will not be affected by the addition of

ENTEROBAL to the diet.

A surveillance program could be beneficial herein. Currently, informal post-market

surveillance is scheduled.

1.8 ABSORPTION, DISTRIBUTION, METABOLISM AND EXCRETION (ADME)

Traditional metabolic and pharmacokinetic studies observe the metabolic state of single

compounds (C-carbon backbones) within the body and are not applicable to complex

chemical mixtures like foods / whole animals.

It is described and has been shown that the eggs (live embryonated Trichuris suis eggs) and

live hatched larvae remain within bowel lumen.

Resorption does not take place before larvae die away.

1.9 NUTRITIONAL INFORMATION

The novel food ENTEROBAL contains live embryonated eggs of the porcine whipworm

Trichuris suis in different concentrations as a preserved (E 202) and phosphate buffered

suspension at pH 3.0.

Such eggs are built up by the same constituent classes as found frequently in similar non-

vertebrate eggs, i.e. protein, fat, carbohydrate, water and also micronutrients. The shell of the

eggs is built of chitin. Eggs and larvae can be regarded like eggs or meat and are digested like

eggs or meat, i.e. can be cleaved (hydrolysed) and resorbed.

Table 6. Nutrition declaration of ENTEROBAL 2’500

ENTEROBAL 2,500 Nutrition declaration per 100 ml per portion (15 ml vial)

Energy value 4 kJ / 16 kcal 0.6 kJ / 2.4 kcal

Fat 0.1 g < 0.1 g

thereof - saturates n.a. *) n.a. *)

Carbohydrate 0.7 g 0.1 g

thereof -sugar < 0.1 g < 0.1 g

Protein < 0.1 g < 0.1 g

Salt 0.92 g 0.14 g

*) not available




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Its potential intake cannot affect significantly and/or negatively the overall nutrition status of

users, particularly, as it will be offered and ingested as a food supplement, a product the

purpose of which is to supplement the general diet only.

Thus it is not expected that the consumption pattern may show a major change when

ENTEROBAL is accessible as a novel food and included in the diet. Human nutritional status

will not be affected. The applicant believes a surveillance program would be beneficial herein.

1.10 TOXICOLOGICAL INFORMATION

There is no information, reported, found or observed, on any single toxic inherent

constituent(s) in Trichuris suis eggs. Such substances would impede or prevent hatching and

(transient) colonisation. The eggs contain a complex mixture of biological macro- and micro-

constituents and provide (little) nutrients of the substance classes protein, fat, carbohydrate as

well as those of the micronutrient substance classes, like minerals, vitamins, etc.

Any effects of processing are neither likely nor applicable as the food supplement is

intended to be consumed as it is without further consumer preparation, though may become

an ingredient in another food product such as yogurt or kefir, if mixed.

A general preclinical toxicology testing pattern as applicable for clinical development

programs has been completed. Particularly, acute (single dose) toxicity, repeated dose

(subchronic) toxicity, and reproductive and development toxicity, have been completed and

are provided with the application, supplemented by further information on local tolerance and

on irritation and sensitisation.

Animal feeding studies using maximum load were neither considered meaningful nor

performed. ENTEROBAL is intended to be offered and marketed as a food supplement. Thus

only restricted intakes are anticipated, as recommended on the product label. Further, high

load feeding studies using ENTEROBAL to feed more or less high amounts of water with low

nutrient concentration have not been completed. Problems frequently encountered by

nutritional imbalance in maximum load animal feeding studies were not an issue.

Traditional metabolic and pharmacokinetic studies as well as use of mutagenicity tests are not

directly applicable to complex chemical mixtures like foods and have not been initiated.

Different preclinical studies were performed using pigs (the natural host of Trichuris suis),

rabbits, rats and cynomolgus monkeys (a dead-end host similar to humans).

Single dose toxicity studies employed various Trichuris suis ova doses up to 25’000 Trichuris

suis ova/kg in monkeys and up to 33’000 Trichuris suis ova/kg in rabbits. Trichuris suis ova

were well tolerated and did not provoke any signs of systemic toxicity. Recorded

observations refer to expected reactions of the immune system of the hosts. In some of the

administered animals, small (size usually 2 mm) focal alterations were observed in the

otherwise unaffected mucosa of the cecum and colon.




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No signs of administration related toxicity were noted during a 13-week subchronic toxicity

study with Trichuris suis ova in cynomolgus monkeys. The typical immunological host

response was noted in a dose dependent manner. As described just above, in animals of all

groups small focal alterations were observed in the otherwise unaffected mucosa of the

cecum and colon. Two of the female animals of the high dose group showed increased

mesenteric lymph node weights.

In the two cases of studies in rabbits and monkeys, none of the findings was considered to be

of pathological and/or toxicological relevance. There have been no adverse effects observed

or reported in animals studied.

The described 13-week subchronic study (repeated administration) in cynomolgus monkey is

regarded as feeding study. Six animals per gender and group were orally administered every

two week and received placebo, 500 Trichuris suis ova/kg b.w. or 2’500 Trichuris suis ova/kg

b.w.

According to the mean body weight, the female animals were given in total 7 doses of 1’500

Trichuris suis ova or 7’500 Trichuris suis ova, while the male animals were given 7 doses of

2’250 Trichuris suis ova or 11’500 Trichuris suis ova, respectively. The study was aimed to

mimic the administration schedule in a clinical trial in humans exceeding the duration of

treatment by one week and number of Trichuris suis ova doses by one dose.

Scaled on a dose per body weight base, the highest dose in this subchronic toxicity study

was more than 20-fold above the highest dose in humans of a human clinical (TSU-2)

study (7’500 Trichuris suis ova or 107 Trichuris suis ova/kg b.w.).

Administration with Trichuris suis ova was generally well tolerated in studies with pigs.

Alterations of the mucosa of cecum and colon were limited to small focal areas. As the

mucosa was otherwise unaffected and as no general inflammation of larger parts of the

mucosa were observed even when the animals were given high doses of 25’000 or 33’000

Trichuris suis ova/kg b.w., the focal alterations were not considered to be local intolerance

reactions.

The fact that Trichuris suis ova and hatched Trichuris suis larvae are restricted to the intestine

rules out a systemic exposure. Irritancy is, therefore, regarded as unlikely.

A reproductive toxicity study in rabbits (TSB-22/Preclin) was conducted to obtain

information on the influence of the administration with Trichuris suis ova on the fertility and

embryo-foetal development by oral administration to the animals of the F0 generation (24

animals per group and gender). Trichuris suis ova was administered every second week

during the pre-mating and the mating periods at dose levels of 500, 2’500 and 7’500 Trichuris

suis ova/animal corresponding to 167, 833, and 2’500 Trichuris suis ova /kg. During gestation

the female animals were dosed weekly to detect any acute and/or short-term effects of the

administration on the critical phase of organogenesis.

Administration of Trichuris suis ova induced the expected host reactions, but no adverse

effects in the parent generation. Administration of Trichuris suis ova had no effect on fertility

and embryo-foetal development and did not express teratogenic properties. The NOAEL on




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the parent generation and the foetuses was > 2’500 Trichuris suis ova/kg (> 7,500 Trichuris

suis ova /animal).

In addition to study TSB-22/Preclin, fertility parameters were tested in the subchronic toxicity

study in monkeys. No Trichuris suis ova-related effect on the estrus cycle was noted and the

sperm analysis did not reveal test-item related changes. No verum-related histopathological

changes were observed in the reproductive organs of male and female monkeys of the

Trichuris suis ova-groups.

During the preceding clinical development phase, experiences with intended exposure of

humans to the novel food ENTEROBAL have been collected.

In three studies with Trichuris suis conducted with patients suffering from inflammatory

bowel disease, no subject experienced any adverse effect that was attributed to the consumed

verum (novel food). In these inflammatory bowel disease studies, more than 180 subjects

have received more than 2’000 doses of Trichuris suis eggs (some subjects for more than 4

years) without reported detrimental or adverse effect. No clinically significant change in the

blood count or hepatic tests occurred in any subject at any time during the study.

The publication of Bager et al. (2009), deviating at first sight, is discussed and explained:

Apparent adverse events are an outcome not replicable from the raw data (participants’

diaries).

No cases of symptomatic or pathologic invasion or infestation of Trichuris suis were

described in humans. Such concerns or suspicions are published occasionally but are without

scientific substantiation.

One isolated case of maturation of Trichuris suis egg to adult whipworm in the intestine was

reported. However, the worm may have been already in the midst of being expelled.

It is highly unlikely that Trichuris suis causes prolonged colonisation of humans. During

studies administering Trichuris suis ova to ulcerative colitis and Crohn’s disease patients,

colonoscopy occasionally detected helminths of variable size and maturity. Eggs were never

detected. Even if eggs would be produced, they would be immature (non-embryonated) and

thus not capable of becoming helminths.

The current application addresses these issues sufficiently. ENTEROBAL is a safe novel food.

1.11 ALLERGENICITY (HERE ANTI-ALLERGENICITY)

Secretory-excretory antigen (peptides or proteins) is secreted to communicate with the

potential host. These substances trigger reactions of the immune system in the host. However,

these reactions are expected and welcome, keep within physiological ranges and are not

indicative of a toxic effect.

Studies on cross-reactivity between Trichuris suis antigens and allergens are not available.




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1.12 CONCLUSION

Overall, it can be concluded that the novel food / novel food ingredient of application,

ENTEROBAL, is safe and suitable for human consumption. There are no relevant sources of

uncertainties.

ENTEROBAL does not present a danger for the consumer and does not mislead the consumer

(see Art. 7 Reg. (EC) No. 2015/2283 on novel foods).

Summary of Application for Authorisation as Novel Food acc ... · Enteron Science GmbH ENTEROBAL...

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