3-hydroxy-2-methyl-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 maltol was mutagenic in the strain S. typhimurium TA 100 in the presence and absence of Phenobarbital-induced rat liver 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): This study is waived as an in vivo mammalian Comet assay is available.

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

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 3 mg/plate.

GLP compliance:

no

Remarks:

pre-GLP

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Chemical Division, Pfizer, New York, NY

Target gene:

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:

Phenobarbital -induced rat liver S9

Test concentrations with justification for top dose:

Initial study: 10 µg, 100 µg, 1 mg, 10 mg/plate
Main study: 0.5, 1, 1.5, 2 and 3 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

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): 3

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 3 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 3 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 3 mg/plate

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Cytotoxicity was not observed for maltol up to the highest concentration tested (3 mg/plate).

Conclusions:

Under the conditions of this test, a dose related mutagenic effect was observed for Maltol against tester strain TA100 but not in strain TA98. Based on this result, the test substance is considered mutagenic.

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 Maltol at concentrations of 0.5, 1, 1.5, 2 and 3 mg/plate (plate incorporation) in the presence and absence of mammalian metabolic activation (Phenobarbital-induced rat liver 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 cytogenicity / chromosome aberration study in mammalian cells

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

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro gene mutation study in mammalian cells does not need to be conducted because adequate data from a reliable in vivo mammalian gene mutation test are available

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Cytogenicity (mammalian erythrocyte micronucleus test): Maltol was found to be negative in an in vivo micronucleus assay (OECD 474/GLP).

Gene mutation (mammalian comet assay): Maltol was found to be negative in an in vivo mammalian comet assay (equivalent or similar to OECD 489/GLP)

Bioanalysis of plasma: Rats dosed with Maltol at 700 mg/kg bw/day were systemically exposed, with peak plasma levels occurring 0.5 to 1 hour after the final dose administration (Refer to 7.1.1)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic and germ cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 March 2012 - 14 February 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

See Principles of Method

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Principles of method if other than guideline:

When the study was conducted, there was no guideline available for the Comet assay. However the protocol was designed in accordance with the recommendations of the Comet and
IWGT workshop (Tice et al., 2000; Hartmann et al., 2003 and Burlinson et al., 2007), Japanese Center for the Validation of Alternative Methods (JaCVAM) and current literature (Hartmann et al., 2004; Smith et al., 2008).

Smith C. C., Adkins D.J., Martin E. A., O'Donovan M.R., (2008), Recommendations for design of the rat comet assay. Mutagenesis vol. 23 No.3 pp 233-240

Tice R R, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, Miyamae Y, Rojas E, Ryu J-C, Sasaki Y F (2000). Single cell gel/Comet assay: Guidelines for in vitro and in vivo genetic toxicology testing. Environmental and Molecular Mutagenesis, 35, 206-221

Hartmann A, Agurell E, Beevers C, Brendler-Schwaab S, Burlinson B, Clay P, Collins A, Smith A, Speit G, Thybaud V, Tice, RR (2003). Recommendations for conducting the in vivo alkaline Comet assay. Mutagenesis 18 (1) 45-51

Hartmann A, Schumacher M, Plappert-Helbig U, Lowe P, Suter W and Mueller L (2004) Use of the alkaline in vivo Comet assay for mechanistic genotoxicity investigations. Mutagenesis 19 51-59

Burlinson B, Tice RR, Speit G, Agurell E, Brendler-Schwaab SY, Collins AR, Escobar P, Honma M, Kumaravel TS, Nakajima M, Sasaki YF, Thybaud V, Uno Y, Vasquez M, Hartmann A (2007). In Vivo Comet Assay Workgroup, part of the Fourth International Workgroup on Genotoxicity Testing. Mutation Research 627(1):31-5

GLP compliance:

yes

Type of assay:

mammalian comet assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
Sigma Aldrich, Germany; STBB9440V
- Expiration date of the lot/batch:
February 2016.
- Purity: >99.9%


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
15-25°C, protected from light.

Species:

rat

Strain:

other: Han Wistar (HsdHan:WIST)

Details on species / strain selection:

The rat was selected as there is a large volume of background data in both end points for this species and because rats were the rodent species of choice for the toxicological evaluation of Maltol.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan UK Ltd., Oxon, UK.
- Age at study initiation: 6-10 weeks old for DRF; 6-8 weeks for main study
- Weight at study initiation: 201-309 g males or 161-221 g females for DRF; 196 - 241g for males only in main study
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Fasting period before study: No
- Housing: The animals were housed in groups of up to six, of the same sex. Bedding was provided on a weekly basis to each cage by use of clean European softwood bedding (Datesand Ltd, Manchester.
- Diet: SQC Rat and Mouse Maintenance Diet No 1, Expanded (Special Diets Services Ltd. Witham) ad libitum
- Water: Mains water was provided ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24°C
- Humidity (%): 45 to 65%
- Air changes (per hr): 15-20 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light (0600 to 1800) and 12 hours dark.

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 0.5% (w/v) aqueous methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Dosing preparations were freshly prepared prior to each dosing occasion by formulating Maltol in 0.5% (w/v) aqueous methylcellulose to give the concentrations specified in Table 4 below. The test article was weighed into suitable containers and transferred to a mortar and pestle. The container was rinsed using a small volume of vehicle, which was then added to the test article to form a smooth paste. The mixture was transferred to the formulation bottle and the mortar and pestle rinsed with the vehicle, which was subsequently added (together with any remaining vehicle) to the formulation bottle to achieve the final volume. Formulations were then mixed using a Silverson until visibly homogenous. To ensure homogeneity, dose bottles were stirred continuously (on a magnetic stirrer) immediately before and throughout dosing.

Duration of treatment / exposure:

Dosed at 0 (Day 1), 24 (Day 2) and 45 (Day 3) hours.

Frequency of treatment:

Daily

Post exposure period:

Post-dosing observation times were as follows:
Day 1: Immediate, 1.0, 2.0, 4.0 and 8.0 hours post dose
Day 2: Pre-dose, immediate, 1.0, 2.0, 4.0 and 8.0 hours post dose
Day 3:3 Pre-dose, immediate and prior to necropsy

Individual body weights were recorded as follows: Day -1 (at study set-up), Day 1 (prior to dosing) and Day 3 (prior to necropsy)

Animals were sampled at 48 hours, i.e. 3 hours after the final administration.

Dose / conc.:

70 mg/kg bw/day (nominal)

Dose / conc.:

350 mg/kg bw/day (nominal)

Dose / conc.:

700 mg/kg bw/day (nominal)

No. of animals per sex per dose:

6 males per dose

Positive control(s):

The positive control was ethyl methanesulfonate (EMS), freshly prepared in purified water on each day of use. EMS (150 mg/kg bw) was administered by oral gavage, at 0, 24 and 45 hours and animals were sampled at 48 hours (3 hours after the final administration).

Tissues and cell types examined:

Liver for Comet Assay

Histopathology samples
A sample of liver from vehicle-control and test article treated animals only was removed, immediately preserved in neutral buffered formalin in uniquely labelled pots, and stored at room temperature. No histopathology samples were preserved for the positive control animals. Preserved samples were embedded in wax blocks and section at 5 μm nominal. Slides were stained with haemolysin and eosin and examined by the study pathologist.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The rat oral LD50 Tox data for ethyl maltol is reported to be 1440 mg/kg (males only). Maltol gave effects in a 90-day at 1000 mg/kg bw/day, although the findings were considered to be tolerable. Based on this information an initial dose of 1500 mg/kg/day was tested in a Range-Finder Experiment. Groups of three male and three female animals were given up to three administrations of Maltol, (at 0, 24 and 45 hours) at 360, 500, 700, 1000, 1500 and 2000 mg/kg bw/day.  

At doses of 1000 mg/kg bw/day and above, severe clinical signs (including piloerection, ataxia and bradypnoea) resulting in either death or early termination due to poor condition, were observed, confirming that these dose levels exceeded a maximum tolerated dose level (Appendix 2). No clinical signs of toxicity were observed at 360 mg/kg bw/day. Dose levels of 500 and 700 mg/kg bw/day caused decreased activity and/or piloerection in all animals solely after the first administration. No clinical signs of toxicity were observed on Day 2 or Day 3 following administration of 500 or 700 mg/kg bw/day. Reductions in individual bodyweights were noted following dosing at 500 mg/kg bw/day and above (Appendix 2).  

From these results, 700 mg/kg bw/day was considered an appropriate estimate of the maximum tolerated dose (MTD). No substantial difference in toxicity was observed between males and females in the Range-Finder, therefore male animals only were used in the Main Experiment.  Based on these data doses of 70, 350 and 700 mg/kg bw/day were selected for testing in the Main Experiment.

DETAILS OF SLIDE PREPARATION:
Tissue processing
The Comet liver samples from all control and test article treated animals were washed thoroughly in Merchants solution and placed in fresh buffer. The samples were cut into small pieces in Merchants solution and the pieces of liver were then pushed through bolting cloth (pore size of 150 μm) with approximately 4 mL of Merchants solution to produce single cell suspensions. Cell suspensions were held on ice for no longer than 2 hours, until slides were prepared.

Slide preparation
Four slides, labelled A, B, C and D, were prepared per single cell suspension. Slides were labelled with the study number, appropriate animal tag number and tissue. Slides were dipped in molten normal melting point agarose (NMA) such that all of the clear area of the slide and at least part of the frosted area was coated. The underside of the slides was wiped clean and the slides allowed to dry. 40 μL of each single cell suspension was added to 400 μL of 0.7% low melting point agarose (LMA) at approximately 37°C. 100 μL of cell suspension/agarose mix was placed on to each slide. The slides were then cover- slipped and allowed to gel on ice.

Cell lysis
Once gelled the coverslips were removed and all slides placed in lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH adjusted to pH 10 with NaOH, 1% Triton X-100, 10% DMSO) at 2-8°C protected from light. Slides A, B and C were lysed overnight; D slides were incubated for 1 hour).

Unwinding and electrophoresis
Following lysis, slides were washed in purified water for 5 minutes. A, B and C slides for each tissue and animal were transferred to electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH>13) at 2-8°C and the DNA unwound for 30 minutes. At the end of the unwinding period the slides were electrophoresed in the same buffer at 0.7 V/cm for 40 minutes. As not all slides could be processed at the same time a block design was employed for the unwinding and electrophoretic steps in order to avoid excessive variation across the groups for each electrophoretic run; i.e. for all animals the same number of triplicate slides was processed at a time.

Neutralisation
At the end of the electrophoresis period, slides were neutralised in 0.4 M Tris, pH 7.0 (3 x 5 minute washes). After neutralisation the slides were dried and stored at room temperature prior to scoring. These slides were used for comet analysis. After the lysis step the D slides from each tissue and animal were placed in 0.4 M Tris, pH 7.0 for approximately 3 x 5 minutes and then dried and stored as described above. This ‘halo’ slide was used to estimate the degree of highly damaged cells in the cell suspensions.
Retention of single cell suspensions Once all comet slides had been prepared, the remaining single cell suspensions from animals in Groups 1-4 were frozen at <-50°C. Prior to freezing the concentration of cells in each suspension was estimated.

Slide analysis
Scoring was carried out using fluorescence microscopy at an appropriate magnification and with suitable filters for the stains used. Slides from the EMS-treated rats were checked first to ensure the system was operating satisfactorily. The slides from all groups were coded (to enable blinded scoring) and analysed by an individual not connected with the dosing phase of the study. Slide analysis was performed by competent analysts trained in the applicable Covance Laboratories Harrogate (CLEH) standard operating procedures.

METHOD OF ANALYSIS:
Immediately prior to scoring, the slides were stained with 100 μL of 2 μg/mL ethidium bromide and cover slipped. Comet scoring was performed using Perceptive Instruments 'Comet Assay IV' image analysis system. Measurements of tail moment and tail intensity (% DNA in tail) were obtained from 100 cells/animal/tissue. In general this was achieved by scoring 50 cells on each of slides A and B. If required the C slide was also scored to achieve a total of 100 cells/animal. Each slide was examined for possible indications of cytotoxicity. The number of 'clouds' (a morphology indicative of highly damaged cells often associated with severe cytotoxicity, necrosis or apoptosis) out of 100 cells was scored for each slide. To avoid the risk of false positive results 'clouds' were not used for comet analysis. Each slide was scanned starting to the left of the centre of the slide.
The following criteria were used for analysis of slides:
1. only clearly defined non overlapping cells were scored
2. clouds were not scored
3. cells with unusual staining artefacts were not scored
4. all slides were scored blind (coded).

Halo (D) slides were scored by assessing 100 cells/slide and recording the number with a clear halo of DNA around the nucleus.

OTHER:
As the experimental unit of exposure for in vivo studies is the animal, all analysis was based on individual animal response. The following were calculated for each animal per tissue:

• Olive tail moment
• tail intensity (i.e. % DNA in the tail).
Animals with >30% clouds and/or >30% cells with halos were to be excluded from analysis. High levels of ‘clouds’ or cells with halos indicated the nuclear complex had been significantly fragmented and was considered evidence of excessive DNA damage. Such damage may be due to the cytotoxic nature of the treatment or due to excessive mechanical disruption during cell isolation, which had the potential to interfere with Comet analysis. Animals with >30% clouds and/or >30% cells with halos may have been excluded from data analysis in order to avoid any interference due to excessive cytotoxicity. Exclusion of animals or acceptance of data where >30% clouds and/or cells with halos observed are discussed.

Evaluation criteria:

The test article was considered positive in this study if all the criteria listed below were met.

The test article was considered negative in this study if none of the following criteria were met.

For valid data, the test article was considered to induce DNA damage if:
1. A dose related change in tail moment or tail intensity was observed in any tissue between the vehicle and test article groups, or

2. A marked change in tail moment or tail intensity was observed in any tissue between the vehicle and at least a single dose group.

Statistics:

For each Comet parameter assessed, the data were treated as follows:
1. Individual cell nuclei data from the replicate slides for each animal were collated
2. The median value for each animal was calculated
3. The mean of the medians and standard error of the mean was calculated for each group.

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
The assay data was considered valid as:
1. The vehicle control data were comparable with the laboratory's historical vehicle control ranges
2. At least five animals/group were available for analysis, and
3. The positive control chemical (EMS) induced a marked increase in Comet parameters
4. For Comet analysis there were ≤30% clouds and/or ≤30% cells with halos in the vehicle control
5. The high dose was considered to be the MTD, the maximum recommended dose, the maximum practicable dose or one that demonstrated cytotoxicity to the target cells.

The supplementary cytotoxicity data ('cloud' assessment and halo slide analysis) for animals treated with Maltol demonstrated little or no cytotoxicity, necrosis or apoptosis in the cell suspensions (Table 11).

Groups of rats treated with Maltol exhibited tail moments and tail intensities that were similar to the concurrent vehicle control group (Table 11) and which fell within the laboratory’s historical control range for this tissue (Appendix 5). Group mean comet parameters for Group 2 (70 mg/kg) and Group 4 (700 mg/kg) were slightly elevated compared to the concurrent vehicle control. However, there were only two animals (one in each group, animal 11 and animal 19) with a % tail intensity and tail moment that was notably higher than the vehicle control animals. However, in both cases these animals fell within the historical control data and there is no dose-dependency associated with these values. The elevated Comet values for these animals were therefore considered to be of no biological relevance.

These data confirmed Maltol did not induce DNA damage in liver under the conditions of this study.

No clinical signs of toxicity were observed in any animal following treatments with vehicle, Maltol (at 70, 350 or 700 mg/kg bw/day) or the positive control (EMS). A mean decrease of approximately 5 g was observed in the body weight of animals dosed at 700 mg/kg bw/day (equivalent to an approximately 2% reduction in body weight; Appendix 1). On necropsy, no clearly treatment-related changes in gross morphology or unusual coloration were observed.  Clinical chemistry results are presented in Appendix 4. In general there are no marked changes between the measured parameters between vehicle control and Maltol treated animals.  

In histopathology assessments of liver samples from animals treated with vehicle or Maltol, in the liver there was a generally dose-related minor reduction in the level of glycogen vacuolation recorded in rats treated with Maltol compared with concurrent controls.  

Plasma samples were stored frozen at <–50°C for possible future proof of exposure and toxicokinetic analysis, so no specific results were available in the report.

Although no clinical signs of toxicity were observed in the Main Experiment, the mortality observed in the Range-Finder Experiment at 1000 mg/kg/day and above, was considered sufficient justification for limiting the maximum dose to 700 mg/kg bw/day. In addition, a small but dose-related loss of body weight was observed in the Main Experiment, following dosing at 700 mg/kg bw/day.  

Conclusions:

In an in vivo Comet assay in male Han Wistar rats, Maltol did not induce DNA damage in liver, under the conditions of this study.

Executive summary:

In a Han Wistar rat comet assay (8262049), groups of 6 male rats were treated by oral gavage with Maltol (>99.9%) in 0.5% (w/v) aqueous methylcellulose at doses of 70, 350 and 700 mg/kg bw/day. The animals were dosed 3 times (0, 24, 45 hrs) and livers were removed at 48 hours. The positive control was ethyl methanesulfonate (150 mg/kg bw/day). For each animal, 100 cells (50 cells/slide from 2 slides) were scored for comets (tail intensity and tail moment).

No clinical signs of toxicity were observed in the main experiment. Mortality was observed in a range-finder experiment (360, 500, 700, 1000, 1500 and 2000 mg/kg bw/day) at 1000 mg/kg bw/day and above, was considered sufficient justification for limiting the maximum dose in the main experiment to the MTD, 700 mg/kg bw/day. In addition, a small but dose-related loss of body weight was observed in the main experiment, following dosing at 700 mg/kg bw/day.  In general, there were no marked changes between the measured parameters between vehicle control and Maltol treated animals.  In histopathology assessments of liver samples from animals treated with vehicle or Maltol, in the liver there was a generally dose-related minor reduction in the level of glycogen vacuolation recorded in rats treated with Maltol compared with concurrent controls.  Plasma samples were stored frozen for possible future proof of exposure and toxicokinetic analysis, so no specific results on bioavailability were available in the report.

The positive control group gave the appropriate response. The cytotoxicity data ('cloud'/hedgehog assessment and halo slide analysis) for animals treated with Maltol demonstrated little or no cytotoxicity, necrosis or apoptosis in the cell suspensions. Groups of rats treated with Maltol exhibited mean tail intensities that were similar to the concurrent vehicle control group (1.31±0.20 control compared to 1.83±0.47 at 700 mg/kg bw/day) and which fell within the laboratory’s historical control range for this tissue (0.30-8.15, n=59; 95% CI for Median % Tail Intensity). Group mean comet parameters for Group 2 (70 mg/kg) and Group 4 (700 mg/kg) were slightly elevated compared to the concurrent vehicle control. However, there were only two animals (one in each group, animal 11 and animal 19) with a % tail intensity and tail moment that was notably higher than the vehicle control animals. However, in both cases these animals fell within the historical control data and there is no dose-dependency associated with these values. The elevated Comet values for these animals were therefore considered to be of no biological relevance. These data confirmed Maltol did not induce DNA damage in liver under the conditions of this study.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 March 2012 - 14 February 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Conforms to 1997 guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997; superseded where appropriate by ICH-S2R1, 2011.

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sigma Aldrich, Germany; STBB9440V
- Expiration date of the lot/batch: February 2016.
- Purity: >99.9%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 15-25°C, protected from light.

Species:

rat

Strain:

other: Han Wistar (HsdHan:WIST)

Details on species / strain selection:

The rat was selected as there is a large volume of background data in both end points for this species and because rats were the rodent species of choice for the toxicological evaluation of Maltol.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan UK Ltd., Oxon, UK.
- Age at study initiation: 6-10 weeks old for DRF; 6-8 weeks for main study
- Weight at study initiation: 201-309 g males or 161-221 g females for DRF; 196 - 241g for males only in main study
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Fasting period before study: No
- Housing: The animals were housed in groups of up to six, of the same sex. Bedding was provided on a weekly basis to each cage by use of clean European softwood bedding (Datesand Ltd, Manchester.
- Diet: SQC Rat and Mouse Maintenance Diet No 1, Expanded (Special Diets Services Ltd. Witham) ad libitum
- Water: Mains water was provided ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24°C
- Humidity (%): 45 to 65%
- Air changes (per hr): 15-20 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light (0600 to 1800) and 12 hours dark.

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 0.5% (w/v) aqueous methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Dosing preparations were freshly prepared prior to each dosing occasion by formulating Maltol in 0.5% (w/v) aqueous methylcellulose to give the concentrations specified in Table 4 below. The test article was weighed into suitable containers and transferred to a mortar and pestle. The container was rinsed using a small volume of vehicle, which was then added to the test article to form a smooth paste. The mixture was transferred to the formulation bottle and the mortar and pestle rinsed with the vehicle, which was subsequently added (together with any remaining vehicle) to the formulation bottle to achieve the final volume. Formulations were then mixed using a Silverson until visibly homogenous. To ensure homogeneity, dose bottles were stirred continuously (on a magnetic stirrer) immediately before and throughout dosing.

Duration of treatment / exposure:

Dosed at 0 (Day 1), 24 (Day 2) and 45 (Day 3) hours.

Frequency of treatment:

Daily

Post exposure period:

Post-dosing observation times were as follows:
Day 1: Immediate, 1.0, 2.0, 4.0 and 8.0 hours post dose
Day 2: Pre-dose, immediate, 1.0, 2.0, 4.0 and 8.0 hours post dose
Day 3:3 Pre-dose, immediate and prior to necropsy

Individual body weights were recorded as follows: Day -1 (at study set-up), Day 1 (prior to dosing) and Day 3 (prior to necropsy)

Animals were sampled at 48 hours, i.e. 3 hours after the final administration.

Dose / conc.:

70 mg/kg bw/day (nominal)

Dose / conc.:

350 mg/kg bw/day (nominal)

Dose / conc.:

700 mg/kg bw/day (nominal)

No. of animals per sex per dose:

6 males per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

The positive control was ethyl methanesulfonate (EMS), freshly prepared in purified water on each day of use. EMS (150 mg/kg bw) was administered by oral gavage, at 0, 24 and 45 hours and animals were sampled at 48 hours (3 hours after the final administration).

Tissues and cell types examined:

Isolated femurs/bone marrow for micronucleus assay

Histopathology samples
A sample of liver from vehicle-control and test article treated animals only was removed, immediately preserved in neutral buffered formalin in uniquely labelled pots, and stored at room temperature. No histopathology samples were preserved for the positive control animals. Preserved samples were embedded in wax blocks and section at 5 μm nominal. Slides were stained with haemolysin and eosin and examined by the study pathologist.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The rat oral LD50 Tox data for ethyl maltol is reported to be 1440 mg/kg (males only). Maltol gave effects in a 90-day at 1000 mg/kg bw/day, although the findings were considered to be tolerable. Based on this information an initial dose of 1500 mg/kg/day was tested in a Range-Finder Experiment. Groups of three male and three female animals were given up to three administrations of Maltol, (at 0, 24 and 45 hours) at 360, 500, 700, 1000, 1500 and 2000 mg/kg bw/day.  

At doses of 1000 mg/kg bw/day and above, severe clinical signs (including piloerection, ataxia and bradypnoea) resulting in either death or early termination due to poor condition, were observed, confirming that these dose levels exceeded a maximum tolerated dose level (Appendix 2). No clinical signs of toxicity were observed at 360 mg/kg bw/day. Dose levels of 500 and 700 mg/kg bw/day caused decreased activity and/or piloerection in all animals solely after the first administration. No clinical signs of toxicity were observed on Day 2 or Day 3 following administration of 500 or 700 mg/kg bw/day. Reductions in individual bodyweights were noted following dosing at 500 mg/kg bw/day and above (Appendix 2).  

From these results, 700 mg/kg bw/day was considered an appropriate estimate of the maximum tolerated dose (MTD). No substantial difference in toxicity was observed between males and females in the Range-Finder, therefore male animals only were used in the Main Experiment.  Based on these data doses of 70, 350 and 700 mg/kg bw/day were selected for testing in the Main Experiment.

DETAILS OF SLIDE PREPARATION: The isolated femurs were cleaned of adherent tissue and the ends removed from the shanks. Using a syringe and needle, bone marrows were flushed from the marrow cavity with 2 mL foetal bovine serum into appropriately labelled centrifuge tubes. No filtration of bone marrow aspirates was performed. A further 3 mL of foetal bovine serum was added to the tubes followed by gentle resuspension of the cell pellet. Following a second centrifugation step at 200 g for approximately five minutes, the serum was aspirated to leave one or two drops and the cell pellet. The pellet was mixed into the small volume of serum in each tube by using a Pasteur pipette, and from each tube one drop of suspension was placed on the end of each of two slides labelled with the appropriate study number, sampling time, sex, date of preparation and animal number. A smear was made from the drop by drawing the end of a clean slide along the labelled slide. At least two slides were prepared from each femur removed. Slides were air-dried and then fixed for 10 minutes in absolute methanol. Slides were allowed to dry and stored at room temperature until required for staining.

METHOD OF ANALYSIS: Scoring was carried out using fluorescence microscopy at an appropriate magnification and with suitable filters for the stains used. Slides from the EMS-treated rats were checked first to ensure the system was operating satisfactorily. The slides from all groups were coded (to enable blinded scoring) and analysed by an individual not connected with the dosing phase of the study. Slide analysis was performed by competent analysts trained in the applicable Covance Laboratories Harrogate (CLEH) standard operating procedures.

Slides were air-dried prior fixing for 10 minutes in absolute methanol. Once fixed the slides were rinsed several times in distilled water and air-dried. One slide per animal was stored at room temperature for subsequent staining. On the day of staining the slides were fixed and washed again (as described above) and immediately stained for 5 minutes in 12.5 μg/mL acridine orange made up in 0.1 M phosphate buffer pH 7.4. Stained slides were rinsed in phosphate buffer, then dried and stored protected from light at room temperature prior to analysis. The dried, unstained slides were initially stored at <-10°C with desiccant. Once final results were confirmed the reserve slides were transferred to storage at room temperature. Initially the relative proportions of polychromatic erythrocytes (PCE), seen as bright orange enucleate cells, and normochromatic erythrocytes (NCE), seen as smaller dark green enucleate cells, were determined until a total of at least 1000 cells (PCE plus NCE) had been analysed. Then at least 2000 PCE/animal were examined for the presence of micronuclei (MN).

The following criteria were used for analysis of slides:
1. cells were of normal cell morphology
2. areas where erythrocytes overlapped were ignored
3. a micronucleus was round or oval in shape
4. a cell containing more than one micronucleus was scored as a single micronucleated cell
5. micronuclei which were refractive, improperly stained or not in the focal plane of the cell were judged to be artefacts and were not scored
6. all slides were scored blind (coded).

Evaluation criteria:

The test article was considered positive in this study if all the criteria listed below were met.

The test article was considered negative in this study if none of the following criteria were met.

For valid data, the test article was considered to induce clastogenic / aneugenic damage if:
1. A statistically significant increase in the frequency of MN PCE occurred at one or more dose levels
2. The incidence and distribution of MN PCE in individual animals at such a point exceeded the laboratory’s historical vehicle control data
3. The group mean MN PCE value at such a point exceeded the 95% calculated confidence interval for the man historical vehicle control data
4. A dose-response trend in the proportion of MN PCE (where more than two dose levels are analysed) was observed.

Statistics:

1.% PCE for each animal and the mean for each group.
2. Frequency of MN PCE (i.e. MN/2000 PCE) and % MN PCE for each animal and the group mean % MN PCE (± standard deviation).

For each group, inter-individual variation in the numbers of MN PCE was estimated by means of a heterogeneity chi-square calculation.

The numbers of MN PCE in each treated group were compared with the numbers in vehicle control groups by using the non-parametric Wilcoxon rank sum test.

Probability values of p ≤0.05 were accepted as significant.

A further statistical test (for linear trend) was used to evaluate possible dose-response relationships.

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
Micronucleus values (observed PCE, NCE and MN PCE numbers together with calculated %PCE and % MN PCE values) for individual animals are presented in Appendix 3.

The current micronucleus historical vehicle and positive control ranges for the laboratory are presented in Appendix 5.

Groups of rats treated with Maltol exhibited %PCE values that were similar to vehicle controls (Table 10) and which fell within acceptable ranges (Appendix 5). There was no evidence of any test article-induced toxicity to the bone marrow (as would usually be indicated by a notable decrease in %PCE values compared to the vehicle control group or dose dependant decrease). Group mean frequencies of MN PCE were similar to and not statistically different from those seen in concurrent vehicle controls for all dose groups (Table 10). Although a significant linear trend was observed, individual frequencies of MN PCE for all treated animals were generally consistent with historical vehicle control distribution data (Appendix 5) and similar to frequencies observed in the concurrent controls (Appendix 3). Only one outlying animal was observed (animal 23 at 700 mg/kg bw/day) with a frequency of 9 MN PCE, which resulted in significant heterogeneity at this dose level. As only one outlying animal was observed, this result was considered to be a chance occurrence and of no biological relevance.

These data confirmed Maltol did not induce micronuclei under the conditions of this study.

No clinical signs of toxicity were observed in any animal following treatments with vehicle, Maltol (at 70, 350 or 700 mg/kg bw/day) or the positive control (EMS). A mean decrease of approximately 5 g was observed in the body weight of animals dosed at 700 mg/kg bw/day (equivalent to an approximately 2% reduction in body weight; Appendix 1). On necropsy, no clearly treatment-related changes in gross morphology or unusual coloration were observed.  Clinical chemistry results are presented in Appendix 4. In general there are no marked changes between the measured parameters between vehicle control and Maltol treated animals.  In histopathology assessments of liver samples from animals treated with vehicle or Maltol, in the liver there was a generally dose-related minor reduction in the level of glycogen vacuolation recorded in rats treated with Maltol compared with concurrent controls.  

Plasma samples were stored frozen at <–50°C for possible future proof of exposure and toxicokinetic analysis, so no specific results were available in the report.

Although no clinical signs of toxicity were observed in the Main Experiment, the mortality observed in the Range-Finder Experiment at 1000 mg/kg/day and above, was considered sufficient justification for limiting the maximum dose to 700 mg/kg bw/day. In addition, a small but dose-related loss of body weight was observed in the Main Experiment, following dosing at 700 mg/kg bw/day.  

Conclusions:

In an in vivo micronucleus assay in male Han Wistar rats, Maltol did not induce micronuclei, under the conditions of this study.

Executive summary:

In a Han Wistar rat bone marrow micronucleus assay (8262049), groups of 6 male rats were treated by oral gavage with Maltol (>99.9%) in 0.5% (w/v) aqueous methylcellulose at doses of 70, 350 and 700 mg/kg bw/day. The animals were dosed 3 times (0, 24, 45 hrs) and bone marrow was sampled at 48 hours. The positive control was ethyl methanesulfonate (150 mg/kg bw/day). 1000 total erythrocytes cells (PCE plus NCE) were analysed; at least 2000 immature erythrocytes(PCE)/animal were examined for the presence of micronuclei.

No clinical signs of toxicity were observed in the main experiment. Mortality was observed in a range-finder experiment (360, 500, 700, 1000, 1500 and 2000 mg/kg bw/day) at 1000 mg/kg bw/day and above, was considered sufficient justification for limiting the maximum dose in the main experiment to the MTD, 700 mg/kg bw/day. In addition, a small but dose-related loss of body weight was observed in the main experiment, following dosing at 700 mg/kg bw/day.  In general, there were no marked changes between the measured parameters between vehicle control and Maltol treated animals.  In histopathology assessments of liver samples from animals treated with vehicle or Maltol, in the liver there was a generally dose-related minor reduction in the level of glycogen vacuolation recorded in rats treated with Maltol compared with concurrent controls.  Plasma samples were stored frozen for possible future proof of exposure and toxicokinetic analysis, so no specific results on bioavailability were available in the report.

The positive control induced the appropriate response.  Groups of rats treated with Maltol exhibited %PCE values that were similar to vehicle controls and which fell within acceptable ranges. There was no evidence of any test article-induced toxicity to the bone marrow (as would usually be indicated by a notable decrease in %PCE values compared to the vehicle control group or dose dependant decrease). Group mean frequencies of MN PCE were similar to and not statistically different from those seen in concurrent vehicle controls for all dose groups. Although a significant linear trend was observed, individual frequencies of MN PCE for all treated animals were generally consistent with historical vehicle control distribution data and similar to frequencies observed in the concurrent controls. Only one outlying animal was observed (at 700 mg/kg bw/day) with a frequency of 9 MN PCE, which resulted in significant heterogeneity at this dose level. As only one outlying animal was observed, this result was considered to be a chance occurrence and of no biological relevance. These data confirmed Maltol did not induce micronuclei under the conditions of this study.    

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro studies

There is one in vitro bacterial reverse mutation/Ames test avaialble. There are no in vitro cytogenicity/micronucleus studies or mammalian cell gene mutation assays available.

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 Maltol at concentrations of 0.5, 1, 1.5, 2 and 3 mg/plate (plate incorporation) in the presence and absence of mammalian metabolic activation (Phenobarbital-induced rat liver 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):

This study is waived as an in vivo mammalian Comet assay is available.

In vivo studies

There are two mammalian erythrocyte micronucleus tests and one mammalian comet assay available.

Cytogenicity (mammalian erythrocyte micronucleus test):

In the ddY mice bone marrow micronucleus supporting study (no guideline), groups of 6 male rats were treated by IP injection with Maltol in olive oil at doses of 125, 250, and 500 mg/kg bw. The animals were dosed once and bone marrow was sampled at 24 hours. The positive control was Mitomycin C.

The result of the positive control was not specified. There were no mortalities in Maltol treated animals. Maltol had a clear dose-dependent statistically significant effect after a single IP injection, with a maximum effect occurring at 500 mg/kg (1.67 ±0.43%) compared to vehicle control (0.18±0.12%).

Intraperitoneal injection is generally not recommended since it is not an intended route of human exposure and no justification was provided so this study is considered a supporting RL4 study.

In the Han Wistar rat bone marrow micronucleus key study (OECD 474/GLP), groups of 6 male rats were treated by oral gavage with Maltol (>99.9%) in 0.5% (w/v) aqueous methylcellulose at doses of 70, 350 and 700 mg/kg bw/day. The animals were dosed 3 times (0, 24, 45 hrs) and bone marrow was sampled at 48 hours. The positive control was ethyl methanesulfonate (150 mg/kg bw/day). 1000 total erythrocytes cells (PCE plus NCE) were analysed; at least 2000 immature erythrocytes(PCE)/animal were examined for the presence of micronuclei.

No clinical signs of toxicity were observed in the main experiment. Mortality was observed in a range-finder experiment (360, 500, 700, 1000, 1500 and 2000 mg/kg bw/day) at 1000 mg/kg bw/day and above, was considered sufficient justification for limiting the maximum dose in the main experiment to the MTD, 700 mg/kg bw/day. In addition, a small but dose-related loss of body weight was observed in the main experiment, following dosing at 700 mg/kg bw/day.  In general, there were no marked changes between the measured parameters between vehicle control and Maltol treated animals.  In histopathology assessments of liver samples from animals treated with vehicle or Maltol, in the liver there was a generally dose-related minor reduction in the level of glycogen vacuolation recorded in rats treated with Maltol compared with concurrent controls.  Plasma samples were stored frozen for possible future proof of exposure and toxicokinetic analysis, so no specific results on bioavailability were available in the report.

The positive control induced the appropriate response.  Groups of rats treated with Maltol exhibited %PCE values that were similar to vehicle controls and which fell within acceptable ranges. There was no evidence of any test article-induced toxicity to the bone marrow (as would usually be indicated by a notable decrease in %PCE values compared to the vehicle control group or dose dependant decrease). Group mean frequencies of MN PCE were similar to and not statistically different from those seen in concurrent vehicle controls for all dose groups. Although a significant linear trend was observed, individual frequencies of MN PCE for all treated animals were generally consistent with historical vehicle control distribution data and similar to frequencies observed in the concurrent controls. Only one outlying animal was observed (at 700 mg/kg bw/day) with a frequency of 9 MN PCE, which resulted in significant heterogeneity at this dose level. As only one outlying animal was observed, this result was considered to be a chance occurrence and of no biological relevance. These data confirmed Maltol did not induce micronuclei under the conditions of this study.    

Gene mutation (mammalian comet assay):

In a Han Wistar rat comet assay (Equivalent or similar to OECD 489/GLP), groups of 6 male rats were treated by oral gavage with Maltol (>99.9%) in 0.5% (w/v) aqueous methylcellulose at doses of 70, 350 and 700 mg/kg bw/day. The animals were dosed 3 times (0, 24, 45 hrs) and livers were removed at 48 hours. The positive control was ethyl methanesulfonate (150 mg/kg bw/day). For each animal, 100 cells (50 cells/slide from 2 slides) were scored for comets (tail intensity and tail moment).

No clinical signs of toxicity were observed in the main experiment. Mortality was observed in a range-finder experiment (360, 500, 700, 1000, 1500 and 2000 mg/kg bw/day) at 1000 mg/kg bw/day and above, was considered sufficient justification for limiting the maximum dose in the main experiment to the MTD, 700 mg/kg bw/day. In addition, a small but dose-related loss of body weight was observed in the main experiment, following dosing at 700 mg/kg bw/day.  In general, there were no marked changes between the measured parameters between vehicle control and Maltol treated animals.  In histopathology assessments of liver samples from animals treated with vehicle or Maltol, in the liver there was a generally dose-related minor reduction in the level of glycogen vacuolation recorded in rats treated with Maltol compared with concurrent controls.  Plasma samples were stored frozen for possible future proof of exposure and toxicokinetic analysis, so no specific results on bioavailability were available in the report.

The positive control group gave the appropriate response. The cytotoxicity data ('cloud'/hedgehog assessment and halo slide analysis) for animals treated with Maltol demonstrated little or no cytotoxicity, necrosis or apoptosis in the cell suspensions. Groups of rats treated with Maltol exhibited mean tail intensities that were similar to the concurrent vehicle control group (1.31±0.20 control compared to 1.83±0.47 at 700 mg/kg bw/day) and which fell within the laboratory’s historical control range for this tissue (0.30-8.15, n=59; 95% CI for Median % Tail Intensity). Group mean comet parameters for Group 2 (70 mg/kg bw/day) and Group 4 (700 mg/kg bw/day) were slightly elevated compared to the concurrent vehicle control. However, there were only two animals (one in each group, animal 11 and animal 19) with a % tail intensity and tail moment that was notably higher than the vehicle control animals. However, in both cases these animals fell within the historical control data and there is no dose-dependency associated with these values. The elevated Comet values for these animals were therefore considered to be of no biological relevance. These data confirmed Maltol did not induce DNA damage in liver under the conditions of this study.

All studies are acceptable for use in the human health risk assessment.

Conclusion:

The EFSA reviewed the Beevers 2013 in vivo studies during FGE.213Rev1 (1) and requested plasma analysis from study samples for proof of exposure. The registrant does not have access to the study report but the EFSA concluded the results were inconclusive. Considering that Maltol has been shown to induce micronuclei in mouse bone marrow after intraperitoneal injection (Hayashi et al., 1988), the EFSA concluded that negative findings observed in the combined bone marrow micronucleus test and Comet assay in the liver of treated rats could not rule out the concern for genotoxicity for Maltol since the data provided to prove systemic availability were considered inconclusive due to the inconsistency of the data.

Following the EFSAs conclusion in FGE.213Rev1, the International Organisation of the Flavor Industry submitted a new plasma analysis (Beevers, 2015) performed on the same strain of rats and using the same dosing regimen of the combined micronucleus test and comet assay (Beevers, 2013). A robust study summary is prepared in 7.1.1 Toxicokinetics. This data was evaluated by the EFSA in FGE.213Rev2 (2). In summary, the EFSA accepted that it now seems justifiable to assume that the animals were systemically exposed to Maltol and that the relevant tissues were exposed in both assay.

The JECFA also re-evaluated Maltol in 2019 [3] and concluded “For two previously evaluated flavouring agents in this group, maltol (No. 1480) and maltyl isobutyrate (No. 1482), additional studies of acute toxicity (No. 1482) and genotoxicity (Nos 1480 and 1482) were available for the present evaluation. These additional data raised no safety concerns and support the previous safety evaluations".

Therefore Maltol is not considered to be genotoxic.

1. EFSA Journal 2014; 12(5):3661

2. EFSA Journal 2015;13(9):4244

3. JECFA (2019). Evaluation of certain food additives. WHO Technical Report Series 1014. Prepared by the Eighty-sixth report of the Joint FAO/WHO Expert Committee on Food Additives (JECFA). World Health Organization, Geneva 2019.

Justification for classification or non-classification

Based on the available information in the dossier, the substance Maltol (CAS No. 118-71-8) does not need to be classified for germ cell mutagenicity when the criteria outlined in Annex I of 1272/2008/EC are applied.