The EFSA Journal (2004) 67 1-27

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Opinion of the Scientific Panel on Food Additives

Flavourings Processing Aids and Materials in Contact with Food on a request from the Commission related to

Furfural and Furfural Diethylacetal

Question number EFSA-Q-2003-236

Adopted by written procedure on 2 June 2004

SUMMARY The Panel on Food Additives Flavourings Processing Aids and Materials in Contact with Food is asked to evaluate substances used as flavourings or present in flavourings or present in other food ingredients with flavouring properties In particular the Panel is asked to advise the Commission on the implications for human health of furfural and furfural diethylacetal in the diet The Panel noted that the Scientific Committee on Food (SCF) had expressed an opinion on furfural and furfural diethylacetal in December 2002 and took cognizance of the documentation used in arriving at that opinion The Panel also considered additional data on the potential genotoxicity of furfural not available at the time of the SCF opinion The new toxicological data on the genotoxic potential of furfural addressed the reservation of the SCF concerning the mechanism by which furfural induced tumours (hepatocellular adenomas and carcinomas) and whether this was a thresholded process The Panel concluded that the new study in a transgenic mouse strain demonstrated that furfural did not induce gene mutations in vivo Based on this together with earlier studies reviewed by the SCF the Panel concluded that the tumour induction was secondary to chronic toxic liver injury and was probably thresholded Since furfural diethylacetal is rapidly converted to furfural at physiological pH the Panel established an Acceptable Daily Intake (ADI) for furfural and the furfural component of furfural diethylacetal of 05 mgkg bw based on a NOAEL (no-observed-adverse-effect level) for hepatotoxicity in a 90-day study in rats of 54 mgkg bwday to which a safety factor of 100 was applied The Panel notes that the available estimates of intake from all sources (natural and use as a chemically defined flavouring substance) suggest that intakes in consumers may approach the ADI and that refined estimates of intake in the EU are desirable

KEYWORDS Furfural furfural diethylacetal flavouring substances

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BACKGROUND The European Food Safety Authority (EFSA) has received a request to evaluate the safety of furfural and furfural diethylacetal used as flavouring substances Previous evaluations Furfural was evaluated most recently by the SCF in 2002 but the Committee was unable to allocate an ADI as it was of the opinion that the data were not totally convincing in demonstrating that the carcinogenicity of furfural was mediated via a thresholded mechanism The Committee noted that it was aware of an ongoing study in transgenic mice of the potential of furfural to induce gene mutations in vivo and wished to re-evaluate furfural in the light of the results of that study This study has now been completed and is evaluated by the panel in this opinion The Panel noted the content of the previous SCF opinion and took cognizance of the documentation used in arriving at that opinion For completeness the earlier SCF opinion is attached in its entirety as Appendix 1 and this also contains details of evaluations by the Council of Europersquos Committee of Experts on Flavouring Substances (CEFS) and the Joint FAOWHO Expert Committee on Food Additives (JECFA) Current Regulatory Status in the EU Furfural and furfural diethylacetal are listed in the register of chemically defined flavouring substances (EC 2002) TERMS OF REFERENCE The European Food Safety Authority is asked to advise the Commission on substances used as flavouring substances or present in flavourings or present in other food ingredients with flavouring properties for which existing toxicological data indicate that restrictions of use or presence might be necessary to ensure safety for human health In particular the Panel is asked to advise the Commission on the implications for human health of furfural and furfural diethylacetal in the diet ASSESSMENT The Panel has given consideration to the database evaluated by the SCF (SCF 2002) as well as a new study in transgenic mice Chemistry Name Furfural Synonyms Furfuraldehyde 2-Furancarboxaldehyde Fural 2-Formylfuran 2-

Furaldehyde Pyromucic aldehyde 2-Furylcarboxaldehyde FL No 13018

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CAS No 98-01-1 FEMA No 2489 CoE No 2014 EINECS 202-627-7 Structure Name Furfural diethylacetal Synonyms 2-furaldehyde diethylacetal FL No 13126 CAS No 13529-27-6 FEMA No - CoE No - EINECS 236-872-6 Structure Exposure

Furfural together with the corresponding furfuryl alcohol occurs naturally in many fruits and in tea coffee and cocoa It is produced during the acid hydrolysis or heating of polysaccharides containing hexose or pentose fragments (Maarse et al 1994) and highest concentrations are found in cocoa and coffee (55-255 mgkg) alcoholic beverages (1-33 mgkg) and whole grain bread (up to 26 mgkg) The estimated daily per capita intake from use as a flavouring agent in Europe (consumers only) is approx 10 microgkg bwday (IOFI 1995) The intake from natural sources has been estimated to be about 300 microgkg bwday in the United States of America (Stofberg amp Grundschober 1987)

Furfural is used as a flavouring substance ingredient in many food categories The average use levels in the USA according to Burdock (2002) are in the range from 4 mgkg in gravies to 63 mgkg in meat products The theoretical maximum daily intake (assumption consumers consume at all times all flavoured foods with maximum concentrations) has been estimated to be 136 microgkg bw (CEFS 1998) Absorption distribution metabolism and excretion

O C

EtO OEt

H

O CHO

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Furfural is converted to furfuryl alcohol by enteric bacteria which can also be formed by hydrolysis of esters of furfuryl alcohol (Boopathy et al 1993 Grundschober 1977 Buck 2000) Furfural and the corresponding alcohol are rapidly absorbed from the gastrointestinal tract at doses of 01 to 200 mgkg bw and virtually totally excreted mainly in urine within 24 hours (Nomeir et al 1992) After absorption furfuryl alcohol is oxidised to furfural and further to 2-furoic acid Furoic acid is then conjugated with glycine or converted to 2-furanacryloyl-CoA and then to 2-furanacrylic acid and its glycine conjugate (Parkash amp Caldwell 1994) Some conversion to carbon dioxide (CO2) occurs in rodents (presumably from decarboxylation of furoic acid) but not in humans In rodents the decarboxylation may be preceded by epoxidation or hydroxylation of the furan ring yielding reactive intermediates (eg furfural-23-epoxide acetyl acrolein or alpha-ketoglutaric acid) which may be decarboxylated or conjugated with glutathione (Ramsdell amp Eaton 1990 Koenig amp Andreesen 1990 Gupta et al 1991 Mishra et al 1991) These latter pathways appear to be insignificant in humans at low levels of exposure (Flek amp Sedivec 1978) Special studies on genotoxicity In a new study not previously evaluated by the SCF furfural was examined for its potential to induce gene mutations of the λlacZ-gene in vivo in the liver of male transgenic mice (CD2F1(BALBc x DBA2) strain 406 with lacZ-genes as reporter genes) The study was carried out under GLP As formal technical guidelines for this type of study are not available the study protocol was designed in conformity with principles for transgenic studies identified by international expert groups (Gorelick and Mirsalis 1996 Heddle et al 2000) The study was conducted in 5 groups three of which received furfural by gavage in corn oil one negative control group received vehicle alone and one positive control group received ethylnitrosourea (ENU) The furfural and negative control groups each comprised 13 mice plus 2 back up animals the positive control group comprised 8 mice plus 2 reserves The furfural groups were given doses of 75 150 or 300 mg furfuralkg bw in corn oil by gavage for 28 consecutive days ENU was given to the positive control group by intraperitoneal injection in saline on days 5-9 of the study at a dose of 50 mgkg bwd On day 28 three animals from each of the furfural and negative control groups were sacrificed for assessment of hepatotoxicity by clinical chemistry and histological examination In addition organ and body weights were monitored throughout After a manifestation period of 34-35 days (days 62-63 of the study) the livers and samples of gastrointestinal tract tissues were fixed for mutation analysis Mutation analysis was carried out on livers of 8 animals per group At least 5000 (preferably 120000) plaque-forming units (PFU) were examined (one PFU corresponding to one recovered copy of the λgt10lacZ shuttle vector)

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There were three early decedents in the highest furfural dose group two during treatment with no clinical signs and one during the manifestation period One animal from the low-dose group died during the manifestation period The cause of death could not be ascertained Body weights in the furfural-treated groups showed a dose related increase compared to negative controls during the first week of treatment In the post-treatment period the difference between control and two lower dose groups disappeared but the body weight of the group treated with 300 mg furfuralkg bw remained higher Evaluation of the clinical chemistry and gross and histopathology of the liver of the treated animals sacrificed at the end of the treatment period showed an increase in blood triglycerides increased liver weight and centrilobular hypertrophy This was interpreted by the authors as some evidence of hepatotoxicity These changes did not persist until the end of the manifestation period 34-35 days after the last dose The mutation frequency in DNA extracted from the livers of the negative control group was similar to historical data There was no significant difference in mutation frequency between negative controls and the furfural-treated groups the positive control group showed a significant increase in mutation frequency It was concluded that oral administration of furfural in corn oil at levels of up to 300 mgkg bwday is not associated with an increase in the induction of mutations in liver cells of λlacZ transgenic mice (CIVOTNO 2003) Negative or weakly positive results have been obtained for most bacterial tests for genotoxicity In particular positive results were obtained in three out of several assays for reverse mutation in Salmonella typhimurium at relatively high concentrations in the absence of metabolic activation Furfural was found to be clearly genotoxic in cultured mammalian cells at the gene and chromosome level in the absence of metabolic activation It induced Sister Chromatid Exchange (SCE) in cultured Chinese Hamster Ovary (CHO) cells and human lymphocytes It was genotoxic in Drosophila in somatic cells (Wing spot test by inhalation) and germ cells (sex-chromosome loss by injection) It did not induce reciprocal translocations and sex-linked recessive lethal mutations with only a doubtful increase in one study in Drosophila Furfural was not genotoxic in any in vivo mammalian assays for chromosome aberrations SCE or Unscheduled DNA Synthesis (UDS) and the study in transgenic mice confirms that furfural does not induce gene mutations in vivo

Discussion The Panel noted the metabolic and toxicity data previously reviewed by the SCF together with the new results of the genotoxicity study in transgenic mice in vivo Furfural was negative in the in vivo genotoxicity assay and this corroborated earlier negative in vivo studies at the chromosome level and in a UDS assay

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In view of the absence of genotoxicity in vivo the tumours observed in the long-term toxicitycarcinogenicity studies in male but not female mice are considered to arise as a consequence of chronic hepatotoxicity (hepatocellular necrosis) which was more marked in male animals An increased tumour incidence was only observed at the highest dose level and at a dose higher than the minimal hepatotoxic dose It should be noted that no hepatocellular tumours were seen in the long-term rat study However liver toxicity was seen in this study (see SCF opinion Appendix 1) and the rat was considered more sensitive to liver toxicity The hepatotoxicity of furfural is dose-dependent but a NOEL was not established in the long-term studies However the short-term (90-day) study in rats was conducted to establish a NOEL for hepatotoxicity which was determined to be 54 mgkg bw The Panel noted that because of possible formulation (corn oil) and dose regimen (bolus dose) effects observed in the gavage studies the dietary administration studies were more appropriate for identifying a NOAEL The Panel conscluded that the NOAEL of 54 mgkg bwday for hepatic changes from the 90 day dietary study was appropriate and noted that the effects observed with doses up to threefold higher were of doubtful toxicological relevance Therefore the Panel concluded that a safety factor of 100 would be sufficient in establishing an ADI from this subchronic study (see SCF opinion) Conclusion and Recommendation The Panel concluded that furfural did not exhibit genotoxicity in vivo in male mice the species and sex which displayed an increased tumour incidence in long-term studies and that the tumours arose by a secondary mechanism consequent on hepatotoxicity which is dose dependent displays a threshold and is seen in both rats and mice It was therefore considered that the NOEL for hepatotoxicity in the rat could be used to derive an ADI for furfural An ADI for furfural was established at 05 mgkg bw based on the NOEL of 54 mgkg bw from the 90-day rat study to which a 100 fold safety factor was applied Since furfural diethylacetal is rapidly converted to furfural at physiological pH the ADI applies also to the furfural component of furfural diethylacetal since furfural is readily liberated from the acetal in vivo The Panel notes that the available estimates of intake from all sources (natural and use as a chemically defined flavouring substance) suggest that intakes in consumers may approach the ADI and that refined estimates of intake in the EU are desirable

References

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Boopathy R Bokang H amp Daniels L 1993 Biotransformation of furfural and 5-hydroxymethyl furfural by enteric bacteria J Ind Microbiol 11 147-150 Buck NR 2000 The hydrolysis of cinnamyl and furfuryl esters Unpublished report to FEMA by University of Southampton Burdock GA (2002) Fenaroliacutes handbook of flavour ingredients 4th edition CRC Press Boca Raton Fl pp 637 CEFS 1998 Council of Europe Committee of Experts on Flavouring Substances RD 424-42 Detailed and publication datasheet on furfural Brussels 20-24 April 1998 CIVOTNO (2003) Summary of in vivo genotoxicity test with furfural in MUTAregMOUSE Unpublished report submitted to EFSA EC (European Commission) 2002 Commission Decision 2002113EC amending Decision 1999217EC as regards the register of flavouring substances used in or on foodstuffs Official Journal of the European Communities 20022002 L 491

Flek J amp Sedivec V1978 The absorption metabolism and excretion of furfural in man Arch Occup Environ Hlth 41 159-168

Gorelick NJ amp Mirsalis JC 1996 A strategy for the application of Transgenic Rodent Mutagenesis Assays Environ Mol Mutagen 28 434-442

Grundschober F 1977 Toxicological assessment of flavoring esters Toxicol 8 387-390 Gupta GD Misra A amp Agarwal DK 1991 Inhalation toxicity of furfural vapours an assessment of biochemical response in rat lungs J Appl Toxicol 11 343-347 Heddle JA Dean S Nohmi T Boerrigter M Casciano D Douglas GR Glickman BW Gorelick NJ Mirsalis JC Martus H-J Skopek TR Thybaud V Tindall KR amp Yajima N 2000 In vivo transgenic mutation assays Environ Mol Mutagen 35 253-259

IOFI 1995 European inquiry on volume of use Private communication to the Flavour and Extract Manufacturersrsquo Association of the United States by International Organisation of the Flavour Industry

Koenig K amp Andreesen JR 1990 Xanthine dehydrogenase and 2-furoyl-coenzyme A dehydrogenase from Pseudomonas p Fu1 two molybdenum-containing dehydrogenases of novel structural composition J Bacteriol 172 5999-6009

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Maarse H Visscher CA Willimsens LC et al (Eds) 1994 Volatile Compounds in Food Qualitative and Quantitative Data 7th Edition Vol III Centraal Instituut voor Voedingsonderzoek TNO Zeist Mishra A Dwivedi PD Verma AS Sinha M Mishra J Lal K Pandya KPamp Dutta KK 1991 Pathological and biochemical alterations induced by inhalation of furfural vapor in rat lung Bull Envir Contam Toxicol 47 668-674 Nomeir AA Siveria DM McComish MF amp Chadwick M 1992 Comparative metabolism and disposition of furfural and furfuryl alcohol in rats Drug Metab Disposit 20 198-204 Parkash MK amp Caldwell J 1994 Metabolism and excretion of [14C]-furfural in the rat and the mouse Food Chem Toxicol 32 887-895 Ramsdell HS amp Eaton DL 1990 Species susceptibility to aflatoxin B1 carcinogenesis comparative kinetics of microsomal biotransformation Cancer Res 50 615 Scientific Committee on Food 2002 Opinion of the Scientific Committee on Food on furfural and furfural diethylacetal (expressed on 2 December 2002) Stofberg J amp Grundschober F 1987 Consumption ratio and food predominance of flavoring materials Perfumer Flavorist 12 27 SCIENTIFIC PANEL MEMBERS Robert Anton Sue Barlow Dimitrios Boskou Laurence Castle Riccardo Crebelli Wolfgang Dekant Karl-Heinz Engel Stephen Forsythe Werner Grunow John Chr Larsen Catherine Leclercq Wim Mennes Maria Rosaria Milana Ivonne Rietjens Kettil Svensson Paul Tobback Fidel Toldraacute ACKNOWLEDGEMENTS The Scientific Panel on Food Additives Flavourings Processing Aids and Materials in Contact with Food wishes to thank Ron Walker for his contribution to the draft opinion

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Appendix 1 Previous SCF opinion from 2003 The text below is from the Secretariat archives the published version of the opinion can be found at httpeuropaeuintcommfoodfsscscfout160_enpdf

EUROPEAN COMMISSION HEALTH amp CONSUMER PROTECTION DIRECTORATE-GENERAL Directorate C - Scientific Opinions C2 - Management of scientific committees II scientific co-operation and networks

Scientific Committee on Food Working Group on Flavourings

SCFCSFLAVFLAVOUR11 ADD1 Final 22 January 2003

Opinion

of the Scientific Committee on Food

on furfural and furfural diethylacetal

(expressed on 2 December 2002)

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CAS No 98-01-1 FEMA No 2489 CoE No 2014 EINECS 202-627-7 Structure Name Furfural diethylacetal Synonyms 2-furaldehyde diethylacetal FL No 13126 CAS No 13529-27-6 FEMA No - CoE No - EINECS 236-872-6 Structure Exposure

Furfural together with the corresponding furfuryl alcohol occurs naturally in many fruits and in tea coffee and cocoa It is produced during the acid hydrolysis or heating of polysaccharides containing hexose or pentose fragments (Maarse et al 1994) and highest concentrations are found in cocoa and coffee (55-255 mgkg) alcoholic beverages (1-33 mgkg) and whole grain bread (up to 26 mgkg) The estimated daily per capita intake from use as a flavouring agent in Europe (consumers only) is approx 10 microgkg bwday (IOFI 1995) The intake from natural sources has been estimated to be about 300 microgkg bwday in the United States of America (Stofberg amp Grundschober 1987)

Furfural is used as a flavouring substance ingredient in many food categories The average use levels in the USA according to Burdock (2002) are in the range from 4 mgkg in gravies to 63 mgkg in meat products The theoretical maximum daily intake (assumption consumers consume at all times all flavoured foods with maximum concentrations) has been estimated to be 136 microgkg bw (CEFS 1998) Absorption distribution metabolism and excretion

O C

EtO OEt

H

O CHO

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Furfural is converted to furfuryl alcohol by enteric bacteria which can also be formed by hydrolysis of esters of furfuryl alcohol (Boopathy et al 1993 Grundschober 1977 Buck 2000) Furfural and the corresponding alcohol are rapidly absorbed from the gastrointestinal tract at doses of 01 to 200 mgkg bw and virtually totally excreted mainly in urine within 24 hours (Nomeir et al 1992) After absorption furfuryl alcohol is oxidised to furfural and further to 2-furoic acid Furoic acid is then conjugated with glycine or converted to 2-furanacryloyl-CoA and then to 2-furanacrylic acid and its glycine conjugate (Parkash amp Caldwell 1994) Some conversion to carbon dioxide (CO2) occurs in rodents (presumably from decarboxylation of furoic acid) but not in humans In rodents the decarboxylation may be preceded by epoxidation or hydroxylation of the furan ring yielding reactive intermediates (eg furfural-23-epoxide acetyl acrolein or alpha-ketoglutaric acid) which may be decarboxylated or conjugated with glutathione (Ramsdell amp Eaton 1990 Koenig amp Andreesen 1990 Gupta et al 1991 Mishra et al 1991) These latter pathways appear to be insignificant in humans at low levels of exposure (Flek amp Sedivec 1978) Special studies on genotoxicity In a new study not previously evaluated by the SCF furfural was examined for its potential to induce gene mutations of the λlacZ-gene in vivo in the liver of male transgenic mice (CD2F1(BALBc x DBA2) strain 406 with lacZ-genes as reporter genes) The study was carried out under GLP As formal technical guidelines for this type of study are not available the study protocol was designed in conformity with principles for transgenic studies identified by international expert groups (Gorelick and Mirsalis 1996 Heddle et al 2000) The study was conducted in 5 groups three of which received furfural by gavage in corn oil one negative control group received vehicle alone and one positive control group received ethylnitrosourea (ENU) The furfural and negative control groups each comprised 13 mice plus 2 back up animals the positive control group comprised 8 mice plus 2 reserves The furfural groups were given doses of 75 150 or 300 mg furfuralkg bw in corn oil by gavage for 28 consecutive days ENU was given to the positive control group by intraperitoneal injection in saline on days 5-9 of the study at a dose of 50 mgkg bwd On day 28 three animals from each of the furfural and negative control groups were sacrificed for assessment of hepatotoxicity by clinical chemistry and histological examination In addition organ and body weights were monitored throughout After a manifestation period of 34-35 days (days 62-63 of the study) the livers and samples of gastrointestinal tract tissues were fixed for mutation analysis Mutation analysis was carried out on livers of 8 animals per group At least 5000 (preferably 120000) plaque-forming units (PFU) were examined (one PFU corresponding to one recovered copy of the λgt10lacZ shuttle vector)

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There were three early decedents in the highest furfural dose group two during treatment with no clinical signs and one during the manifestation period One animal from the low-dose group died during the manifestation period The cause of death could not be ascertained Body weights in the furfural-treated groups showed a dose related increase compared to negative controls during the first week of treatment In the post-treatment period the difference between control and two lower dose groups disappeared but the body weight of the group treated with 300 mg furfuralkg bw remained higher Evaluation of the clinical chemistry and gross and histopathology of the liver of the treated animals sacrificed at the end of the treatment period showed an increase in blood triglycerides increased liver weight and centrilobular hypertrophy This was interpreted by the authors as some evidence of hepatotoxicity These changes did not persist until the end of the manifestation period 34-35 days after the last dose The mutation frequency in DNA extracted from the livers of the negative control group was similar to historical data There was no significant difference in mutation frequency between negative controls and the furfural-treated groups the positive control group showed a significant increase in mutation frequency It was concluded that oral administration of furfural in corn oil at levels of up to 300 mgkg bwday is not associated with an increase in the induction of mutations in liver cells of λlacZ transgenic mice (CIVOTNO 2003) Negative or weakly positive results have been obtained for most bacterial tests for genotoxicity In particular positive results were obtained in three out of several assays for reverse mutation in Salmonella typhimurium at relatively high concentrations in the absence of metabolic activation Furfural was found to be clearly genotoxic in cultured mammalian cells at the gene and chromosome level in the absence of metabolic activation It induced Sister Chromatid Exchange (SCE) in cultured Chinese Hamster Ovary (CHO) cells and human lymphocytes It was genotoxic in Drosophila in somatic cells (Wing spot test by inhalation) and germ cells (sex-chromosome loss by injection) It did not induce reciprocal translocations and sex-linked recessive lethal mutations with only a doubtful increase in one study in Drosophila Furfural was not genotoxic in any in vivo mammalian assays for chromosome aberrations SCE or Unscheduled DNA Synthesis (UDS) and the study in transgenic mice confirms that furfural does not induce gene mutations in vivo

Discussion The Panel noted the metabolic and toxicity data previously reviewed by the SCF together with the new results of the genotoxicity study in transgenic mice in vivo Furfural was negative in the in vivo genotoxicity assay and this corroborated earlier negative in vivo studies at the chromosome level and in a UDS assay

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In view of the absence of genotoxicity in vivo the tumours observed in the long-term toxicitycarcinogenicity studies in male but not female mice are considered to arise as a consequence of chronic hepatotoxicity (hepatocellular necrosis) which was more marked in male animals An increased tumour incidence was only observed at the highest dose level and at a dose higher than the minimal hepatotoxic dose It should be noted that no hepatocellular tumours were seen in the long-term rat study However liver toxicity was seen in this study (see SCF opinion Appendix 1) and the rat was considered more sensitive to liver toxicity The hepatotoxicity of furfural is dose-dependent but a NOEL was not established in the long-term studies However the short-term (90-day) study in rats was conducted to establish a NOEL for hepatotoxicity which was determined to be 54 mgkg bw The Panel noted that because of possible formulation (corn oil) and dose regimen (bolus dose) effects observed in the gavage studies the dietary administration studies were more appropriate for identifying a NOAEL The Panel conscluded that the NOAEL of 54 mgkg bwday for hepatic changes from the 90 day dietary study was appropriate and noted that the effects observed with doses up to threefold higher were of doubtful toxicological relevance Therefore the Panel concluded that a safety factor of 100 would be sufficient in establishing an ADI from this subchronic study (see SCF opinion) Conclusion and Recommendation The Panel concluded that furfural did not exhibit genotoxicity in vivo in male mice the species and sex which displayed an increased tumour incidence in long-term studies and that the tumours arose by a secondary mechanism consequent on hepatotoxicity which is dose dependent displays a threshold and is seen in both rats and mice It was therefore considered that the NOEL for hepatotoxicity in the rat could be used to derive an ADI for furfural An ADI for furfural was established at 05 mgkg bw based on the NOEL of 54 mgkg bw from the 90-day rat study to which a 100 fold safety factor was applied Since furfural diethylacetal is rapidly converted to furfural at physiological pH the ADI applies also to the furfural component of furfural diethylacetal since furfural is readily liberated from the acetal in vivo The Panel notes that the available estimates of intake from all sources (natural and use as a chemically defined flavouring substance) suggest that intakes in consumers may approach the ADI and that refined estimates of intake in the EU are desirable

References

Furfural and furfural acetate The EFSA Journal (2004) 67 1-27

7 of 26

Boopathy R Bokang H amp Daniels L 1993 Biotransformation of furfural and 5-hydroxymethyl furfural by enteric bacteria J Ind Microbiol 11 147-150 Buck NR 2000 The hydrolysis of cinnamyl and furfuryl esters Unpublished report to FEMA by University of Southampton Burdock GA (2002) Fenaroliacutes handbook of flavour ingredients 4th edition CRC Press Boca Raton Fl pp 637 CEFS 1998 Council of Europe Committee of Experts on Flavouring Substances RD 424-42 Detailed and publication datasheet on furfural Brussels 20-24 April 1998 CIVOTNO (2003) Summary of in vivo genotoxicity test with furfural in MUTAregMOUSE Unpublished report submitted to EFSA EC (European Commission) 2002 Commission Decision 2002113EC amending Decision 1999217EC as regards the register of flavouring substances used in or on foodstuffs Official Journal of the European Communities 20022002 L 491

Flek J amp Sedivec V1978 The absorption metabolism and excretion of furfural in man Arch Occup Environ Hlth 41 159-168

Gorelick NJ amp Mirsalis JC 1996 A strategy for the application of Transgenic Rodent Mutagenesis Assays Environ Mol Mutagen 28 434-442

Grundschober F 1977 Toxicological assessment of flavoring esters Toxicol 8 387-390 Gupta GD Misra A amp Agarwal DK 1991 Inhalation toxicity of furfural vapours an assessment of biochemical response in rat lungs J Appl Toxicol 11 343-347 Heddle JA Dean S Nohmi T Boerrigter M Casciano D Douglas GR Glickman BW Gorelick NJ Mirsalis JC Martus H-J Skopek TR Thybaud V Tindall KR amp Yajima N 2000 In vivo transgenic mutation assays Environ Mol Mutagen 35 253-259

IOFI 1995 European inquiry on volume of use Private communication to the Flavour and Extract Manufacturersrsquo Association of the United States by International Organisation of the Flavour Industry

Koenig K amp Andreesen JR 1990 Xanthine dehydrogenase and 2-furoyl-coenzyme A dehydrogenase from Pseudomonas p Fu1 two molybdenum-containing dehydrogenases of novel structural composition J Bacteriol 172 5999-6009

Furfural and furfural acetate The EFSA Journal (2004) 67 1-27

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Maarse H Visscher CA Willimsens LC et al (Eds) 1994 Volatile Compounds in Food Qualitative and Quantitative Data 7th Edition Vol III Centraal Instituut voor Voedingsonderzoek TNO Zeist Mishra A Dwivedi PD Verma AS Sinha M Mishra J Lal K Pandya KPamp Dutta KK 1991 Pathological and biochemical alterations induced by inhalation of furfural vapor in rat lung Bull Envir Contam Toxicol 47 668-674 Nomeir AA Siveria DM McComish MF amp Chadwick M 1992 Comparative metabolism and disposition of furfural and furfuryl alcohol in rats Drug Metab Disposit 20 198-204 Parkash MK amp Caldwell J 1994 Metabolism and excretion of [14C]-furfural in the rat and the mouse Food Chem Toxicol 32 887-895 Ramsdell HS amp Eaton DL 1990 Species susceptibility to aflatoxin B1 carcinogenesis comparative kinetics of microsomal biotransformation Cancer Res 50 615 Scientific Committee on Food 2002 Opinion of the Scientific Committee on Food on furfural and furfural diethylacetal (expressed on 2 December 2002) Stofberg J amp Grundschober F 1987 Consumption ratio and food predominance of flavoring materials Perfumer Flavorist 12 27 SCIENTIFIC PANEL MEMBERS Robert Anton Sue Barlow Dimitrios Boskou Laurence Castle Riccardo Crebelli Wolfgang Dekant Karl-Heinz Engel Stephen Forsythe Werner Grunow John Chr Larsen Catherine Leclercq Wim Mennes Maria Rosaria Milana Ivonne Rietjens Kettil Svensson Paul Tobback Fidel Toldraacute ACKNOWLEDGEMENTS The Scientific Panel on Food Additives Flavourings Processing Aids and Materials in Contact with Food wishes to thank Ron Walker for his contribution to the draft opinion

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Appendix 1 Previous SCF opinion from 2003 The text below is from the Secretariat archives the published version of the opinion can be found at httpeuropaeuintcommfoodfsscscfout160_enpdf

EUROPEAN COMMISSION HEALTH amp CONSUMER PROTECTION DIRECTORATE-GENERAL Directorate C - Scientific Opinions C2 - Management of scientific committees II scientific co-operation and networks

Scientific Committee on Food Working Group on Flavourings

SCFCSFLAVFLAVOUR11 ADD1 Final 22 January 2003

Opinion

of the Scientific Committee on Food

on furfural and furfural diethylacetal

(expressed on 2 December 2002)

Furfural and furfural acetate The EFSA Journal (2004) 67 1-27

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Furfural is converted to furfuryl alcohol by enteric bacteria which can also be formed by hydrolysis of esters of furfuryl alcohol (Boopathy et al 1993 Grundschober 1977 Buck 2000) Furfural and the corresponding alcohol are rapidly absorbed from the gastrointestinal tract at doses of 01 to 200 mgkg bw and virtually totally excreted mainly in urine within 24 hours (Nomeir et al 1992) After absorption furfuryl alcohol is oxidised to furfural and further to 2-furoic acid Furoic acid is then conjugated with glycine or converted to 2-furanacryloyl-CoA and then to 2-furanacrylic acid and its glycine conjugate (Parkash amp Caldwell 1994) Some conversion to carbon dioxide (CO2) occurs in rodents (presumably from decarboxylation of furoic acid) but not in humans In rodents the decarboxylation may be preceded by epoxidation or hydroxylation of the furan ring yielding reactive intermediates (eg furfural-23-epoxide acetyl acrolein or alpha-ketoglutaric acid) which may be decarboxylated or conjugated with glutathione (Ramsdell amp Eaton 1990 Koenig amp Andreesen 1990 Gupta et al 1991 Mishra et al 1991) These latter pathways appear to be insignificant in humans at low levels of exposure (Flek amp Sedivec 1978) Special studies on genotoxicity In a new study not previously evaluated by the SCF furfural was examined for its potential to induce gene mutations of the λlacZ-gene in vivo in the liver of male transgenic mice (CD2F1(BALBc x DBA2) strain 406 with lacZ-genes as reporter genes) The study was carried out under GLP As formal technical guidelines for this type of study are not available the study protocol was designed in conformity with principles for transgenic studies identified by international expert groups (Gorelick and Mirsalis 1996 Heddle et al 2000) The study was conducted in 5 groups three of which received furfural by gavage in corn oil one negative control group received vehicle alone and one positive control group received ethylnitrosourea (ENU) The furfural and negative control groups each comprised 13 mice plus 2 back up animals the positive control group comprised 8 mice plus 2 reserves The furfural groups were given doses of 75 150 or 300 mg furfuralkg bw in corn oil by gavage for 28 consecutive days ENU was given to the positive control group by intraperitoneal injection in saline on days 5-9 of the study at a dose of 50 mgkg bwd On day 28 three animals from each of the furfural and negative control groups were sacrificed for assessment of hepatotoxicity by clinical chemistry and histological examination In addition organ and body weights were monitored throughout After a manifestation period of 34-35 days (days 62-63 of the study) the livers and samples of gastrointestinal tract tissues were fixed for mutation analysis Mutation analysis was carried out on livers of 8 animals per group At least 5000 (preferably 120000) plaque-forming units (PFU) were examined (one PFU corresponding to one recovered copy of the λgt10lacZ shuttle vector)

Furfural and furfural acetate The EFSA Journal (2004) 67 1-27

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There were three early decedents in the highest furfural dose group two during treatment with no clinical signs and one during the manifestation period One animal from the low-dose group died during the manifestation period The cause of death could not be ascertained Body weights in the furfural-treated groups showed a dose related increase compared to negative controls during the first week of treatment In the post-treatment period the difference between control and two lower dose groups disappeared but the body weight of the group treated with 300 mg furfuralkg bw remained higher Evaluation of the clinical chemistry and gross and histopathology of the liver of the treated animals sacrificed at the end of the treatment period showed an increase in blood triglycerides increased liver weight and centrilobular hypertrophy This was interpreted by the authors as some evidence of hepatotoxicity These changes did not persist until the end of the manifestation period 34-35 days after the last dose The mutation frequency in DNA extracted from the livers of the negative control group was similar to historical data There was no significant difference in mutation frequency between negative controls and the furfural-treated groups the positive control group showed a significant increase in mutation frequency It was concluded that oral administration of furfural in corn oil at levels of up to 300 mgkg bwday is not associated with an increase in the induction of mutations in liver cells of λlacZ transgenic mice (CIVOTNO 2003) Negative or weakly positive results have been obtained for most bacterial tests for genotoxicity In particular positive results were obtained in three out of several assays for reverse mutation in Salmonella typhimurium at relatively high concentrations in the absence of metabolic activation Furfural was found to be clearly genotoxic in cultured mammalian cells at the gene and chromosome level in the absence of metabolic activation It induced Sister Chromatid Exchange (SCE) in cultured Chinese Hamster Ovary (CHO) cells and human lymphocytes It was genotoxic in Drosophila in somatic cells (Wing spot test by inhalation) and germ cells (sex-chromosome loss by injection) It did not induce reciprocal translocations and sex-linked recessive lethal mutations with only a doubtful increase in one study in Drosophila Furfural was not genotoxic in any in vivo mammalian assays for chromosome aberrations SCE or Unscheduled DNA Synthesis (UDS) and the study in transgenic mice confirms that furfural does not induce gene mutations in vivo

Discussion The Panel noted the metabolic and toxicity data previously reviewed by the SCF together with the new results of the genotoxicity study in transgenic mice in vivo Furfural was negative in the in vivo genotoxicity assay and this corroborated earlier negative in vivo studies at the chromosome level and in a UDS assay

Furfural and furfural acetate The EFSA Journal (2004) 67 1-27

6 of 26

In view of the absence of genotoxicity in vivo the tumours observed in the long-term toxicitycarcinogenicity studies in male but not female mice are considered to arise as a consequence of chronic hepatotoxicity (hepatocellular necrosis) which was more marked in male animals An increased tumour incidence was only observed at the highest dose level and at a dose higher than the minimal hepatotoxic dose It should be noted that no hepatocellular tumours were seen in the long-term rat study However liver toxicity was seen in this study (see SCF opinion Appendix 1) and the rat was considered more sensitive to liver toxicity The hepatotoxicity of furfural is dose-dependent but a NOEL was not established in the long-term studies However the short-term (90-day) study in rats was conducted to establish a NOEL for hepatotoxicity which was determined to be 54 mgkg bw The Panel noted that because of possible formulation (corn oil) and dose regimen (bolus dose) effects observed in the gavage studies the dietary administration studies were more appropriate for identifying a NOAEL The Panel conscluded that the NOAEL of 54 mgkg bwday for hepatic changes from the 90 day dietary study was appropriate and noted that the effects observed with doses up to threefold higher were of doubtful toxicological relevance Therefore the Panel concluded that a safety factor of 100 would be sufficient in establishing an ADI from this subchronic study (see SCF opinion) Conclusion and Recommendation The Panel concluded that furfural did not exhibit genotoxicity in vivo in male mice the species and sex which displayed an increased tumour incidence in long-term studies and that the tumours arose by a secondary mechanism consequent on hepatotoxicity which is dose dependent displays a threshold and is seen in both rats and mice It was therefore considered that the NOEL for hepatotoxicity in the rat could be used to derive an ADI for furfural An ADI for furfural was established at 05 mgkg bw based on the NOEL of 54 mgkg bw from the 90-day rat study to which a 100 fold safety factor was applied Since furfural diethylacetal is rapidly converted to furfural at physiological pH the ADI applies also to the furfural component of furfural diethylacetal since furfural is readily liberated from the acetal in vivo The Panel notes that the available estimates of intake from all sources (natural and use as a chemically defined flavouring substance) suggest that intakes in consumers may approach the ADI and that refined estimates of intake in the EU are desirable

References

Furfural and furfural acetate The EFSA Journal (2004) 67 1-27

7 of 26

Boopathy R Bokang H amp Daniels L 1993 Biotransformation of furfural and 5-hydroxymethyl furfural by enteric bacteria J Ind Microbiol 11 147-150 Buck NR 2000 The hydrolysis of cinnamyl and furfuryl esters Unpublished report to FEMA by University of Southampton Burdock GA (2002) Fenaroliacutes handbook of flavour ingredients 4th edition CRC Press Boca Raton Fl pp 637 CEFS 1998 Council of Europe Committee of Experts on Flavouring Substances RD 424-42 Detailed and publication datasheet on furfural Brussels 20-24 April 1998 CIVOTNO (2003) Summary of in vivo genotoxicity test with furfural in MUTAregMOUSE Unpublished report submitted to EFSA EC (European Commission) 2002 Commission Decision 2002113EC amending Decision 1999217EC as regards the register of flavouring substances used in or on foodstuffs Official Journal of the European Communities 20022002 L 491

Flek J amp Sedivec V1978 The absorption metabolism and excretion of furfural in man Arch Occup Environ Hlth 41 159-168

Gorelick NJ amp Mirsalis JC 1996 A strategy for the application of Transgenic Rodent Mutagenesis Assays Environ Mol Mutagen 28 434-442

Grundschober F 1977 Toxicological assessment of flavoring esters Toxicol 8 387-390 Gupta GD Misra A amp Agarwal DK 1991 Inhalation toxicity of furfural vapours an assessment of biochemical response in rat lungs J Appl Toxicol 11 343-347 Heddle JA Dean S Nohmi T Boerrigter M Casciano D Douglas GR Glickman BW Gorelick NJ Mirsalis JC Martus H-J Skopek TR Thybaud V Tindall KR amp Yajima N 2000 In vivo transgenic mutation assays Environ Mol Mutagen 35 253-259

IOFI 1995 European inquiry on volume of use Private communication to the Flavour and Extract Manufacturersrsquo Association of the United States by International Organisation of the Flavour Industry

Koenig K amp Andreesen JR 1990 Xanthine dehydrogenase and 2-furoyl-coenzyme A dehydrogenase from Pseudomonas p Fu1 two molybdenum-containing dehydrogenases of novel structural composition J Bacteriol 172 5999-6009

Furfural and furfural acetate The EFSA Journal (2004) 67 1-27

8 of 26

Maarse H Visscher CA Willimsens LC et al (Eds) 1994 Volatile Compounds in Food Qualitative and Quantitative Data 7th Edition Vol III Centraal Instituut voor Voedingsonderzoek TNO Zeist Mishra A Dwivedi PD Verma AS Sinha M Mishra J Lal K Pandya KPamp Dutta KK 1991 Pathological and biochemical alterations induced by inhalation of furfural vapor in rat lung Bull Envir Contam Toxicol 47 668-674 Nomeir AA Siveria DM McComish MF amp Chadwick M 1992 Comparative metabolism and disposition of furfural and furfuryl alcohol in rats Drug Metab Disposit 20 198-204 Parkash MK amp Caldwell J 1994 Metabolism and excretion of [14C]-furfural in the rat and the mouse Food Chem Toxicol 32 887-895 Ramsdell HS amp Eaton DL 1990 Species susceptibility to aflatoxin B1 carcinogenesis comparative kinetics of microsomal biotransformation Cancer Res 50 615 Scientific Committee on Food 2002 Opinion of the Scientific Committee on Food on furfural and furfural diethylacetal (expressed on 2 December 2002) Stofberg J amp Grundschober F 1987 Consumption ratio and food predominance of flavoring materials Perfumer Flavorist 12 27 SCIENTIFIC PANEL MEMBERS Robert Anton Sue Barlow Dimitrios Boskou Laurence Castle Riccardo Crebelli Wolfgang Dekant Karl-Heinz Engel Stephen Forsythe Werner Grunow John Chr Larsen Catherine Leclercq Wim Mennes Maria Rosaria Milana Ivonne Rietjens Kettil Svensson Paul Tobback Fidel Toldraacute ACKNOWLEDGEMENTS The Scientific Panel on Food Additives Flavourings Processing Aids and Materials in Contact with Food wishes to thank Ron Walker for his contribution to the draft opinion

Furfural and furfural acetate The EFSA Journal (2004) 67 1-27

9 of 26

Appendix 1 Previous SCF opinion from 2003 The text below is from the Secretariat archives the published version of the opinion can be found at httpeuropaeuintcommfoodfsscscfout160_enpdf

EUROPEAN COMMISSION HEALTH amp CONSUMER PROTECTION DIRECTORATE-GENERAL Directorate C - Scientific Opinions C2 - Management of scientific committees II scientific co-operation and networks

Scientific Committee on Food Working Group on Flavourings

SCFCSFLAVFLAVOUR11 ADD1 Final 22 January 2003

Opinion

of the Scientific Committee on Food

on furfural and furfural diethylacetal

(expressed on 2 December 2002)

Furfural and furfural acetate The EFSA Journal (2004) 67 1-27

5 of 26

There were three early decedents in the highest furfural dose group two during treatment with no clinical signs and one during the manifestation period One animal from the low-dose group died during the manifestation period The cause of death could not be ascertained Body weights in the furfural-treated groups showed a dose related increase compared to negative controls during the first week of treatment In the post-treatment period the difference between control and two lower dose groups disappeared but the body weight of the group treated with 300 mg furfuralkg bw remained higher Evaluation of the clinical chemistry and gross and histopathology of the liver of the treated animals sacrificed at the end of the treatment period showed an increase in blood triglycerides increased liver weight and centrilobular hypertrophy This was interpreted by the authors as some evidence of hepatotoxicity These changes did not persist until the end of the manifestation period 34-35 days after the last dose The mutation frequency in DNA extracted from the livers of the negative control group was similar to historical data There was no significant difference in mutation frequency between negative controls and the furfural-treated groups the positive control group showed a significant increase in mutation frequency It was concluded that oral administration of furfural in corn oil at levels of up to 300 mgkg bwday is not associated with an increase in the induction of mutations in liver cells of λlacZ transgenic mice (CIVOTNO 2003) Negative or weakly positive results have been obtained for most bacterial tests for genotoxicity In particular positive results were obtained in three out of several assays for reverse mutation in Salmonella typhimurium at relatively high concentrations in the absence of metabolic activation Furfural was found to be clearly genotoxic in cultured mammalian cells at the gene and chromosome level in the absence of metabolic activation It induced Sister Chromatid Exchange (SCE) in cultured Chinese Hamster Ovary (CHO) cells and human lymphocytes It was genotoxic in Drosophila in somatic cells (Wing spot test by inhalation) and germ cells (sex-chromosome loss by injection) It did not induce reciprocal translocations and sex-linked recessive lethal mutations with only a doubtful increase in one study in Drosophila Furfural was not genotoxic in any in vivo mammalian assays for chromosome aberrations SCE or Unscheduled DNA Synthesis (UDS) and the study in transgenic mice confirms that furfural does not induce gene mutations in vivo

Discussion The Panel noted the metabolic and toxicity data previously reviewed by the SCF together with the new results of the genotoxicity study in transgenic mice in vivo Furfural was negative in the in vivo genotoxicity assay and this corroborated earlier negative in vivo studies at the chromosome level and in a UDS assay

Furfural and furfural acetate The EFSA Journal (2004) 67 1-27

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In view of the absence of genotoxicity in vivo the tumours observed in the long-term toxicitycarcinogenicity studies in male but not female mice are considered to arise as a consequence of chronic hepatotoxicity (hepatocellular necrosis) which was more marked in male animals An increased tumour incidence was only observed at the highest dose level and at a dose higher than the minimal hepatotoxic dose It should be noted that no hepatocellular tumours were seen in the long-term rat study However liver toxicity was seen in this study (see SCF opinion Appendix 1) and the rat was considered more sensitive to liver toxicity The hepatotoxicity of furfural is dose-dependent but a NOEL was not established in the long-term studies However the short-term (90-day) study in rats was conducted to establish a NOEL for hepatotoxicity which was determined to be 54 mgkg bw The Panel noted that because of possible formulation (corn oil) and dose regimen (bolus dose) effects observed in the gavage studies the dietary administration studies were more appropriate for identifying a NOAEL The Panel conscluded that the NOAEL of 54 mgkg bwday for hepatic changes from the 90 day dietary study was appropriate and noted that the effects observed with doses up to threefold higher were of doubtful toxicological relevance Therefore the Panel concluded that a safety factor of 100 would be sufficient in establishing an ADI from this subchronic study (see SCF opinion) Conclusion and Recommendation The Panel concluded that furfural did not exhibit genotoxicity in vivo in male mice the species and sex which displayed an increased tumour incidence in long-term studies and that the tumours arose by a secondary mechanism consequent on hepatotoxicity which is dose dependent displays a threshold and is seen in both rats and mice It was therefore considered that the NOEL for hepatotoxicity in the rat could be used to derive an ADI for furfural An ADI for furfural was established at 05 mgkg bw based on the NOEL of 54 mgkg bw from the 90-day rat study to which a 100 fold safety factor was applied Since furfural diethylacetal is rapidly converted to furfural at physiological pH the ADI applies also to the furfural component of furfural diethylacetal since furfural is readily liberated from the acetal in vivo The Panel notes that the available estimates of intake from all sources (natural and use as a chemically defined flavouring substance) suggest that intakes in consumers may approach the ADI and that refined estimates of intake in the EU are desirable

References

Furfural and furfural acetate The EFSA Journal (2004) 67 1-27

7 of 26

Boopathy R Bokang H amp Daniels L 1993 Biotransformation of furfural and 5-hydroxymethyl furfural by enteric bacteria J Ind Microbiol 11 147-150 Buck NR 2000 The hydrolysis of cinnamyl and furfuryl esters Unpublished report to FEMA by University of Southampton Burdock GA (2002) Fenaroliacutes handbook of flavour ingredients 4th edition CRC Press Boca Raton Fl pp 637 CEFS 1998 Council of Europe Committee of Experts on Flavouring Substances RD 424-42 Detailed and publication datasheet on furfural Brussels 20-24 April 1998 CIVOTNO (2003) Summary of in vivo genotoxicity test with furfural in MUTAregMOUSE Unpublished report submitted to EFSA EC (European Commission) 2002 Commission Decision 2002113EC amending Decision 1999217EC as regards the register of flavouring substances used in or on foodstuffs Official Journal of the European Communities 20022002 L 491

Flek J amp Sedivec V1978 The absorption metabolism and excretion of furfural in man Arch Occup Environ Hlth 41 159-168

Gorelick NJ amp Mirsalis JC 1996 A strategy for the application of Transgenic Rodent Mutagenesis Assays Environ Mol Mutagen 28 434-442

Grundschober F 1977 Toxicological assessment of flavoring esters Toxicol 8 387-390 Gupta GD Misra A amp Agarwal DK 1991 Inhalation toxicity of furfural vapours an assessment of biochemical response in rat lungs J Appl Toxicol 11 343-347 Heddle JA Dean S Nohmi T Boerrigter M Casciano D Douglas GR Glickman BW Gorelick NJ Mirsalis JC Martus H-J Skopek TR Thybaud V Tindall KR amp Yajima N 2000 In vivo transgenic mutation assays Environ Mol Mutagen 35 253-259

IOFI 1995 European inquiry on volume of use Private communication to the Flavour and Extract Manufacturersrsquo Association of the United States by International Organisation of the Flavour Industry

Koenig K amp Andreesen JR 1990 Xanthine dehydrogenase and 2-furoyl-coenzyme A dehydrogenase from Pseudomonas p Fu1 two molybdenum-containing dehydrogenases of novel structural composition J Bacteriol 172 5999-6009

Furfural and furfural acetate The EFSA Journal (2004) 67 1-27

8 of 26

Maarse H Visscher CA Willimsens LC et al (Eds) 1994 Volatile Compounds in Food Qualitative and Quantitative Data 7th Edition Vol III Centraal Instituut voor Voedingsonderzoek TNO Zeist Mishra A Dwivedi PD Verma AS Sinha M Mishra J Lal K Pandya KPamp Dutta KK 1991 Pathological and biochemical alterations induced by inhalation of furfural vapor in rat lung Bull Envir Contam Toxicol 47 668-674 Nomeir AA Siveria DM McComish MF amp Chadwick M 1992 Comparative metabolism and disposition of furfural and furfuryl alcohol in rats Drug Metab Disposit 20 198-204 Parkash MK amp Caldwell J 1994 Metabolism and excretion of [14C]-furfural in the rat and the mouse Food Chem Toxicol 32 887-895 Ramsdell HS amp Eaton DL 1990 Species susceptibility to aflatoxin B1 carcinogenesis comparative kinetics of microsomal biotransformation Cancer Res 50 615 Scientific Committee on Food 2002 Opinion of the Scientific Committee on Food on furfural and furfural diethylacetal (expressed on 2 December 2002) Stofberg J amp Grundschober F 1987 Consumption ratio and food predominance of flavoring materials Perfumer Flavorist 12 27 SCIENTIFIC PANEL MEMBERS Robert Anton Sue Barlow Dimitrios Boskou Laurence Castle Riccardo Crebelli Wolfgang Dekant Karl-Heinz Engel Stephen Forsythe Werner Grunow John Chr Larsen Catherine Leclercq Wim Mennes Maria Rosaria Milana Ivonne Rietjens Kettil Svensson Paul Tobback Fidel Toldraacute ACKNOWLEDGEMENTS The Scientific Panel on Food Additives Flavourings Processing Aids and Materials in Contact with Food wishes to thank Ron Walker for his contribution to the draft opinion

Furfural and furfural acetate The EFSA Journal (2004) 67 1-27

9 of 26

Appendix 1 Previous SCF opinion from 2003 The text below is from the Secretariat archives the published version of the opinion can be found at httpeuropaeuintcommfoodfsscscfout160_enpdf

EUROPEAN COMMISSION HEALTH amp CONSUMER PROTECTION DIRECTORATE-GENERAL Directorate C - Scientific Opinions C2 - Management of scientific committees II scientific co-operation and networks

Scientific Committee on Food Working Group on Flavourings

SCFCSFLAVFLAVOUR11 ADD1 Final 22 January 2003

Opinion

of the Scientific Committee on Food

on furfural and furfural diethylacetal

(expressed on 2 December 2002)

Furfural and furfural acetate The EFSA Journal (2004) 67 1-27

6 of 26

In view of the absence of genotoxicity in vivo the tumours observed in the long-term toxicitycarcinogenicity studies in male but not female mice are considered to arise as a consequence of chronic hepatotoxicity (hepatocellular necrosis) which was more marked in male animals An increased tumour incidence was only observed at the highest dose level and at a dose higher than the minimal hepatotoxic dose It should be noted that no hepatocellular tumours were seen in the long-term rat study However liver toxicity was seen in this study (see SCF opinion Appendix 1) and the rat was considered more sensitive to liver toxicity The hepatotoxicity of furfural is dose-dependent but a NOEL was not established in the long-term studies However the short-term (90-day) study in rats was conducted to establish a NOEL for hepatotoxicity which was determined to be 54 mgkg bw The Panel noted that because of possible formulation (corn oil) and dose regimen (bolus dose) effects observed in the gavage studies the dietary administration studies were more appropriate for identifying a NOAEL The Panel conscluded that the NOAEL of 54 mgkg bwday for hepatic changes from the 90 day dietary study was appropriate and noted that the effects observed with doses up to threefold higher were of doubtful toxicological relevance Therefore the Panel concluded that a safety factor of 100 would be sufficient in establishing an ADI from this subchronic study (see SCF opinion) Conclusion and Recommendation The Panel concluded that furfural did not exhibit genotoxicity in vivo in male mice the species and sex which displayed an increased tumour incidence in long-term studies and that the tumours arose by a secondary mechanism consequent on hepatotoxicity which is dose dependent displays a threshold and is seen in both rats and mice It was therefore considered that the NOEL for hepatotoxicity in the rat could be used to derive an ADI for furfural An ADI for furfural was established at 05 mgkg bw based on the NOEL of 54 mgkg bw from the 90-day rat study to which a 100 fold safety factor was applied Since furfural diethylacetal is rapidly converted to furfural at physiological pH the ADI applies also to the furfural component of furfural diethylacetal since furfural is readily liberated from the acetal in vivo The Panel notes that the available estimates of intake from all sources (natural and use as a chemically defined flavouring substance) suggest that intakes in consumers may approach the ADI and that refined estimates of intake in the EU are desirable

References

Furfural and furfural acetate The EFSA Journal (2004) 67 1-27

7 of 26

Boopathy R Bokang H amp Daniels L 1993 Biotransformation of furfural and 5-hydroxymethyl furfural by enteric bacteria J Ind Microbiol 11 147-150 Buck NR 2000 The hydrolysis of cinnamyl and furfuryl esters Unpublished report to FEMA by University of Southampton Burdock GA (2002) Fenaroliacutes handbook of flavour ingredients 4th edition CRC Press Boca Raton Fl pp 637 CEFS 1998 Council of Europe Committee of Experts on Flavouring Substances RD 424-42 Detailed and publication datasheet on furfural Brussels 20-24 April 1998 CIVOTNO (2003) Summary of in vivo genotoxicity test with furfural in MUTAregMOUSE Unpublished report submitted to EFSA EC (European Commission) 2002 Commission Decision 2002113EC amending Decision 1999217EC as regards the register of flavouring substances used in or on foodstuffs Official Journal of the European Communities 20022002 L 491

Flek J amp Sedivec V1978 The absorption metabolism and excretion of furfural in man Arch Occup Environ Hlth 41 159-168

Gorelick NJ amp Mirsalis JC 1996 A strategy for the application of Transgenic Rodent Mutagenesis Assays Environ Mol Mutagen 28 434-442

Grundschober F 1977 Toxicological assessment of flavoring esters Toxicol 8 387-390 Gupta GD Misra A amp Agarwal DK 1991 Inhalation toxicity of furfural vapours an assessment of biochemical response in rat lungs J Appl Toxicol 11 343-347 Heddle JA Dean S Nohmi T Boerrigter M Casciano D Douglas GR Glickman BW Gorelick NJ Mirsalis JC Martus H-J Skopek TR Thybaud V Tindall KR amp Yajima N 2000 In vivo transgenic mutation assays Environ Mol Mutagen 35 253-259

IOFI 1995 European inquiry on volume of use Private communication to the Flavour and Extract Manufacturersrsquo Association of the United States by International Organisation of the Flavour Industry

Koenig K amp Andreesen JR 1990 Xanthine dehydrogenase and 2-furoyl-coenzyme A dehydrogenase from Pseudomonas p Fu1 two molybdenum-containing dehydrogenases of novel structural composition J Bacteriol 172 5999-6009

Furfural and furfural acetate The EFSA Journal (2004) 67 1-27

8 of 26

Maarse H Visscher CA Willimsens LC et al (Eds) 1994 Volatile Compounds in Food Qualitative and Quantitative Data 7th Edition Vol III Centraal Instituut voor Voedingsonderzoek TNO Zeist Mishra A Dwivedi PD Verma AS Sinha M Mishra J Lal K Pandya KPamp Dutta KK 1991 Pathological and biochemical alterations induced by inhalation of furfural vapor in rat lung Bull Envir Contam Toxicol 47 668-674 Nomeir AA Siveria DM McComish MF amp Chadwick M 1992 Comparative metabolism and disposition of furfural and furfuryl alcohol in rats Drug Metab Disposit 20 198-204 Parkash MK amp Caldwell J 1994 Metabolism and excretion of [14C]-furfural in the rat and the mouse Food Chem Toxicol 32 887-895 Ramsdell HS amp Eaton DL 1990 Species susceptibility to aflatoxin B1 carcinogenesis comparative kinetics of microsomal biotransformation Cancer Res 50 615 Scientific Committee on Food 2002 Opinion of the Scientific Committee on Food on furfural and furfural diethylacetal (expressed on 2 December 2002) Stofberg J amp Grundschober F 1987 Consumption ratio and food predominance of flavoring materials Perfumer Flavorist 12 27 SCIENTIFIC PANEL MEMBERS Robert Anton Sue Barlow Dimitrios Boskou Laurence Castle Riccardo Crebelli Wolfgang Dekant Karl-Heinz Engel Stephen Forsythe Werner Grunow John Chr Larsen Catherine Leclercq Wim Mennes Maria Rosaria Milana Ivonne Rietjens Kettil Svensson Paul Tobback Fidel Toldraacute ACKNOWLEDGEMENTS The Scientific Panel on Food Additives Flavourings Processing Aids and Materials in Contact with Food wishes to thank Ron Walker for his contribution to the draft opinion

Furfural and furfural acetate The EFSA Journal (2004) 67 1-27

9 of 26

Appendix 1 Previous SCF opinion from 2003 The text below is from the Secretariat archives the published version of the opinion can be found at httpeuropaeuintcommfoodfsscscfout160_enpdf

EUROPEAN COMMISSION HEALTH amp CONSUMER PROTECTION DIRECTORATE-GENERAL Directorate C - Scientific Opinions C2 - Management of scientific committees II scientific co-operation and networks

Scientific Committee on Food Working Group on Flavourings

SCFCSFLAVFLAVOUR11 ADD1 Final 22 January 2003

Opinion

of the Scientific Committee on Food

on furfural and furfural diethylacetal

(expressed on 2 December 2002)

Furfural and furfural acetate The EFSA Journal (2004) 67 1-27

7 of 26

Boopathy R Bokang H amp Daniels L 1993 Biotransformation of furfural and 5-hydroxymethyl furfural by enteric bacteria J Ind Microbiol 11 147-150 Buck NR 2000 The hydrolysis of cinnamyl and furfuryl esters Unpublished report to FEMA by University of Southampton Burdock GA (2002) Fenaroliacutes handbook of flavour ingredients 4th edition CRC Press Boca Raton Fl pp 637 CEFS 1998 Council of Europe Committee of Experts on Flavouring Substances RD 424-42 Detailed and publication datasheet on furfural Brussels 20-24 April 1998 CIVOTNO (2003) Summary of in vivo genotoxicity test with furfural in MUTAregMOUSE Unpublished report submitted to EFSA EC (European Commission) 2002 Commission Decision 2002113EC amending Decision 1999217EC as regards the register of flavouring substances used in or on foodstuffs Official Journal of the European Communities 20022002 L 491

Flek J amp Sedivec V1978 The absorption metabolism and excretion of furfural in man Arch Occup Environ Hlth 41 159-168

Gorelick NJ amp Mirsalis JC 1996 A strategy for the application of Transgenic Rodent Mutagenesis Assays Environ Mol Mutagen 28 434-442

Grundschober F 1977 Toxicological assessment of flavoring esters Toxicol 8 387-390 Gupta GD Misra A amp Agarwal DK 1991 Inhalation toxicity of furfural vapours an assessment of biochemical response in rat lungs J Appl Toxicol 11 343-347 Heddle JA Dean S Nohmi T Boerrigter M Casciano D Douglas GR Glickman BW Gorelick NJ Mirsalis JC Martus H-J Skopek TR Thybaud V Tindall KR amp Yajima N 2000 In vivo transgenic mutation assays Environ Mol Mutagen 35 253-259

IOFI 1995 European inquiry on volume of use Private communication to the Flavour and Extract Manufacturersrsquo Association of the United States by International Organisation of the Flavour Industry

Koenig K amp Andreesen JR 1990 Xanthine dehydrogenase and 2-furoyl-coenzyme A dehydrogenase from Pseudomonas p Fu1 two molybdenum-containing dehydrogenases of novel structural composition J Bacteriol 172 5999-6009

Furfural and furfural acetate The EFSA Journal (2004) 67 1-27

8 of 26

Maarse H Visscher CA Willimsens LC et al (Eds) 1994 Volatile Compounds in Food Qualitative and Quantitative Data 7th Edition Vol III Centraal Instituut voor Voedingsonderzoek TNO Zeist Mishra A Dwivedi PD Verma AS Sinha M Mishra J Lal K Pandya KPamp Dutta KK 1991 Pathological and biochemical alterations induced by inhalation of furfural vapor in rat lung Bull Envir Contam Toxicol 47 668-674 Nomeir AA Siveria DM McComish MF amp Chadwick M 1992 Comparative metabolism and disposition of furfural and furfuryl alcohol in rats Drug Metab Disposit 20 198-204 Parkash MK amp Caldwell J 1994 Metabolism and excretion of [14C]-furfural in the rat and the mouse Food Chem Toxicol 32 887-895 Ramsdell HS amp Eaton DL 1990 Species susceptibility to aflatoxin B1 carcinogenesis comparative kinetics of microsomal biotransformation Cancer Res 50 615 Scientific Committee on Food 2002 Opinion of the Scientific Committee on Food on furfural and furfural diethylacetal (expressed on 2 December 2002) Stofberg J amp Grundschober F 1987 Consumption ratio and food predominance of flavoring materials Perfumer Flavorist 12 27 SCIENTIFIC PANEL MEMBERS Robert Anton Sue Barlow Dimitrios Boskou Laurence Castle Riccardo Crebelli Wolfgang Dekant Karl-Heinz Engel Stephen Forsythe Werner Grunow John Chr Larsen Catherine Leclercq Wim Mennes Maria Rosaria Milana Ivonne Rietjens Kettil Svensson Paul Tobback Fidel Toldraacute ACKNOWLEDGEMENTS The Scientific Panel on Food Additives Flavourings Processing Aids and Materials in Contact with Food wishes to thank Ron Walker for his contribution to the draft opinion

Furfural and furfural acetate The EFSA Journal (2004) 67 1-27

9 of 26

Appendix 1 Previous SCF opinion from 2003 The text below is from the Secretariat archives the published version of the opinion can be found at httpeuropaeuintcommfoodfsscscfout160_enpdf

EUROPEAN COMMISSION HEALTH amp CONSUMER PROTECTION DIRECTORATE-GENERAL Directorate C - Scientific Opinions C2 - Management of scientific committees II scientific co-operation and networks

Scientific Committee on Food Working Group on Flavourings

SCFCSFLAVFLAVOUR11 ADD1 Final 22 January 2003

Opinion

of the Scientific Committee on Food

on furfural and furfural diethylacetal

(expressed on 2 December 2002)

Furfural and furfural acetate The EFSA Journal (2004) 67 1-27

8 of 26

Maarse H Visscher CA Willimsens LC et al (Eds) 1994 Volatile Compounds in Food Qualitative and Quantitative Data 7th Edition Vol III Centraal Instituut voor Voedingsonderzoek TNO Zeist Mishra A Dwivedi PD Verma AS Sinha M Mishra J Lal K Pandya KPamp Dutta KK 1991 Pathological and biochemical alterations induced by inhalation of furfural vapor in rat lung Bull Envir Contam Toxicol 47 668-674 Nomeir AA Siveria DM McComish MF amp Chadwick M 1992 Comparative metabolism and disposition of furfural and furfuryl alcohol in rats Drug Metab Disposit 20 198-204 Parkash MK amp Caldwell J 1994 Metabolism and excretion of [14C]-furfural in the rat and the mouse Food Chem Toxicol 32 887-895 Ramsdell HS amp Eaton DL 1990 Species susceptibility to aflatoxin B1 carcinogenesis comparative kinetics of microsomal biotransformation Cancer Res 50 615 Scientific Committee on Food 2002 Opinion of the Scientific Committee on Food on furfural and furfural diethylacetal (expressed on 2 December 2002) Stofberg J amp Grundschober F 1987 Consumption ratio and food predominance of flavoring materials Perfumer Flavorist 12 27 SCIENTIFIC PANEL MEMBERS Robert Anton Sue Barlow Dimitrios Boskou Laurence Castle Riccardo Crebelli Wolfgang Dekant Karl-Heinz Engel Stephen Forsythe Werner Grunow John Chr Larsen Catherine Leclercq Wim Mennes Maria Rosaria Milana Ivonne Rietjens Kettil Svensson Paul Tobback Fidel Toldraacute ACKNOWLEDGEMENTS The Scientific Panel on Food Additives Flavourings Processing Aids and Materials in Contact with Food wishes to thank Ron Walker for his contribution to the draft opinion

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Appendix 1 Previous SCF opinion from 2003 The text below is from the Secretariat archives the published version of the opinion can be found at httpeuropaeuintcommfoodfsscscfout160_enpdf

EUROPEAN COMMISSION HEALTH amp CONSUMER PROTECTION DIRECTORATE-GENERAL Directorate C - Scientific Opinions C2 - Management of scientific committees II scientific co-operation and networks

Scientific Committee on Food Working Group on Flavourings

SCFCSFLAVFLAVOUR11 ADD1 Final 22 January 2003

Opinion

of the Scientific Committee on Food

on furfural and furfural diethylacetal

(expressed on 2 December 2002)

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Appendix 1 Previous SCF opinion from 2003 The text below is from the Secretariat archives the published version of the opinion can be found at httpeuropaeuintcommfoodfsscscfout160_enpdf

EUROPEAN COMMISSION HEALTH amp CONSUMER PROTECTION DIRECTORATE-GENERAL Directorate C - Scientific Opinions C2 - Management of scientific committees II scientific co-operation and networks

Scientific Committee on Food Working Group on Flavourings

SCFCSFLAVFLAVOUR11 ADD1 Final 22 January 2003

Opinion

of the Scientific Committee on Food

on furfural and furfural diethylacetal

(expressed on 2 December 2002)

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SCFCSFLAVFLAVOUR11 ADD1 Final

Opinion of the Scientific Committee on Food on

furfural and furfural diethylacetal

(expressed on 2 December 2002)

Terms of Reference The Committee is asked to advise the Commission on substances used as flavouring substances or present in flavourings or present in other food ingredients with flavouring properties for which existing toxicological data indicate that restrictions of use or presence might be necessary to ensure safety for human health In particular the Committee is asked to advise the Commission on the implications for human health of the presence of furfural in the diet Introduction Beside furfural furfural diethylacetal is listed in the register of chemically defined flavouring substances laid down in Commission Decision 1999217EC (EC 1999) as last amended by Commission Decision 2002113EC (EC 2002) The Committee was aware that diethylacetals are readily hydrolysed to the corresponding aldehyde and ethanol under acid conditions such as occur in the human stomach Since furfural diethylacetal would undergo such hydrolysis to furfural the conclusions of this opinion are also valid for furfural diethylacetal Previous evaluations In an earlier evaluation by the Scientific Committee on Food (SCF 1995) furfural was placed in category 4 (unsuitable for use due to evidence of toxicity) The Committee of Experts on Flavouring Substances of the Council of Europe evaluated furfural as follows (CEFS 1998) Many short term and long term studies have shown the hepatoxicity of furfural Carcinogenic effects occurred in male rats and male mice at the highest dosage (60 mgkg in rat 175 mgkg in mice)hellipFurfural should be considered as a non-genotoxic compound and the highest hepatoxic dose levels administered to male rat or male mice could lead to cell proliferation and cell death and after prolonged exposure to liver tumours If the Committee considers furfural as a non-genotoxic compound a NOEL can be set at 30 mgkg bw Using a safety factor of 100 a TDI of 300 microgkgday is proposed

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In the 45th CEFS meeting a majority decision was taken to delete furfural from the list of active principles and consider it as a special case because it occurs only in trace amounts in natural sources of flavourings In addition it was deleted from the 4th edition of the Blue Book (Vol 1) (CEFS 1999) The Joint FAOWHO Expert Committee on Food Additives (JECFA) was unable to allocate an ADI to furfural at its thirty-ninth meeting (JECFA 1992) and its fifty-first meeting (JECFA 1999) due to concern about tumours observed in male mice given furfural and the fact that no NOEL was established for hepatotoxicity in rats At its Fifty-fifth meeting (JECFA 2000) the JECFA concluded that new data from a rat toxicity study enabled a NOEL to be determined and a UDS study by Edwards (1999) was conducted in response to a request by the JECFA for a study on the formation of DNA adducts in order to clarify whether the tumours seen in the National Toxicology Program (NTP) study in mice arose from a genotoxic mechanism or were secondary to the increased incidence of liver necrosis Although the UDS assay was not a direct assay for adduct formation the JECFA considered that its requirements for additional studies to establish a NOEL and clarify the potential genotoxicity had been met It concluded that the evidence provided indicated a non-genotoxic mechanism for tumour formation in the mouse liver and hence an ADI could be established The hydrolysis of furfuryl esters and interconversion of furfuryl alcohol and furfural with subsequent oxidation to furoic acid provided the basis for the JECFA allocating a Group ADI of 0-05 mgkg bw for furfural furfuryl alcohol furfuryl acetate furfuryl propionate furfuryl pentanoate furfuryl octanoate furfuryl 3-methylbutanoate methyl 2-furoate propyl 2-furoate amyl 2-furoate hexyl 2-furoate and octyl 2-furoate

Current Regulatory Status Furfural and furfural diethylacetal are listed in the register of chemically defined flavouring substances (EC 2002) Furfural has Flavour and Essence Manufacturers Association (FEMA) GRAS status in the United States

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Chemical characterisation Name Furfural Synonyms Furfuraldehyde 2-Furancarboxaldehyde Fural 2-Formylfuran 2-

Furaldehyde Pyromucic aldehyde 2-Furylcarboxaldehyde FL No 13018 CAS No 98-01-1 FEMA No 2489 CoE No 2014 EINECS 202-627-7 Structure Name Furfural diethylacetal Synonyms 2-furaldehyde diethylacetal FL No 13126 CAS No 13529-27-6 FEMA No - CoE No - EINECS 236-872-6 Structure

Exposure assessment

Furfural together with the corresponding furfuryl alcohol occurs naturally in many fruits and in tea coffee and cocoa It is produced during the acid hydrolysis or heating of polysaccharides containing hexose or pentose fragments (Maarse et al 1994) and highest concentrations are found in cocoa and coffee (55-255 mgkg) alcoholic beverages (1-33

O C

EtO OEt

H

O CHO

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mgkg) and whole grain bread (up to 26 mgkg) The estimated daily per capita intake from use as a flavouring agent in Europe (eaters only) is approx 10 microgkg bwday (IOFI 1995) The intake from natural sources has been estimated to be about 300 microgkg bwday in the United States of America (Stofberg amp Grundschober 1987)

Furfural is used as a flavouring substance ingredient in many food categories The average use levels according to Fenaroli (1995) are in the range from 4 mgkg in gravies to 63 mgkg in meat products The theoretical maximum daily intake (assumption consumers consume at all times all flavoured foods with maximum concentrations) has been estimated to be 136 microgkg bw (CEFS 1998)

Hazard identificationcharacterisation

Absorption metabolism and excretion Furfural is converted to furfuryl alcohol by enteric bacteria which can also be formed by hydrolysis of esters of furfuryl alcohol (Boopathy et al 1993 Grundschober 1977 Buck 2000) Furfural and the corresponding alcohol are rapidly absorbed from the gi tract at doses of 01 to 200 mgkg bw and virtually totally excreted mainly in urine within 24 hours (Nomeir et al 1992) After absorption furfuryl alcohol is oxidised to furfural and further to 2-furoic acid Furoic acid is then conjugated with glycine or converted to 2-furanacryloyl-CoA and then to 2-furanacrylic acid and its glycine conjugate (Parkash amp Caldwell 1994) Some conversion to CO2 occurs in rodents (presumably from decarboxylation of furoic acid) but not in humans In rodents the decarboxylation may be preceded by epoxidation or hydroxylation of the furan ring yielding reactive intermediates (eg furfural-23-epoxide acetyl acrolein or alpha-ketoglutaric acid) which may be decarboxylated or conjugated with glutathione (Ramsdell amp Eaton 1990 Koenig amp Andreesen 1990 Gupta et al 1991 Mishra et al 1991) These latter pathways appear to be insignificant in humans at low levels of exposure (Flek amp Sedivec 1978) Acute toxicity The oral LD50 for furfural was reported to be 127 mgkg bw in rats (Jenner et al 1964) and 333 mgkg bw in mice (Boyland et al 1940) Sub-acutesub-chronic toxicity Groups of five male and five female Fischer rats that received furfural by gavage at doses of 0 15 30 60 120 or 240 mgkg bw for a total of 12 doses over 16 days did not display any gross or histopathological abnormalities at termination although eight of ten rats at the top

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dose were reported to have died due to gavaging accidents In a study using a similar protocol groups of five male and five female B6C3F1 mice were given 12 doses of furfural by gavage at doses of 0 25 50 100 200 or 400 mgkg bw No gross or histopathological abnormalities were reported but one male of the top dose group and two females were stated to have died from gavaging accidents In a 13 week study groups of 10 male and 10 female mice were given furfural by gavage in corn oil at doses of 0 75 150 300 600 or 1200 mgkg bw per day on five days per week All the mice in the top dose group and all but one of each sex in the 600 mgkg bw groups died before the end of the study Females at 75-300 mgkg bw and males at 300 mgkg bw per day showed increased liver weight Most males in the two highest dose groups and one in each of the 300 and 150 mgkg bw dose groups showed centrilobular coagulative necrosis of hepatocytes only two females in the highest group showed similar lesions The lesion was accompanied by inflammation with mild mononuclear inflammatory cell infiltrate (NTP 1990) In a similar 13-week study in rats furfural was administered at doses of 0 11 22 45 90 or 180 mgkg bw per day by gavage on 5 days per week to groups of 10 males and 10 females Only one male in the top dose group survived to termination The liver and kidney weights of males in the two highest dose groups were increased and there was an increase in cytoplasmic vacuolation in hepatocytes in all groups of treated males No lesions were reported in the kidneys (NTP 1990) In a study of unusual design rats treated with furfural for 90 days at doses varying from 20 mgkg diet to 40 mgkg diet showed marked cholangiofibrosis with fibrous widening of Glissonrsquos sheath bile duct proliferation and parenchymal damage consisting of necrosis and hydropic degeneration of hepatocytes However furfural did not cause hepatocellular hyperplastic changes (Shimizu amp Kanisawa 1986 Shimizu et al1989)

A study was conducted to examine the mode of action by which furfural was hepatotoxic Rats were dosed with furfural in the diet at approx 1200 mgkg bw for 30 days and 1700 mgkg bw thereafter The furfural was removed after 15 30 60 90 120 or 150 days and the animals killed 14 days later Increased duration of exposure was associated with increased numbers and size of foci positive for placental type glutathione-S-transferase (Pickett amp Lu 1988) In response to a JECFA requirement for the establishment of a NOEL for hepatotoxicity a 14 day pilot study and a subsequent 90-day study were conducted in F344N rats in which the animals were given microencapsulated furfural in the diet to provide target dose levels of 030 60 90 120 and 180 mgkg bw per day In the pilot study a NOEL of 120 mgkg bwday was established At the higher dose there was an increase in liver weight (Jonker 2000a) In the 90 day study 10 animals of each sex were used at each dose level There were no clinical

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signs of toxicity in any treatment group and food consumption and body weight gain were unaffected Some haematological changes (decrease in erythrocytes in males in the highest dose group with increased cell volume and mean corpuscular haemoglobin in the top two dose groups males were observed In females there was a decrease in serum alkaline phosphatase an increase in gamma-glutamyltransferase and an increase in plasma albumin in the highest dose group In males in the high dose group there was a decrease in ALAT an increase in plasma albumin and albuminglobulin ratio At necropsy an increase in liver weight was observed in the top dose group only with no gross pathological changes Slight histopathological changes occurred in liver of males of the 90 and 180 mgkg bw dose group in the perilobular region characterised by cells with less coarse cytoplasm with eosinophilic clumps and a less dense periphery and more prominent nucleoli No such effects were seen in females and there was no sign of hepatotoxic effects such as degeneration necrosis or inflammation nor of bile duct proliferation The NOEL established from this study was 54 mgkg bwday (Jonker 2000b)

Long-term studies of chronic toxicity and carcinogenicity

In a two-year study in B6C3F1 mice 50 animals of each sex received doses of 0 50 100 or 175 mgkg bw by gavage in corn oil on 5 days per week At termination there was no significant effect of furfural on body weight or survival Histological examination showed chronic inflammation necrosis and pigmentation in the liver of males in the two highest dose groups and in females of the top dose group only Hepatocellular adenomas and carcinomas were observed in all dose groups including controls but these tumours occurred with significantly increased incidence only in males of the top dose group The incidence of carcinomas was similar in high dose females and controls Tumours of other organs occurred only with low incidence and with no dose response relationship Slight increases in the incidence of hyperplasia and papillomas in the forestomach of female mice were considered by the authors to be due to the irritating effect of gavage administration and not of toxicological significance none of the animals had malignant lesions of the forestomach (NTP 1990)

In a similar study in Fischer 344N rats furfural was administered at doses of 0 30 or 60 mgkg bw by gavage in corn oil to 50 animals of each sex Mild centrilobular hepatocellular necrosis occurred in all groups but the incidence did not appear to be dose related particularly in females where the incidence was inversely related to dose Bile duct hyperplasia occurred with high incidence in all groups including controls and did not appear to be treatment related Focal bile duct dysplasia was seen in one male at the intermediate dose level and bile duct hyperplasia accompanied by fibrosis occurred in two males in the top dose group One control male had a hepatocellular adenoma and two males in the high dose group had a cholangiocarcinoma (a rare tumour in historical controls) It was considered by the authors

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that although the incidence of this lesion was not statistically significant it offered some evidence of carcinogenicity

Special studies on genotoxicity

Negative or weakly positive results have been obtained for most bacterial tests for genotoxicity In particular positive results were obtained in three out of several assays for reverse mutation in Salmonella typhimurium at relatively high concentrations in the absence of metabolic activation Furfural was found to be clearly genotoxic in cultured mammalian cells at the gene and chromosome level in the absence of metabolic activation It induced SCE in cultured CHO cells and human lymphocytes It was gentoxic in Drosophila in somatic cells (Wing spot test by inhalation) and germ cells (sex-chromosome loss by injection) It did not induce reciprocal translocations and sex-linked recessive lethal mutations with only a doubtful increase in one study in Drosophila Furfural was not genotoxic in any in vivo mammalian assays for chromosome aberrations SCE or UDS The results of genotoxicity studies on furfural are summarised in Table 1

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Table 1 Special studies on genotoxicity

End-Point

Test object

Concentration

Result

Reference

In vitro

Reverse mutation

Styphimurium TA100 TA98 TA1535

Styphimurium TA100 TA98 TA102

Styphimurium TA100 TA98

Styphimurium TA102 TA104

Styphimurium TA98 TA100TA1535 TA1537

Styphimurium TA98 TA100

Styphimurium TA98 TA100

Styphimurium TA100

Styphimurium TA98 TA100 TA104

EcoliWP2uvrAPKM101

Styphimurium TA104

005-60 micromolplate

le12 mmolplate

0165-0660 micromolplate

5-500 microgplate

333-6666 micromolplate

1-15 microLplate

7 microLplate

444 micromolplate

20 microLplate

1 micromol (max non-toxic dose)

Weakly positive (TA100)b

Negativea

Negativea

Positive (TA104)

Negativea

Positivea (TA100)

Negativea

Negativea

Negativea

Negativeb

Loquet et al 1981

Aeschbacher et al1989

Shinohara et al 1986

Shane et al1988

Mortelmans et al1986

Zdzienicka et al1978

Sasaki amp Endo 1978

Osawa amp Namiki 1982

McMahon et al1979

Marnett et al 1985

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Umu gene expression

Rec assay

Forward mutation

Chromosomal aberration

Sister chromatid exchange

Unscheduled DNA synthesis

Styphimurium TA1535pSK002

Bsubtilis H17 M45

Bsubtilis H17 M45

L5178Ytk+- mouse lymphoma cells

Chinese hamster ovary cells

Chinese hamster ovary cells

Chinese hamster ovary cells

Chinese hamster V79 cells

Chinese hamster ovary cells

Human peripheral lymphocytes

Human liver slices

1932 microgmL

17-17 mgdisk

1 mgdisk

25-800 microgmL

10-40 mM

200-1230 microgmL

15-5000 microgmL

500-2000 microgmL

117-3890 microgmL

35-14x10-5 M

014 mmolL

0-25 mmolL

Negativea

Positivea

Negativea

Positiveb

Positivea

Positivea

Positivea

Positivea

Positivea

Positiveb

Negative

Nakamura et al 1987

Shinohara et al 1986

Matsui et al 1989

McGregor et al1988

Stich 1981a 1981b

Galloway et al 1985

Gudi amp Schadly 1996

Nishi et al 1989

Galloway et al 1985

Gomez-Arroyo amp Souza 1985

Lake et al 1998

In vivo

Sex-linked recessive lethal

test

Dmelanogaster

Dmelanogaster

1000 ppm in diet

100 ppm by injection

Negative

Positive

Woodruff et al 1985

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Wing spot test

Sex-chromosome loss

Reciprocal translocation

Sister chromatid exchange chromosomal aberration

Somatic chromosomal aberration

Sperm head abnormalities

Unscheduled DNA synthesis

Dmelanogaster

Dmelanogaster

Dmelanogaster

B6C3F1 mouse bone marrow cells

Swiss albino mouse bone marrow cells

Swiss albino mouse

Fischer 344 rat hepatocytes

B6C3F1 mouse hepatocytes

3750-7500 ppm by aerial exposure

3750-5000 ppm in diet and by injection

1000 ppm in diet

50-200 mgkg bw once ip

4000 ppm for 5 days in diet

4000 ppm for 5 weeks in diet

50 167 or 50 mgkg bw orally

50 175 or 320 mgkg bw orally

Positive

Positive on injection

Negative

Negative

Negative

Negative

Negative

Negative

Rodriguez-Arnaiz et al 1989

Rodriguez-Arnaiz et al 1989 1992

Woodruff et al 1985

National Toxicology Program 1990

Subramanyam et al 1989+

Subramanyam et al 1989+

Phillipset al 1997

Edwards 1999

a with and without metabolic activation b without metabolic activation

c with metabolic activation +abstract only no details available

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Reproductive and Developmental toxicity

There are few data on the reproductive toxicity of furfural In one limited study five groups of eight pregnant rats received furfural by gavage in water at doses of 10 50 100 500 or 1000 mgkg bwday on days 6-15 of gestation Excessive deaths occurred at the two top doses and further groups were given 150 250 and 350 mgkg bw per day Because of excessive mortality all animals receiving doses of 250 mgkg bw and higher were killed after 6 days All other females survived except one that died within 1 hour of dosing probably as a result of a dosing accident Intrauterine growth and survival were not affected at doses of 10 ndash 150 mgkg bw and no developmental variations were noted (US EPA 1997)

Summary of the hazard identificationcharacterisation

Furfural is rapidly absorbed and metabolised by oxidation of the aldehydic function to furoic acid most of which is conjugated with glycine and excreted A small amount is condensed with CoA to form furanacroylCoA The resultant furanacryloic acid is conjugated with glycine and excreted in urine Since furfuryl alcohol is oxidised in vivo to furfural and then follows the same metabolic pathway as furfural the alcohol and its esters that are hydrolysed by intestinal enzymes may be considered together with furfural

Furfural is clearly hepatotoxic in short-term studies at daily doses of 90 mgkg bw and above in male rats and 150 mgkg bw and above in mice The toxicity may have been exaggerated by the vehicle used in gavage studies (corn oil) as a dose of 150 mgkg bw was the highest dose tolerated in the rat in the developmental study where the vehicle was water

Furfural was essentially negative in Salmonella reverse mutation assays and other bacterial assays but was directly genotoxic in cultured mammalian cells at the gene and chromosome levels Furfural was genotoxic in Drosophila it was negative in vivo in mammalian assays at the chromosome level and in a UDS assay

In long-term toxicitycarcinogenicity studies the combined incidence of hepatocellular adenomas and carcinomas was increased at the highest dose of 175mgkg bw in male mice but not females Hepatotoxicity (focal and multifocal necrosis and chronic inflammation) occurred in all groups including controls but was markedly more frequent in the high dose mice

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Long-term studies in rats led to bile duct hyperplasia and cholangiofibrosis in all animals (controls had the highest incidence) Mild hepatocellular necrosis was seen in all groups but at higher rates in males at the high dose

The recent short-term study in rats was conducted in order to establish a NOEL for the hepatotoxicity which was determined to be 54 mgkg bw

Conclusion

The Committee was of the opinion that the data were not totally convincing in demonstrating that the carcinogenicity of furfural was mediated via a thresholded mechanism and hence was unable to allocate an ADI to furfural at the present time It was aware that a study in transgenic mice of the potential of furfural to induce gene mutations in vivo was in progress The results were expected to be available in the near future and the Committee would wish to re-evaluate furfural in the light of the results of this study

References Aeschbacher HU Wolleb U Loliger J Spadone JC amp Liardon R 1989 Contribution of coffee aroma constituents to the mutagenicity of coffee Food ChemToxicol 27 227-232 Boopathy R Bokang H amp Daniels L 1993 Biotransformation of furfural and 5-hydroxymethyl furfural by enteric bacteria J Ind Microbiol 11 147-150 Boyland E 1940 Experiments on the chemotherapy of cancer 4 Further experiments with aldehydes and their derivatives Biochem J 34 1196-1201 Buck NR 2000 The hydrolysis of cinnamyl and furfuryl esters Unpublished report to FEMA by University of Southampton CEFS 1998 Council of Europe Committee of Experts on Flavouring Substances RD 424-42 Detailed and publication datasheet on furfural Brussels 20-24 April 1998 CEFS 1999 Council of Europe Committee of Experts on Flavouring Substances Record of 45th meeting Zurich 11 ndash 15 October 1999 EC (European Commission) 1999 Commission Decision 1999217EC adopting a register of flavouring substances used in or on foodstuffs drawn up in application of Regulation (EC) No

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223296 of the European Parliament and of the Council of 28 October 1996 Official Journal of the European Communities 2731999 L841-137

EC (European Commission) 2002 Commission Decision 2002113EC amending Decision 1999217EC as regards the register of flavouring substances used in or on foodstuffs Official Journal of the European Communities 20022002 L 491

Edwards A 1999 An in vivo unscheduled DNA synthesis assay in the mouse with furfural Report No 33891199 BIBRA International Carshalton UK Flek J amp Sedivec V1978 The absorption metabolism and excretion of furfural in man Arch Occup Environ Hlth 41 159-168 Galloway SM Bloom AD Resnick M Margolin BH Nakamura F Archer P amp Zeiger E 1985 Development of standard protocol for in vitro cytogenetic testing with Chinese hamster ovary cells comparison of results for 22 compounds in two laboratories Environ Mutagen 7 1-51 Gomez-Arroyo S amp Souza V 1985 In vitro and occupational induction of sister-chromatid exchanges in human lymphocytes with furfuryl alcohol and furfural Mutat Res 156 233-238

Grundschober F 1977 Toxicological assessment of flavoring esters Toxicol 8 387-390 Gudi R amp Schadly EH 1996 In vitro mammalian cytogenetic test with an independent repeat assay Microbiological Associates Inc Maryland Laboratory Study Number G96AS33335 Gupta GD Misra A amp Agarwal DK 1991 Inhalation toxicity of furfural vapours an assessment of biochemical response in rat lungs J Appl Toxicol 11 343-347

IOFI 1995 European inquiry on volume of use Private communication to the Flavour and Extract Manufacturersrsquo Association of the United States by International Organisation of the Flavour Industry JECFA 1992 Evaluation of Certain Food Additives and Naturally Occurring Toxicants 39th Report of the Joint FAOWHO Expert Committee on Food Additives WHO Technical Report Series No 828 World Health Organisation Geneva JECFA 1999 Evaluation of Certain Food Additives 51st Report of the Joint FAOWHO Expert Committee on Food Additives WHO Technical Report Series No 891 World Health Organisation Geneva

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JECFA 2000 Evaluation of Certain Food Additives and Contaminants 55th Report of the Joint FAOWHO Expert Committee on Food Additives WHO Technical Report Series No901 World Health Organization Geneva

Jenner PM Hagan EC Taylor JM Cook El amp Fitzhugh OG 1964 Food flavourings and compounds of related structure I Acute oral toxicity Food Cosmet Toxicol 2 327-343 Jonker D 2000a Dose range finding study (14-day) with micro-encapsulated furfural in F344 rats Unpublished report V981173 from TNO Zeist Netherlands Submitted to WHO by the Flavor and Extract Manufacturersacute Association of the United States Jonker D 2000b Sub-chronic (13-week) oral toxicity study in rats with micro-encapsulated furfural Unpublished report V99520 from TNO Zeist Netherlands Submitted to WHO by the Flavor and Extract Manufacturersacute Association of the United States

Koenig K amp Andreesen JR 1990 Xanthine dehydrogenase and 2-furoyl-coenzyme A dehydrogenase from Pseudomonas p Fu1 two molybdenum-containing dehydrogenases of novel structural composition J Bacteriol 172 5999-6009 Lake BG Adams TB Beamad JA Price RJ Ford RA amp Goodman JI 1998 An investigation of the effect of furfural on the unscheduled DNA synthesis in cultured human liver slices Report to FEMA Loquet C Toussant G amp LeTalaer JY 1981 Studies on mutagenic constituents of apple brandy and various alcoholic beverages collected in western France a high incidence area for oesophageal cancer MutatRes 88 155-164 Maarse H Visscher CA Willimsens LC et al (Eds) 1994 Volatile Compounds in Food Qualitative and Quantitative Data 7th Edition Vol III Centraal Instituut voor Voedingsonderzoek TNO Zeist McMahon RE Cline JC amp Thompson CZ 1979 Assay of 855 test chemicals in ten tester strains using a new modification of the Ames test for bacterial mutagens Cancer Res 39 682-693 Marnett LJ Hurd HK HollsteinMC Levin DE Esterbauer Hamp Ames BN 1985 Naturally occurring carbonyl compounds are mutagens in Salmonella tester strain TA104 MutatRes 148 25-34 Matsui S Yamamoto R amp Yamada H (1989) The Bacillus subtilismicrosome rec-assay for detection of DNA-damaging substances which may occur in chlorinated and ozonated waters WatSciTech 21 875-887

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McGregor DB Brown A Cattanach P Edwards I McBride D and Caspary WJ 1988 Responses of the L5178Ytk+tk- Mouse Lymphoma Cell Forward Mutation Assay II 18 coded chemicals Environ Molec Mutag 11 91-118 Mishra A Dwivedi PD Verma AS Sinha M Mishra J Lal K Pandya KPamp Dutta KK 1991 Pathological and biochemical alterations induced by inhalation of furfural vapor in rat lung Bull Envir Contam Toxicol 47 668-674 Mortelmans K Haworth S Lawlor T Speck W Trainer B amp Zeiger E 1986 Salmonella mutagenicity tests II Results from the testing of 270 chemicals EnvironMutagen 8 1-119 Nakamura S-I Oda Y Shimada T Oki I amp Sugimoto K 1987 SOS-inducing activity of chemical carcinogens and mutagens in Salmonella typhimurium TA1535pSK1002 Examination with 151 chemicals MutatRes 192 239-246 Nishi Y Miyakawa Y amp Kato K 1989 Chromosome aberrations induced by pyrolysates of carbohydrates in Chinese hamster V79 cells Mutat Res 227 117-123 Nomeir AA Siveria DM McComish MF amp Chadwick M 1992 Comparative metabolism and disposition of furfural and furfuryl alcohol in rats Drug Metab Disposit 20 198-204 NTP (National Toxicology Program) 1990 Toxicology and Carcinogenesis studies of furfural (CAS No 98-01-1) in Fischer 344N rats and B6C3F1 mice (gavage studies) National Toxicology Program Technical Report Series No382 Research Triangle Park North Carolina Osawa T amp Namiki M 1982 Mutagen formation in the reaction of nitrite with the food components analogous to sorbic acid AgricBiolChem 45 2299-2304 Parkash MK amp Caldwell J 1994 Metabolism and excretion of [14C]-furfural in the rat and the mouse Food Chem Toxicol 32 887-895 Phillips BJ Jackson LI Tate B Price RJ Adams TB Ford RA Goodman JI amp Lake BJ 1997 Furfural does not induce unscheduled DNA synthesis (UDS) in the in vivo rat hepatocyte DNA repair assay In Proceedings of the Society of Toxicology Annual Meeting 1997 Academic Press Cincinnati Ohio New York Pickett CB amp Lu AYH 1988 The structure genetics and regulation of soluble glutathione S-transferases In Glutathione Conjugation Mechanisms and Biological Significance (Sies H amp Ketterer B Eds) Academic Press New York p 147

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Appendix 1 httpeuropaeuintcommfoodfsscscfout160_enpdf 25 of 26

Ramsdell HS amp Eaton DL 1990 Species susceptibility to aflatoxin B1 carcinogenesis comparative kinetics of microsomal biotransformation Cancer Res 50 615 Rodriguez-Arnaiz R Morales PR Moctezuma RV amp Salas RMB 1989 Evidence for the absence of mutagenic activity of furfuryl alcohol in tests of germ cells in Drosophila melanogaster Mutat Res 223 309-311 Rodriguez-Arnaiz R Morales PR amp Zimmering S 1992 Evaluation in Drosophila melanogaster of the mutagenic potential of furfural in the mei-9a test for chromosome loss in germ-line cells and the wing spot test for mutational activity in somatic cells Mutat Res 280 75-80 Sasaki Y amp Endo R 1978 Mutagenicity of aldehydes in Salmonella Mutat Res 54 251-252 Scientific Committee on Food 1995 Extracts from First Report on Chemically Defined Flavouring Substances Annex 6 to the minutes of the 98th meeting of the Scientific Committee on Food 21 and 22 September 1995 Shane BS Troxclair AM McMillin DJ amp Henry CB 1988 Comparative mutagenicity of nine brands of coffee to Salmonella typhimurium TA100 TA102 and TA104 EnvironMol Mutagen 11 195-206 Shimizu A amp Kanisawa M 1986 Experimental studies on hepatic cirrhosis and hepatocarcinogenesis I Production of hepatic cirrhosis by furfural administration Acta Pathol Japan 36 1027-1038 Shimizu A Nakamura Y Harada M Ono T Sato K Inoue T amp Kanisawa M 1989 Positive foci of glutathione S-transferase placental form in the liver of rats given furfural by oral administration Jap J Cancer Res 80 608-611 Shinohara K Kim E amp Omura H 1986 Furans as the mutagens formed by amino-carbonyl reactions In Developments in food science 13 amino-carbonyl reactions in food and biological systems Proceedings of the 3rd International Symposium on the Maillard Reaction Susono Shizuoka Japan 1-5 July 1985 (Fujimaki M Namaiki M amp Kato H Eds) Kodansha Ltd Tokyo Stich HF Rosin MP Wu CH amp Powrie WD 1981a Clastogenicity of furans found in food Cancer Letters 13 89-95 Stich HF Rosin MP San RH Wu CH amp Powrie WD 1981b Intake formation and release of mutagens by man In Gastrointestinal Cancer (Banbury Report 7) CSH Press Cold Spring Harbor New York pp 247-266

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Stofberg J amp Grundschober F 1987 Consumption ratio and food predominance of flavoring materials Perfumer Flavorist 12 27 Subramanyam A Sailaja D amp Rathnaprabba D 1989 Genotoxic assay of two dietary furans by some in vivo cytogenetic parameters EMS Abstracts p 239 US EPA (United States Environmental Protection Agency) 1997 Summary report A dose range finding development toxicity study of furfural (TSCA submission 8EHQ-1097-14026) Washington DC Woodruff RC Mason JM Valencia R amp Zimmering S 1985 Chemical mutagenesis testing in Drosophila V Results of 53 coded compounds tested for the National Toxicology Program Environ Mutat 7 677-702 Zdzienicka M Tudek B Zielenska M amp Szymczyk T 1978 Mutagenic activity of furfural in Salmonella typhimurium TA100 Mutat Res 58 205-209