3-hydroxy-2-methyl-4-pyrone

Genetic toxicity: in vivo

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

3-hydroxy-2-methyl-4-pyrone

EC Number:

204-271-8

EC Name:

3-hydroxy-2-methyl-4-pyrone

Cas Number:

118-71-8

Molecular formula:

C6H6O3

IUPAC Name:

3-hydroxy-2-methyl-4H-pyran-4-one

Test material form:

solid

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.

Test animals

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.

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

70 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

350 mg/kg bw/day (nominal)

Doses / concentrations 3

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).

Examinations

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.

Results and discussion

Test results

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.

Any other information on results incl. tables

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.  

Applicant's summary and conclusion

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.