Gelatin Manufacturers Association of Japan

BSE Safety of Gelatin
Study Report on Overseas and Domestic Situations regarding the Validation Studyon the Capacity of Gelatin Manufacturing Process to Inactivate TSE Infectivity（2005/02/15）

The gelatin produced by member companies of Gelatin Manufacturers Association of Japan (GMJ)
is a safe product for which safety against BSE is fully ensured.
Even before the advent of BSE in Japan, GMJ members have complied with relevant food andpharmaceutical regulations and committed to securing safe bovine material fitting for gelatin
production through such measures as clarification of the country of origin of raw material, removal of
BSE risk materials, and contamination control under close partnerships with their raw materialsuppliers.
In addition to this, BSE safety of gelatin is further enhanced by the fact that gelatin is a high-purity
protein manufactured through various refining processes. It has been reported that different stepsof gelatin production including chemical/physical process and heat treatment is capable tosignificantly inactivate BSE infectivity, and even if there is any chance of raw material containing
BSE infectious agent due to cross-contamination, the infectious agent would not remain in the finalproduct.
Gelatin Manufacturers Associations of Europe (GME) has continued conducting a variety ofvalidation studies 1), 2), 3), 4) for ten years in order to verify that the production process of gelatin iscapable to inactivate potential BSE infectivity. As reported earlier, the study report of GME has
been already introduced at "The meeting for the study report on BSE safety of gelatin” 5), 6), which
was organized by GMJ in June 2002.
To further promote the understanding of BSE safety of gelatin manufactured in Japan, domestic
specialists, who are involved in the study of BSE and/or the manufacturing of gelatin, conducted theabove captioned study. The study verified the effectiveness of BSE measures implemented by
GMJ member gelatin manufacturers and the capability of gelatin production process to inactivate
BSE infectivity.
The following is a summary of the study report 7), which we would like to present to further
emphasize the BSE safety of Japanese gelatin.
Please contact GMJ office or member companies for the details of the study report (Japanese only).

-1English Translation by Nitta Gelatin Inc.

I. Investigation Outline
This investigation was conducted as the Ministry of Economy, Trade and Industry’s support project
for the development of leather industry for 2004 referred to as “the overseas information researchand domestic PR operations regarding the capability of gelatin production process to inactivate
potential BSE infectivity”. The following study was conducted by the working party composed of Dr.
Naotaka Ishiguro, Doctor of Veterinary Medicine and Professor of Gifu University, who leads thestudy of BSE in Japan, and Dr. Suguru Sumita (formerly worked for Hyogo Prefectural Institute ofTechnology), Doctor of Engineering and Instructor of technical development in Hyogo Prefecture,
who is familiar with gelatin manufacturing.
A) Evaluate the validity of the validation study carried out with the laboratory models based on
the manufacturing condition adopted by GME was evaluated.
B) Consider whether the production activities of Japanese gelatin manufacturers areequivalent to those of GME members and review the actual situation of the assurance ofBSE safety of Japanese bovine bone gelatin.
The working party visited the Institute for Animal Health in Edinburgh, U.K. on October 1, 2004 andwas able to directly interview Dr. Grobben, who conducted a laboratory scale experiment with aminiature model of the GME study, and Dr. Somerville and Dr. Taylor, who conducted a bioassay
experiment, to get the details of the study. They also visited various regions in Japan, Germany
and Belgium to conduct the research on the actual situation of production activities, targeting bovinebone gelatin plants of the three GMJ member companies in Japan (Nippi Inc., Jellice Co, Ltd., andNitta Gelatin Inc.) and the three establishments of the two GME member companies in Europe (DGF
Stoess AG in Eberbach and Memmingen, Germany and PB Gelatins in Vilvoorde, Belgium).
As a result, Japanese third-party experts confirmed the following.
・ They confirmed the validity of the GME validation study 2), 3), 4) with the test model based on
the manufacturing condition adopted by GME.
・ All the process conditions adopted by Japanese bovine bone gelatin manufactures areequivalent to those adopted by GME. The manufacturing is conducted properly so that it
effectively contribute to the inactivation of BSE infectivity.
・ Quality records of the production process and certificates for the country of origin, animal
health, materials to be used, and SRM free are properly controlled and utilized so that thetraceability of raw material is ensured.
Accordingly, it was further confirmed that Japanese gelatin manufacturers have properly
implemented measures against potential risk of BSE, and therefore, Japanese bovine bone gelatin
is a safe product for which BSE safety is ensured.

II. Abstract of the Study Report
Since gelatin is a product used in a wide variety of food and pharmaceutical products, major
challenges for gelatin manufacturers are to ensure sufficient safety and to verify it. The
requirements of BSE measures for gelatin manufacturing are as follows: 1) Procurement of safe rawmaterial. 2) Standardization and assurance of the production process. 3) Inactivation of potential
TSE infectivity during the production process and verification of it.
-2English Translation by Nitta Gelatin Inc.

1. Inactivation of potential TSE infectivity during the production process and verification of it
The purpose of the validation is whether the production process of gelatin is capable of removing orinactivating TSE infectivity if raw material contaminated by TSE infectious agent enters into theproduction process.
The verification test was conducted with laboratory scale gelatinmanufacturing equipment in order to confirm the degree of the capacity of each manufacturing
process to inactivate high TSE infectivity with which raw material was artificially contaminated 2), 3).

A. Starting Material
Raw material derived from pig or fish has no risk of BSE. Even raw material of bovine origin, if it
originates from bone or hide, has no BSE infectivity, either. The potential risk of BSE in bovine
gelatin is that highly infectious bovine tissues such as brain and spinal cord could contaminatebones. Such cross-contamination in bovine hide is highly unlikely. Accordingly, a starting material
was bovine bone that was artificially contaminated with TSE infectivity (spiking) to set the infectivity
level of the material at 1,000 times worse than the actual situation. This cross-contamination reflecta hypothetical “worst case scenario” which has never and could never occur under realisticconditions.
B. Test with downscaled model of gelatin manufacturing process
Since each manufacturer has different manufacturing conditions and equipment, the test wasdesigned to be conducted according to the minimum conditions that all the manufacturers have incommon, considering the following points 2), 3).
1. Downscale basic conditions of all the process to an accurate laboratory scale model.
2. Also, maintain other conditions than basic ones as much as possible in the laboratory scale
test.
3. Give consideration to those conditions that cannot be maintained in the laboratory test andthat are not the requirements of the test so as not to have any effect on inactivation.
The gelatins produced through alkali and acid processes, both of which are traditional methods ofgelatin manufacturing, were used for the validation. Bovine bone, on which spiking was performed,
was crushed, degreased, and demineralized with diluted hydrochloric acid to produce ossein.
Lime and hydrochloric acid were used for “alkali process” and “acid process”, respectively. As for“acid process”, short-time NaOH pre-treatment, which was a new method, was also examined.
After washing the ossein, gelatin was extracted from it with hot water. The unpurified gelatin went
through filtration, ion-exchange, concentrating, and UHT sterilization processes in order, and thefinal product was then produced.
The unpurified gelatin and the final product, refined gelatin, produced by the laboratory had theequivalent composition and characteristics to those of the gelatin produced by the actual gelatin
production process. It was, therefore, confirmed that the technical aspect of the process was wellmodeled. Although those manufacturing conditions were equivalent to the minimum requirementsof GME, manufacturing conditions adopted by each of the GMJ member manufactures are beyond
this level.
-3English Translation by Nitta Gelatin Inc.

C. Evaluation of the Infectivity
The 301V strain of mouse-passaged BSE agent and the 263K strain of hamster-passaged Scrapie
agent were used for spiking. The both 301V and 263K have constant and high infectivity, and ashort incubation period after which an infection was developed in the rodents that received
intracerebral injections. The two strains also cause apparent pathologic changes after an infection
was developed. The 301V BSE strain has extremely strong thermal resistance. Those are thefeatures of the strains used for spiking 8).
The infectivity present in the samples and remaining infectivity after processing was determined byintracerebral inoculation in VM mice for 301V (20 microliter / head) and hamsters for 263K (50microliter / head), with the samples diluted ten times. After the animals were observed for
maximum 600 days following the inoculation, they were killed to measure whether their tissue slices
show spongy lesion. It can be said that the combination of the strains and the rodents used for thisbioassay is the most sensitive one among TSE test methods.
D. Results
The infectivity was detected in neither limed bone gelatin nor acid bone gelatin, which went throughall the production process 3), 4), 10). Although there are some differences in the capacity of eachprocess to remove the infectivity, each process has such capacity and the effects were cumulative.
The clearance factors for acid and alkali treatments in pretreatment process were 102.6-104.6. The
following refining processes (filtration, ion-exchange, and high-temperature sterilization) showed
higher clearance factors 9). Especially, the clearance factor for the high-temperature sterilizationstood at very high level of 10 3.0 for the 301, and 10 4.1 for the 263 9), and no TSE infectivity wasremained in the acid bone gelatin with short-time NaOH pre-treatment 10), 11). Accordingly, it was
shown that high-temperature and strong alkali treatments contribute to the removal and inactivationof the TSE infectivity.
The contamination level of the strains used for spiking is at least 1000 times higher than theindustrial situation. Furthermore, the bio-assay was conducted using rodents so that there was nospecies barrier effect. It means that the capacity of gelatin production process to remove/inactivate
the infectivity is extremely high. It is, therefore, conceivable that the risk of BSE infection to humanthough gelatin consumption is close to zero 12).

2. Research on the Japanese gelatin production
A. Control of the specified risk material (SRM) and traceability system of bovine bone
All the GMJ members depend completely on overseas for their bovine bone. GMJ members
receive bovine bone originating from fresh bone as GME members do and also obtain from their
suppliers such documents as certificates for the country of origin, animal health, animal tissues to beused, and SRM free. It was confirmed that management conditions of quality records and otherdocuments at the three Japanese manufacturers are appropriate. GME has advised that thetraceability of fresh bone after slaughter house is completely established. It is also reported that thetraceability of CB (Crushed Bone) from outside EU is reportedly established. All the GMJ members-4English Translation by Nitta Gelatin Inc.

ensure traceability for their raw material as GME members do.
EMEA’s “Note for guidance on minimizing the risk of transmitting animal spongiform encephalopathy
agents via human and veterinary medicinal products” 13) requires bovine bone gelatin manufacturers
to comply the following: 1) Clarify the country of origin and establish traceability. 2) Skulls andspinal cord as SRM should be removed from the starting material. The removal of vertebra is also
recommended for bovine bone from high BSE risk countries. 3) Monitor production process basedon HACCP and ISO9000. Japanese manufactures follow the requirements of EMEA as they havebeen certified according to ISO 9000 and/or obtained certificates from EDQM (European Directoratefor the Quality of Medicine). Even though gelatin manufacturers are not required to remove
vertebra from bovine bone originating from BSE free countries including Australia, New Zealand,and India for edible use, GMJ members are increasingly removing vertebra at their own initiative tofurther ensure the safety.
B. Production process that contributes to the inactivation
The study showed that the manufacturing conditions of acid demineralization (osseinfication), alkali
treatment (liming) of raw material, filtrate, ion-exchange, and sterilization at each GMJ member
manufacture 3), 4) were equivalent to those of GME model, which was verified by their inactivation
study. Although the degreasing system of bovine bone of Indian origin 14) is different from that ofGME, it would not cause major difference in the inactivation, considering the effects of suchprocesses as those which raw materials go through after degreasing and high-temperaturesterilization in the gelatin refining process. Further to this, the introduction of hot water washing for
Indian CB would enable the manufacturing conditions of Japanese manufactures to close to those ofEU 15).
The minimum requirements presented in the GME study report for the process of raw material(demineralization and liming) and gelatin production process (filtration, ion-exchange, andsterilization) are less strict than the typical manufacturing conditions adopted by gelatinmanufacturers. It means that the manufacturing conditions adopted by Japanese gelatinmanufacturers fully meet the minimum requirements. Accordingly, the manufacturing conditions ofJapanese manufacturers have an expected capability of inactivating the BSE infectivity.
C. Comparison of the conditions of domestic and overseas gelatin plants.
Comparing the conditions of GME’s laboratory model and the process records and research resultsof production plants of Japanese manufacturers, it turned out, as a whole, that manufacturingconditions of Japanese manufacturers are equivalent to those of GME. It is rather natural to thinkthat there would not be much difference in the manufacturing conditions adopted by gelatinmanufactures if they pursue producing high quality gelatin from the same raw material at the lowest
possible cost.

-5English Translation by Nitta Gelatin Inc.

Reference
1) Gelatin Manufacturers of Europe : The BSE Safety of Pharmaceutical Gelatin from Bovine Raw Material,
Status 28 (1994)
2) Taylor, D.M., Somerville, R.A., Steele, P.J., Grobben, A.H. 2002. Validation of the clearance of TSEagent by the initial steps of the alkaline gelatine manufacturing process. Ref.No.06667/alkaline 301V.
p1-963) Taylor, D.M., Somerville, R.A., Steele, P.J., Grobben, A.H. 2003. Validation of the clearance of TSE
agent by the initial steps of the alkaline gelatine manufacturing process. Ref.No.0667/alkaline 263K.
p1-73.
4) Grobben, A.H., Steele, P. J., Somerville, R.A., Taylor, D.M. 2004. Inactivation of the bovine-spongiformencephalopathy (BSE) agent by the acid and alkaline processes used in the manufacture of bonegelatine. Biotechnol. Appl. Biochem. 39:329-338.
5) http://wwwsoc.nii.ac.jp/jsvs/05_byouki/prion/pf133.html (Zoonosis No. 133, Kazuya Yamanouchi)
6) Gelatin Manufacturers Association of Japan, The meeting for the study report on BSE safety ofgelatin (June 24, 2002)
7) Naotaka Ishiguro, Suguru Sumita, Feb. 15, 2005, Study Report on Overseas and Domestic Situations
regarding the Validation Study on the Capacity of Gelatin Manufacturing Process to Inactivate TSE
Infectivity
8) Taylor, D.M., Fernie, K., Steele, P.J., McConnell, I., Somerville, R.A. 2002. Thermostability of mousepassaged BSE and scrapie is independent of host PrP genotype: implications for the nature of thecausal agents. J. Gen. Viol. 83:3199-3204.
9) Rohwer, R.G., Grobben, A.H., MacAuley, C.M. 2001. Intermediate data on the removal and inactivationof TSE agents by the individual process steps of the finishing unit operations of the gelatine
manufacturing process. (provided in confidence)10) Grobben, A.H., Steele, P.J. 2003. Validation of the clearance of TSE infectivity by the initial steps of theacid bone gelatine manufacturing process with an additional short NaOH treatment. p1-14.
11) Brown, P., Rohwer, R.G. 1986. Newer data on the inactivation of scrapie virus or Creutzfeldt-Jakob
Disease virus in brain tissue. 1986. J. Infect. Dis., 153:1145-1148.
12) European Commission. 2002. Updated opinion on the safety with regard to TSE risks of gelatine
derived from ruminant bones or hides. Adopted by The Scientific Steering Committee at its meeting of5-6 December 2002.
13) OJ No C 24, 28. 1.2004, p. 614) Japan Leather and Leather Goods Industries Association, International Industrial Survey Report in 2003
"Investigative Report on Traceability of Gelatin raw material in India" (March 2004)
15) OJ No L 290, 12.11.1999, p.32

-6English Translation by Nitta Gelatin Inc.