2-ethyl-3-hydroxy-4-pyrone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial Reverse Mutation Assay/Ames test): the substance Ethyl Maltol was mutagenic in the strain S. typhimurium TA 100 in the presence and absence of S9 metabolic activation. (No guideline).

Chromosome aberration (in vitro cytogenicity/micronucleus study): This study is waived as an in vivo mammalian erythrocyte micronucleus test is available.

Gene mutation (mammalian cell gene mutation assay) (Read-across to Kojic acid (CAS No. 501-30-4)): Kojic acid did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells in the absence or presence of Aroclor 1254-induced rat liver S9 (OECD 476/GLP).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Validity of study confirmed by EFSA (FGE.213), Only tested strains TA98 and TA100, Quercetin and sterigmatocystin were used as positive controls. Max dose tested 2 mg/plate.

Qualifier:

according to guideline

Guideline:

other: Ames, B.N., J. McCann and E. Yamasaki, Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test, Mutation Res., 31 (1975) 347-364.

Deviations:

yes

Remarks:

Only tested strains TA98 and TA100, Quercetin and sterigmatocystin were used as positive controls. Max dose tested 2 mg/plate.

GLP compliance:

no

Remarks:

pre-GLP

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine (hisG46, hisD3052)

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Provided by B.N. Ames
- Suitability of cells: commonly used strain to detect frameshift reverse mutation

Additional strain / cell type characteristics:

other: R factor plasmid, pKM101, deep rough coat mutation and uvrB deletion

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Provided by B.N. Ames
- Suitability of cells: commonly used strain to detect base-pair substitution reverse mutation

Additional strain / cell type characteristics:

other: R factor plasmid, pKM101, deep rough coat mutation and uvrB deletion

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

0.5, 1 and 2 mg/plate

Vehicle / solvent:

Filter sterilised water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: Quercetin, Sterigmatocystin

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation)

DURATION
- Exposure duration: 48h

DETERMINATION OF CYTOTOXICITY
- Method: not specified

Rationale for test conditions:

As described by Ames et al. (1975)

Evaluation criteria:

Not specified

Statistics:

The mean number of revertant colonies is calculated from triplicate runs, and the number of spontaneous revertants is subtracted from each value.
Further statistics were not specified.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

max dose tested 2 mg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

max dose tested 2 mg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: the publication did not mention precipitation, also not at the highest dose tested 2 mg/plate

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Cytotoxicity was not observed for ethyl maltol up to the highest concentration tested (2 mg/plate).

Conclusions:

Under the conditions of this test, a dose related mutagenic effect was observed for Ethyl Maltol against tester strain TA100 but not in strain TA98.

Executive summary:

In a reverse gene mutation assay in bacteria (Bjeldanes and Chew, 1979), strains of S. typhimurium TA 98 and TA 100 were exposed to Ethyl Maltol at concentrations of 0.5, 1 and 2 mg/plate (plate incorporation) in the presence and absence of mammalian metabolic activation (S9). Quercetin, sterigmatocystin and benzo[a]pyrene were used as positive controls. The study was judged to be valid by the EFSA (FGE.213) and rated Klimisch 2.

The substance induced a dose-related increase in the number of revertant colonies in S. typhimurium TA100, both in the absence and presence of S9 metabolic activation. No mutagenicity was observed in strain TA98. Under the conditions of this study, the test substance is considered mutagenic.

EFSA Journal 2015;13(9):4244

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19-11-2001 to 28-03-2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

21 July 1997

GLP compliance:

yes

Type of assay:

other: Mammalian cell gene mutation assay

Target gene:

HPRT

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Dr. Donald Clive, Burroughs Wellcome Co.
- Suitability of cells: the mammalian mouse lymphoma L5178Y cell line has been sufficiently well validated for use in in vitro mutation assays.
- Methods for maintenance in cell culture if applicable:
L5178Y TK mouse lymphoma cells were stored as frozen stocks in liquid nitrogen, the original cultures were obtained from Dr Donald Clive, Burroughs Wellcome Co. Each batch of frozen cells was purged of TK", mutants, checked for spontaneous mutant frequency and that it was mycoplasma free. For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated in a humidified atmosphere of 5% v/v CO2 in air. When the cells were growing well, subcultures were established in an appropriate number of flasks.

MEDIA USED
- Type and identity of media: RPMI 1640 medium with Horse Serum, Gentamycin, Amphotericin B and Pluronic.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically 'cleansed' against high spontaneous background: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver post-mitochondrial fraction (S9)

Test concentrations with justification for top dose:

Cytotoxicity range-finding experiment: six doses, ranging from 43.75, 87,5, 175, 350, 700 and 1421 μg/mL (equivalent to 10mM at the highest concentration tested, in accordance with OECDTG476)
Main Experiments: five doses, ranging from 300, 600, 900, 1200 and 1421 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: purified water
- Justification for choice of solvent/vehicle: the test item was soluble in purified water up to at least 27 mg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in suspension
- Cell density at seeding (if applicable): cytotoxicity range finding: 8 cells/mL, main experiment 2 x 10^5 cells/mL

DURATION
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 13 to 14 days

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: 2 independent experiments with 2 replicates each

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Rationale for test conditions:

The test is performed in line with OECDTG476

Evaluation criteria:

The test substance was considered to be mutagenic if:
1. the assay was valid
2. the mutant frequency at one or more doses was significantly greater than that of the negative control
3. there was a significant dose-relationship as indicated by the linear trend analysis
4. the effects described above were reproducible.

Statistics:

Determination of survival or viability
Calculation of the probable number of clones/well (P) on a microtitre plate in which there are empty wells (EW) (total well number TW):
P = -ln(EW/TW)

Plating Efficiency (PE)
PE = P/number of cells plated per well

The percentage relative survival (%RS)
% RS = [PE (test) PE (control)] X 100

Adjusted percentage relative survival values for each dose of test article:
Adjusted %RS = %RS x Post-treatment cell concentration for dose / Post-treatment cell concentration for solvent control

Determination of mutant frequency (MF)
MF = [PE (mutant)/PE (viable)] x 10^6

Assessment of statistical significance of mutant frequencies was carried out according to the UKEMS guidelines. Thus the control log mutant frequency (LMF) was compared with the LMF from each treatment dose, and secondly the data were checked for a linear trend in mutant frequency with treatment dose. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: No cytotoxicity was observed at any dose level tested in the absence or presence of S9. The highest dose tested (1421 µ/mL) yielded 97% relative survival in the absence of S9 and 115% relative survival in the presence of S9.

HISTORICAL CONTROL DATA

Treatment MF MFi-MFc Ratio C/H
Negative control 6.06 1.542
0,1 NQO 31.64 25.58 0.698
0,15 NQO 44.39 38.33 0.759

MF = Mutant Frequency
MFi = Mutant Frequency Induced
MFc = Mutant Frequency Control
Ratio C/H = Current/Historical

A small statistically significant increase in mutant frequency was observed following treatment with Kojic acid at the lowest dose level (300 μg/mL) tested in the presence of S9, in Experiment 2. However, there was no evidence of a dose-related response in this experiment (indicated by a non-significant linear trend) and no other statistically significant increases in mutant frequency were observed at any dose level tested in the absence or presence of S9 in Experiments 1 and 2. This observation was therefore considered to be a chance event of no biological significance. It may be noted that the unusually high heterogeneity values were observed for mutation (at 600 μg/mL in the presence of S9 in Experiment 1 and at 900 μg/mL in the presence of S9 in Experiment 2) and for viability (at 900 μg/mL in the absence of S9 in Experiment 2). The levels of heterogeneity observed in these treatments were sporadic and were not believed to have prejudiced the validity of the study in any way, therefore they were included in the analyses.

Conclusions:

Kojic acid did not induce any toxicologically significant increases in the mutant frequency at the HPRT locus in the L5178Y cells and was therefore not considered to be mutagenic under the conditions of the test.

Executive summary:

In a mammalian cell gene mutation assay (Hprt; 413/45-D6173), mouse lymphoma L5178Y cells cultured in vitro were exposed to Kojic Acid in water at concentrations of  300, 600, 900, 1200 and 1421 µg/mL in the presence and absence of mammalian metabolic activation (Aroclor-induced rat liver S9) for 3 hours.  

There was no cytotoxicity or precipitation but Kojic Acid was tested up to the limit concentration. The positive controls induced the appropriate response.  

A small statistically significant increase in mutant frequency was observed following treatment with Kojic acid at the lowest dose level (300 μg/mL) tested in the presence of S9, in Experiment 2. However, there was no evidence of a dose-related response in this experiment (indicated by a non-significant linear trend) and no other statistically significant increases in mutant frequency were observed at any dose level tested in the absence or presence of S9 in Experiments 1 and 2. This observation was therefore considered to be a chance event of no biological significance. It may be noted that the unusually high heterogeneity values were observed for mutation (at 600 μg/mL in the presence of S9 in Experiment 1 and at 900 μg/mL in the presence of S9 in Experiment 2) and for viability (at 900 μg/mL in the absence of S9 in Experiment 2). The levels of heterogeneity observed in these treatments were sporadic and were not believed to have prejudiced the validity of the study in any way, therefore they were included in the analyses. Therefore, under the conditions in this study, Kojic Acid is not mutagenic in this test system.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Result derived from read across is sufficiently reliable because all Annex XI criteria are met.

Justification for type of information:

The read across justification is presented in the Genotoxicity Endpoint summary. The accompanying files are also attached there.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

Based on the results of the study for read-across substance Kojic acid, Ethyl Maltol is not considered to be mutagenic in mammalian cells.

Reference 4

Endpoint:

in vitro cytogenicity / micronucleus study

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Cytogenicity (mammalian erythrocyte micronucleus test): Ethyl Maltol was found to be negative in an in vivo micronucleus assay (Equivalent or similar to OECD 474).

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Intraperitoneal administration of test substance (not justified), only 4 animals per dose, no toxicity measurements in the bone marrow included.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

Intraperitoneal administration of test substance, sampling at different time intervals after single exposure (24, 48 and 72 hours)

Qualifier:

according to guideline

Guideline:

other: M. Salamone et al. Towards an improved micronucleus test Studies on 3 model agents. In Mutation Research/Environmental Mutagenesis and Related Subjects, Volume 74, Issue 5, 1980, Pages 347-356, ISSN 0165-1161.

GLP compliance:

no

Type of assay:

other: Micronucleus test

Species:

mouse

Strain:

NMRI

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Ivanovas GmbH, Kisslegg
- Age at study initiation: 10-14 weeks
- Diet: Altromin standard chow ad libitum
- Water: ad libitum

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: olive oil

Frequency of treatment:

Two doses (at 0h and 24h) or single dose (at 0h)

Post exposure period:

Groups exposed twice: 30h
Groups exposed once: 24h, 48h, 72h

Dose / conc.:

980 mg/kg bw (total dose)

Remarks:

Administered as single dose (0h) or dosed twice (0h and 24h)

Dose / conc.:

700 mg/kg bw (total dose)

Remarks:

Administered twice (0h and 24h)

Dose / conc.:

420 mg/kg bw (total dose)

Remarks:

Administered twice (0h and 24h)

No. of animals per sex per dose:

4

Control animals:

yes, concurrent vehicle

Positive control(s):

none

Tissues and cell types examined:

polychromatic erythrocytes in the bone marrow

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION:
Bone-marrow smears were prepared 24h, 30h, 48h or 72h after treatment. The smears were stained according to the method of Schmid (1976).

METHOD OF ANALYSIS:
Slides were scored as previously described (Wild, King & Eckhardt, 1980).

Evaluation criteria:

no data

Statistics:

Statistical significance was determined according to the methods of Kastenbaum & Bowman (1970)

Key result

Sex:

female

Genotoxicity:

negative

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Additional information on results:

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no effect
- Ratio of PCE/NCE (for Micronucleus assay) was not examined

Results of micronucleus tests on mouse bone marrow after exposure to Ethyl Maltol

Dose (mg/kg)	Surviving/treated mice	Mean no. of micronucleated PE/1000 PE
0	4/4	1.3
2*980	4/4	3.0
2*700	4/4	3.2
2*420	4/4	2.3
0	4/4	2.7
1* 980 (24 hr)	4/4	1.3
1* 980 (48 hr)	4/4	3.5
1* 980 (72 hr)	4/4	1.8

Conclusions:

Under the conditions of this study, Ethyl Maltol did not induce any increase in micronucleated erythrocytes after peritoneal injection in mice.

Executive summary:

In an NMRI mouse mammalian erythrocyte micronucleus test (Wild et al., 1983), groups of 4 female mice were treated intraperitoneally with Ethyl Maltol in olive oil at doses of 980 mg/kg bw (one dose) or 480, 700, 980 mg/kg bw (two doses 0hrs, 24hrs). Animals were sacrificed 24, 48 or 72 hrs after single treatment, or 30 hours after the second treatment.

Slides were scored for the mean number of micronucleated PE/1000PE (Polychromatic Erythrocytes). After two exposures to 980, 700, 420 or 0 mg/kg bw, the mean numbers of micronucleated PE/100PE were 3.0, 3.2, 2.3 and 1.3 respectively. After a single exposure to 980 mg/kg bw (24, 48, 72 hrs), the number of micronucleated PE/1000PEs were 1.3, 3.5 and 1.8 respectively, with a control value of 2.7. No statistically significant differences were observed. The results of these tests therefore indicate no mutagenic potential of the test substance.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro studies

There is one in vitro bacterial reverse mutation/Ames test available. There are no in vitro cytogenicity/micronucleus studies available. There is one gene mutation (mammalian cell gene mutation assay) available (read-across to Kojic Acid (CAS No. 501-30-4)).

Gene mutation (Bacterial Reverse Mutation Assay/Ames test):

In a reverse gene mutation assay in bacteria (no guideline), strains of S. typhimurium TA 98 and TA 100 were exposed to Ethyl Maltol at concentrations of 0.5, 1 and 2 mg/plate (plate incorporation) in the presence and absence of mammalian metabolic activation (S9). Quercetin, sterigmatocystin and benzo[a]pyrene were used as positive controls. The study was judged to be valid by the EFSA (FGE.213) and rated Klimisch 2.

The substance induced a dose-related increase in the number of revertant colonies in S. typhimurium TA100, both in the absence and presence of S9 metabolic activation. No mutagenicity was observed in strain TA98. Under the conditions of this study, the test substance is considered mutagenic.

EFSA Journal 2015;13(9):4244

Chromosome aberration (in vitro cytogenicity/micronucleus study):

This study is waived as an in vivo mammalian erythrocyte micronucleus test is available.

Gene mutation (mammalian cell gene mutation assay):

In a mammalian cell gene mutation assay (Hprt; OECD 476/GLP), mouse lymphoma L5178Y cells cultured in vitro were exposed to Kojic Acid in water at concentrations of  300, 600, 900, 1200 and 1421 µg/mL in the presence and absence of mammalian metabolic activation (Aroclor-induced rat liver S9) for 3 hours.

There was no cytotoxicity or precipitation but Kojic Acid was tested up to the limit concentration. The positive controls induced the appropriate response.  

A small statistically significant increase in mutant frequency was observed following treatment with Kojic acid at the lowest dose level (300 μg/mL) tested in the presence of S9, in Experiment 2. However, there was no evidence of a dose-related response in this experiment (indicated by a non-significant linear trend) and no other statistically significant increases in mutant frequency were observed at any dose level tested in the absence or presence of S9 in Experiments 1 and 2. This observation was therefore considered to be a chance event of no biological significance. It may be noted that the unusually high heterogeneity values were observed for mutation (at 600 μg/mL in the presence of S9 in Experiment 1 and at 900 μg/mL in the presence of S9 in Experiment 2) and for viability (at 900 μg/mL in the absence of S9 in Experiment 2). The levels of heterogeneity observed in these treatments were sporadic and were not believed to have prejudiced the validity of the study in any way, therefore they were included in the analyses. Therefore, under the conditions in this study, Kojic Acid is not mutagenic in this test system.

Read-across justification:

Introduction and hypothesis for the read across

Ethyl Maltol is a 4-pyranone with an ethyl and hydroxyl group attached at the 2 and 3 position, respectively. For this substance an Ames test, and in vivo mammalian erythrocyte micronucleus test are available but no data regarding in vitro genemutations in mammalian cells is available.In accordance with Article 13 of REACH, lacking information should be generated whenever possible by means other than vertebrate animal tests, i.e. applying alternative methods such as in vitro tests, QSARs, grouping and read-across. For assessing the in vitro genemutations in mammalian cells for Ethyl Maltol the analogue approach is selected because for one closely related analogue, Kojic acid, such in vitro mutagenicity data is available.

Hypothesis:Ethyl Maltolis not expected to be mutagenic to mammalian cells based on analogue information from Kojic acid.

Available information: For Ethyl Maltol a positive Ames test is available (similar to OECD TG 471, Kl2) and a negative in vivo micronucleus test is available (similar to OECD TG 474, Kl2). For Ethyl Maltol also a carcinogenicity study is available (similar to OECD TG 453, Kl2), showing negative results. No data regarding in vitro genemutations in mammalian cells is available. To fill this genetic toxicity endpoint information from Kojic acid is used for read across. Kojic acid is negative for in vitro genemutations in mammalian cells (OECD TG 476, Kl 1) showing negative results. Also other genotoxicity information is available and reviewed by SCCP. Despite some positive effects in in vitro studies the substance is considered negative for in vitro and in vivo genotoxicity (SCCP, 1182/08).

Target and Source chemical(s):

Chemical structure of the target chemical (Ethyl Maltol) is shown in Data matrix, including physico-chemical properties and toxicological information, thought relevant for using the read across.

Purity / Impurities:

The purity and impurities of Ethyl Maltol do not indicate in vitro gene mutation potential other than indicated by the parent substance. The impurities are all below 1 %.

Analogue approach justification

According to REACH Annex XI an analogue approach and structural alert information can be used to replace testing when information from different sources provides sufficient evidence to conclude that this substance has or does not have a particular dangerous property. The result derived should be applicable for C&L and/or risk assessment and be presented with adequate and reliable documentation.

Analogue selection:Analogues identified in the OECD QSAR toolbox by using structural similarity with Tanimoto 80% did not reveal any substances. Therefore analogues were identified in the OECD QSAR toolbox by using structural similarity with Tanimoto 60%, which showed 10 substances. Kojic acid and Maltol were both considered as possible RA candidates. As mutagenicity testing is ongoing for Maltol and a final conclusion cannot be made, Kojic acid is used as source substance.

Structural similarities and differences:Thetarget chemical, Ethyl Maltol and source chemical, Kojic acid both have a3-hydroxy-4-pyranone backbone and functional group. There are two differences between these two substances: Ethyl Maltol has its alcohol group attached to the ring mirrored compared to Kojic acid. The second difference is that Ethyl Maltol has an ethyl group at the ring where Kojic acid has a hydroxymethyl group.

Toxicokinetics:Absorption of Ethyl Maltol and Kojic acid will be alike based on the similarity in being solids, molecular weight are 140 and 142, the water solubilities are9345 and 43850 mg/L, and calculated log Kow values of 0.29 and -1.25, respectively. The log Kow of Ethyl Maltol is somewhat higher due to the extra methyl group instead of an OH group at this spot.

Metabolism: Theoretically the ethyl group in Ethyl Maltol may turn intoalcohol, aldehyde and finally acid. At this spot in the chemical structure Kojic acid already has an alcohol at its methyl group and may turn into an acid. Experimentally Ethyl Maltol was recovered in the urine of dogs as being glucuronic conjugates or as ethereal sulphates (Rennhard et al., 1971). In a report by Sansho Seiyaku Co (2001) to the ADME of Kojic acid in the rat, Kojic acid was detected in the urine and liver. Metabolites in all organs or tissues detected were glucuronate (6.4 – 39.6% of total radioactivity) and mainly sulphate (35.6 –93.7% of total radioactivity) conjugates of Kojic acid. This is comparable to the experimental findings in the study by Rennhard et al. (1971).

Toxicodynamics, reactivity: Ethyl Maltol and Kojic acid have electrophilic features in the form of alpha and beta unsaturated carbonyls, which can promote to Michael addition, and may cause genotoxicity by combination with nucleophilic groups in nucleic acids. Both Ethyl Maltol and Kojic acid have an OH group (electron withdrawing) attached to the ring. The differences in reactivity between these two substance (ethyl and hydroxymethyl group) can be shown by pKa predictions of both substances. Ethyl Maltol has a higher pKa (11.26) and thus a slightly lower reactivity compared to Kojic acid which has a pKa 7.6.Based on the pKa Ethyl Maltol is expected to be slightly less reactive than Kojic acid. This reactivity of both Ethyl Maltol and Kojic acid may have led to the positive results in the Ames test.

Remaining uncertainties:There are no remaining uncertainties because Ethyl Maltol andKojic acidare structurally very close analogues and also their metabolites. Both are positive in the Ames test. Ethyl Maltol is considered slightly less reactive and therefore read across from Kojic acid is conservative.

Data matrix

The relevant information on physico-chemical properties and toxicological characteristics are presented in the data matrix in Table 1 to further support the read across between Ethyl Maltol and Kojic acid.

Conclusionsfor in vitro mammalian gene mutation

For Ethyl Maltol a positive Ames test is available as well as a negative in vivo micronucleus test similar to OECD TG 471 and 474, respectively.To cover the in vitro genemutations in mammalian cells, information from Kojic acid is used to fill this data gaps. Kojic acid is negative in this test and therefore Ethyl Maltol is also expected to be negative in this test.

Final conclusion on hazard and application in the risk assessment:

Ethyl Maltol is negative in the in vitro gene mutations in mammalian cells.

Common names	Ethyl Maltol	Kojic acid
	Target	Source
Chemical name: IUPAC	2-Ethyl-3-hydroxy-4H-pyran-4-one	5-Hydroxy-2-(hydroxymethyl)-4H-pyran-4-one
Chemical structure
CAS no.	4940-11-8	501-30-4
Einecs no	225-582-5	207-922-4
Reach registration	REACH 2018 registration	Not REACH registered
Molecular weight	140	142
Physical state	Solid	Solid
Physico-chemical data
Vapour pressure (Pa)	0.2 (IFF measured)	0.000102 (EpiSuite)
Water solubility (mg/l)	9345 (IFF measured)	43850 (SCCP)
Log Kow	2.9 (IFF measured)	-0.64 (measured from EpiSuite)
Log Kow predicted	0.29	-1.25
pKa	11.26 (SPARC)	7.9 and 8.03 (SCCP)
Human toxicology
Acute oral Toxicity mg/kg bw	1220 (OECD TG 401)	1000-4000  (OECD TG 401, SCCP)
Ames	Positive (OECD TG 471)	Positive (OECD TG 471)
In vitro mutagenicity in genemutations in mammalian cells	Read Across	Negative (OECD TG 476)
In vivo mammalian cytogenicity	Negative (Similar to OECD 474)	Negative (OECD TG 474)
Genotoxic carcinogenicity	Negative (Similar to OECD TG 453)	Negative (SCCP)

References

Rennhard H.H., 1971, Metabolism of ethyl Maltol and Maltol in the dog, J. Agric. Food Chem., 19 (1), pp 152–154

Sansho Seiyaku, Absorption, Distribution, Metabolism, and Excretion (ADME) of Kojic Acid in the Rat - Revision of 2001 Institute of Whole Body metabolism Sansho Seiyaku Co., Ltd. unpublished report

SCCP/1182/08. Opinion on Kojic Acid, adopted by the SCCP during the 17th plenary meeting of 30 September 2008:https://ec.europa.eu/health/ph_risk/committees/04_sccp/docs/sccp_o_148.pdf.

In vivo studies

There is one mammalian erythrocyte micronucleus test available.

Cytogenicity (mammalian erythrocyte micronucleus test):

In an NMRI mouse mammalian erythrocyte micronucleus test ((Equivalent or similar to OECD 474), groups of 4 female mice were treated intraperitoneally with Ethyl Maltol in olive oil at doses of 980 mg/kg bw (one dose) or 480, 700, 980 mg/kg bw (two doses 0hrs, 24hrs). Animals were sacrificed 24, 48 or 72 hrs after single treatment, or 30 hours after the second treatment.

Slides were scored for the mean number of micronucleated PE/1000PE (Polychromatic Erythrocytes). After two exposures to 980, 700, 420 or 0 mg/kg bw, the mean numbers of micronucleated PE/100PE were 3.0, 3.2, 2.3 and 1.3 respectively. After a single exposure to 980 mg/kg bw (24, 48, 72 hrs), the number of micronucleated PE/1000PEs were 1.3, 3.5 and 1.8 respectively, with a control value of 2.7. No statistically significant differences were observed. The results of these tests therefore indicate no mutagenic potential of the test substance.

Overall conclusion:

There is a positive in vitro bacterial reverse mutation assay/Ames test. According to the EFSA expert panel in 2015, the result from the combined chronic toxicity/carcinogenicity OECD 453 study conducted with Ethyl Maltol (Refer to 7.5.1) overrules the mutagenicity observed in bacteria.

There is a negative mammalian cell gene mutation assay (read-across to Kojic Acid (CAS No. 501-30-4)). There is a negative in vivo mammalian erythrocyte micronucleus test. Therefore the overall conclusion on the genotoxic properties is negative.

EFSA Journal 2015;13(9):4244

Justification for classification or non-classification

Based on the available information in the dossier, the substance Ethyl Maltol (CAS No. 4940-11-8) does not need to be classified for germ cell mutagenicity when the criteria outlined in Annex I of 1272/2008/EC are applied.