Hydroxyacetone

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 JUL 2022 - 15 NOV 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: Council Regulation (EU) No 2017/735, Annex Part B, B.62: In vivo Mammalian Alkaline Comet Assay

Version / remarks:

14 February 2017

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

14 February 2017

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

29th July, 2016

Test guideline 4

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

29th July, 2016

Principles of method if other than guideline:

Bowen D.E., Whitwell J.H., Lillford L., Henderson D., Kidd D., McGarry S., Pearce G., Beevers C. and Kirkland D.J.: Evaluation of a multi-endpoint assay in rats, combining the bone-marrow micronucleus test, the Comet assay and the flow-cytometric peripheral blood micronucleus test, Mutation Research 722: 7-19, 2011

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Reference substance name:

Hydroxyacetone

EC Number:

204-124-8

EC Name:

Hydroxyacetone

Cas Number:

116-09-6

Molecular formula:

C3H6O2

IUPAC Name:

1-hydroxypropan-2-one

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Remarks:

HAN:WIST of Wistar origin

Details on species / strain selection:

Hygienic level at arrival: SPF
Hygienic level during the study: Good conventional
Justification of the species: Rats are routinely tested in this test and the chosen Wistar rat was selected due to a wide range of experience with this strain of rat in corresponding toxicity studies and historical control data at TOXI-COOP ZRT.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop ZRT. H-1103 Budapest, Cserkesz u. 90.
- Age at study initiation: 55-60 days (Young adult rats, 8-9 weeks old at the commencement of the treatment).
- Weight at study initiation: 260-276 g, the weight variation of animals involved at the start of the study did not exceed ± 20 %
- Assigned to test groups randomly: yes, under following basis: All animals were sorted according to body weight by computer and grouped according to weight ranges. There were an equal number of animals from each weight group in each of the experimental groups during the randomization. The grouping was controlled by SPSS/PC computer program according to the actual body weight verifying the homogeneity and deviations among the groups and cages.
- Housing: 3 animals / cage
Cage type: Type III polypropylene/polycarbonate (Size: 22 x 32 x 19 cm (width x length x height).
Bedding: Certified laboratory wood bedding (SAFE 3/ 4-S-FASERN produced by J. Rettenmaier & Söhne GmbH+Co. KG; D-73494 Rosenberg Holzmühle 1 Germany). The cages and bedding were changed once during the acclimatization and experimental periods.
- Diet (e.g. ad libitum): received ssniff® SM R/M-Z+H complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany, ad libitum.
- Water (e.g. ad libitum): tap water from watering bottles (from municipal supply, as for human consumption, from 500 mL bottles), ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): In the range of 22 ± 3 °C (the actual values: 19.0-24.4°C).
- Humidity (%): In the range of 30 – 70 % (the actual values: 33-69 %).
- Air changes (per hr): Provided by central air-condition system. The numbers of air changes per hour is higher than 10.
- Photoperiod (hrs dark / hrs light): Artificial light, 12 hours daily, from 6 a.m. to 6 p.m.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: distilled water (Aqua purificata)
- Concentration of test material in vehicle: 200, 100 and 50 mg/mL.
- Amount of vehicle (if gavage or dermal): 2000, 1000 and 500 mg/kg body weight/day
A constant treatment volume of 10 mL formulation/kg body weight was administered in all test item treatment groups, in the vehicle control group and in the positive control groups.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The planned doses were 2000, 1000 and 500 mg/kg body weight/day, consequently the test item was formulated in distilled water (Aqua purificata) in concentrations of 200, 100 and 50 mg/mL.
The formulations were prepared in the laboratory of the Test Facility just before each treatment.
Analysis of formulations (for checking of each concentration and homogeneity) was performed in the Analytical Laboratory of Test Facility according to the validated analytical method.

Duration of treatment / exposure:

45h exposure, 48h sampled

Frequency of treatment:

The test item formulated in the vehicle, was administered in dose by oral gavage three times: once on the day 0, second on the day 1 (24 hours after first treatment) and third on the day 2 (45 hours after the first treatment).

Post exposure period:

3-4 hours after the third treatment (doses and vehicle control) and 3-4 hours after the treatment (EMS positive control). the animals were euthanized and the cells of the target tissues were isolated for comet assay and in parallel bone marrow was sampled for micronucleus test (this meant the bone marrow was sampled at 24 h after the second dose, in the case of CP positive control at 24 h after the treatment).

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

Test item treatment

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Test item treatment

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Test item treatment

Doses / concentrations 4

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Negative control

No. of animals per sex per dose:

5 animals per test item dose or vehicle control or positive control groups.

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive control for comet assay:
Name: Ethyl methanesulfonate (EMS)
Batch Number: BCCH1105
Appearance: Colourless liquid
Expiry Date: May 27, 2023
Storage: At room temperature in tightly closed container, under inert atmosphere
Supplier/Manufacturer: SIGMA-ALDRICH
- Route of administration: oral gavage
- Doses / concentrations: 200 mg/kg bw/d

Positive control for bone marrow micronucleus test:
Name: Cyclophosphamide monohydrate (CP)
Batch Number: MKCN3646
Appearance: White powder
Retest Date: October 31, 2023
Storage: At 2-8°C
Supplier/Manufacturer: SIGMA-ALDRICH

- Route of administration: oral gavage
- Doses / concentrations: 20 mg/kg bw/d

Examinations

Tissues and cell types examined:

Target tissues for sample preparation and analysis: Stomach, liver, duodenum (comet assay); bone marrow (micronucleus test).

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: For dose selection the proposals of corresponding OECD guidelines, the information provided by the Sponsor and the results of non-GLP preliminary dose range finding test (using the same species, strain, sex, and treatment regimen to be used in the main study) were taken into consideration.
In the range-finding test (based on the availably information) two animals (male rats, HAN:WIST of Wistar origin) were treated with the test item by oral administration at 10 mL/kg body weight treatment volume at the concentration of 2000 mg/kg body weight/day. The treatment was performed on three consecutive days with 24h and 21h intervals. At the chosen concentration level neither mortality nor any clinical sign or any suffering of animals were observed. Based on this information the maximum dose was 2000 mg/kg body weight/day, and in addition to the maximum dose two additional doses, 1000 and 500 mg/kg body weight/day were selected.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): In order to cover both the comet assay and micronucleus test designs, the combination protocol-design consisted of animals dosed at 0, 24 and 45 h, and sampled at 48 h (i.e., three hours after the final dose). Using this dosing regimen, only the first two doses (at 0 and 24 h) could impact upon the bone marrow micronucleus endpoint, as the final dose is given too close to the sampling time to have any effect on micronucleated polychromatic erythrocytes (MPCE) production. Bone marrow is sampled effectively at 24 h after the second dose, and is therefore in line with the conditions of OECD 474 recommendations. The bone marrow sample collection was performed 24 hours after the second treatment. As expulsion of the nucleus is known to occur within 5–10 h after the last cell division, with the loss of RNA and maturation to NCE status within a further 10–30-h period, it is currently recommended that sampling of bone marrow and subsequent analysis of the immature PCE population for MN should occur approximately 18–24 h following the final administration of test chemical. For comet assay the sampling time is a critical variable because it is determined by the period needed for the test chemicals to reach maximum concentration in the target tissue and for DNA strand breaks to be induced but before those breaks are removed, repaired or lead to cell death. A suitable compromise for the measurement of genotoxicity is to sample at 2-6 hours after last treatment (based on the experience of the laboratory over the last years the sampling should be performed 3-4 hours after the second (in this case third) treatment. The animals were euthanized (consistent with the effective animal welfare legislation and 3Rs principles using Isofluran CP®) and the cells of the target tissues were isolated and care was taken to necropsy all animal at the same time after the last dose.


DETAILS OF SLIDE PREPARATION:
The bone marrow was obtained from one femur of the exposed rats immediately after sacrificing. The bone marrow was flushed with 5 mL of foetal bovine serum to a 15 mL centrifuge tube. After mixing, the cell suspension was concentrated by centrifugation at 2000 rpm for 10 minutes and the supernatant was discarded. Smears of the cell pellet was made on standard microscope slides. The slides were dried at room temperature overnight. Subsequently the slides were fixed in methanol for 5 minutes and allowed to air-dry. Slides were stained with 10 % Giemsa solution for 25 minutes; thereafter rinsed in distilled water. After drying at room temperature (at least 12 hours), slides were coated with EZ-Mount (TM).
The slide preparation for the Comet assay was conducted within one hour after single cell preparation. Four slides were prepared for each animal for each tissue sample, with six animals per dose group and vehicle control and for the EMS positive control group. In summary 24 slides per treatment per tissue for the test item dose groups and controls (negative, vehicle and positive).
Pre-treatment of Comet slides: According to general procedure, the conventional (superfrost) slides were dipped in hot 0.5 % normal melting point agarose in water. After gently removing, the underside of the slides were wiped in order to remove the excess of agarose. The slides were then laid on a flat surface and allowed to dry.


METHOD OF ANALYSIS:
Analysis of Bone Marrow Slides
Prior to microscopic analysis, the prepared slide from each animal was given a code, which was covered the original animal numbers to ensure that the slides were scored without bias. Four thousand polychromatic erythrocytes (PCEs) were scored per animal to assess the micronucleated cells. The number of micronucleated cells were counted and frequency was expressed as percent of micronucleated cells based on the first 4000 PCEs counted in the optic field. The microscopic analysis was performed by eye.

Visualisation and Analysis of Comet Assay Slides
Every animal was euthanised and cells of the defined organs were isolated. For each animal and each tissue 4 slides were prepared (12 slides per animal, 72 slides per test item dose and negative, vehicle and EMS positive controls). Three of the four slides from five animals per test item treatments and per controls were stained and analysed (9 slides per animal, 45 slides per dose, per controls). Coded slides were stained and blind scored. The slides were examined with an appropriate magnification (200x) using fluorescent microscope (Nikon Upright Microscope Eclipse Ci-S) equipped with Nikon-INTENSILIGHT C-HGFI Precentered Fiber Illuminator an appropriate excitation filter (TRITC) and with an Andor-Zyla sCMOS camera. For image analysis, the Andor Komet GLP 7.1.0 (Andor Technology) was used. For each tissue sample, fifty cells per slide were randomly scored i.e. 150 cells per animal (750 analysed cells per test item treatment, per vehicle control and per EMS positive control). DNA strand breaks in the comet assay were measured by independent endpoints such as %tail DNA, olive tail moment (OTM) and tail length. The tail % DNA (also known as tail intensity) was applied for the evaluation and interpretation of the results and determined by the DNA fragment intensity in the tail expressed as a percentage of the cell’s total intensity. Additionally, the OTM and tail length values were collected. The OTM is expressed in arbitrary units and is calculated by multiplying the percentage of DNA (fluorescence) in the tail by the length of the tail in μm. The tail length is measured between the center of the comet head and the end of the comet tail. In addition, each slide was examined for presence of ghost cells (possible indicator of toxicity and/or apoptosis). Ghost cells results from a total migration of the DNA from the nucleus into the comet tail, reducing the size of the head to a minimum. Ghost cells, also known as clouds or hedgehogs, are morphological indicative of highly damaged cells and their presence is often associated with severe genotoxicity, necrosis and apoptosis. Ghost cells were excluded from the image analysis data collection, however determining of their frequency is useful for the data interpretation. The ghost cells were recorded for each slide per animal, per treatment and per tissue.

Evaluation criteria:

A study is considered as valid when:
The applied concurrent negative control is considered as acceptable for addition of the laboratory historical control databases (for comet assay and for bone marrow micronucleus test parts of this study).
Concurrent positive controls induce responses that are compatible with those generated in the historical control database and produce a statistically significant increase compared with the concurrent negative control.
Adequate numbers of cells and doses were analyzed.
The highest dose was selected according to the criteria required by the OECD 474 and 489 guidelines.
In the case of outlier assay data, they may be acceptable for inclusion in, as long as these data are not extreme outliers and there is evidence of absence of technical or human failure.

Statistics:

The statistical analysis of % tail DNA values, tail length and OTM values; the number of ghost cells; furthermore, the number of micronucleated polychromatic erythrocytes and the proportion of immature among total erythrocytes was carried out using the appropriate statistical methods, using SPSS software.
The heterogeneity of the obtained data was tested. The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. In case of a positive analysis, Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. If the data were not normal distributed, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. In case of a positive analysis result, the inter-group comparisons were performed using Mann-Whitney U-test.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

positive

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: While the investigated test item Hydroxyacetone was not genotoxic in the examined tissues in the comet assay, it showed unequivocally genotoxic activity in the rat bone marrow micronucleus test.

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: the maximum dose was 2000 mg/kg body weight/day, and in addition to the maximum dose two additional doses, 1000 and 500 mg/kg body weight/day were selected.
-Solubility: The test item Hydroxyacetone in distilled water formulations was considered to be homogeneous.
- Clinical signs of toxicity in test animals: At the chosen concentration level neither mortality nor any clinical sign or any suffering of animals were observed.
- Rationale for exposure: The oral route was considered to be the most relevant exposure route and for comet assay the glandular stomach, the duodenum and the liver were selected as the tissues of interest in line with the respective requirements of the EU authorities requesting this test; furthermore, bone marrow samples were obtained for analysis from one femur of the exposed rats.

RESULTS OF DEFINITIVE STUDY
- Ratio of PCE/NCE (for Micronucleus assay): Negative control: 0.56 ± 0.07. test item 500mg/kg bw/day: 0.54 ± 0.06. test item 1000mg/kg bw/day: 0.59 ± 0.03. test item 2000mg/kg bw/day: 0.55 ± 0.05. Positive control: 0.47 ± 0.07
- Evidence of cytotoxicity in tissue analysed: Cytotoxicity was determined (as a first screening part of comet assay) on a small sample of each isolated cell

Any other information on results incl. tables

Mortality and Clinical Observations

No mortality was observed during the treatments and expression period in any dose group up to the highest dose and in the controls (negative and positive). Neither toxic symptoms nor any clinical signs were observed during the study at the observation time points (on day 0 before the start of treatments), 1, 2 and 4 hours after the first and second treatments (day 0 and day 1), just before the second and third treatments on days 1 and 2; furthermore 1 hour after the third treatment and shortly before sampling time (on day 2). At the tissue isolation (opening the abdominal cavity for sample preparation for comet assay) normal appearance and anatomy of stomach, duodenum and liver was observed for all dose levels and controls. No signs of toxicity or local test item effects were observed in the test item treated doses and controls. Slight body weight increases were observed in the test item treated dose groups and in the negative and EMS positive controls when comparing the weight values measured on Day 0 and just before the sacrifice. The body weight increase was 2.18 % in the vehicle control, 1.43 % in the EMS positive control, 1.06 % at 500 and 1000 mg/kg body weight/day, and 0.06 % at the highest dose of 2000 mg/kg body weight/day. At the CP positive control group slight, 2.3 % weight loss was noticed. These weight gains were in the range of the expected changes for such exposure times, were in the normal development range.

Comet Assay Observations: Cytotoxicity, Ghost Cells

In this study a first indication of possible cytotoxicity was estimated by Trypan blue dye exclusion technique. This screening technique provided preliminary information from the effectiveness and success of the single cell preparation. The cell concentrations of the isolated cell suspensions were in the 10⁵‑10⁷ order of magnitude. No significant changes were detected in the screening viability values of stomach, duodenum and liver cell suspensions. All of them remained within the vehicle control range at all test item treatment doses and EMS positive control, no dose related changes were realised. The average screened viability values varied between 74-80 % at the stomach cell preparations, between 74-79 % at the duodenum cell preparations and 95-98 % at the liver preparations. In the duodenum samples, the mean ghost cell values varied between 11-16 %. The percentages of ghost cells showed a clear dose related tendency (confirmed by linear trend analysis). Accordingly, the highest ghost cell percentage (16 %) was obtained at the 2000 mg/kg body weight/day. This percentage as well as the 15 % obtained at the dose 1000 mg/kg body weight/day, differed statistically significantly from that of the negative control value. In the liver preparations (the mean value was 5 % at the vehicle control, 6 % at 500 mg/kg body weight/day, 9 % at 1000 mg/kg body weight/day, and 8 % at 2000 mg/kg body weight/day) the percentages of ghost cells were above the laboratory’s historical control data range in all test item doses, and the higher values differed statistically significantly from the vehicle control value at 1000 and 2000 mg/kg body weight/day. 

% Tail DNA, Tail Length and Olive Tail Moment Comparisons

The test item Hydroxyacetone was investigated at three dose levels 2000, 1000 and 500 mg/kg body weight/day. Animals were treated for three consecutive days. Three target organs (tissues) of each animal were investigated: stomach duodenum and liver. For each tissue sample fifty cells per slide were randomly scored i.e. 150 cells per animal (750 analyzed cells per test item dose and controls (negative, vehicle and positive)). DNA strand breaks in the comet assay were measured by independent endpoints such as % tail DNA, tail length and olive tail moment (OTM). The mean median % tail DNA values (group means) of each dose remained within the vehicle control range for the examined tissues, and did not differ statistically significantly from that of the vehicle control up to the highest dose of 2000 mg/kg body weight/day. The linear trend analysis did not show significance, consequently no dose related increase in % tail DNA values was observed. Most of the group mean % tail DNA values (and most of the individual animal values) of the test item doses in the stomach, duodenum and liver samples were within the corresponding historical control data ranges (95 % confidence intervals, C-charts). The tail length values differed statistically significantly from that of the concurrent negative control for the stomach samples at 2000 mg/kg body weight/day. However, this significance was considered as not relevant for mutagenicity assessment since the significance was observed for the lower value (compared to the concurrent control) of the above dose level. The further tail length and olive tail moment values in the stomach, duodenum and liver samples of the test item dose groups did not differ statistically significant from that of the vehicle control.

Table. Summarized results of Comet assay

Dose (mg/kg body weight/day)	Stomach
	Measured parameters
	% Tail DNA	Tail length (µm)	OTM
Negative (vehicle) control	10.09 ± 5.84	28.79 ± 11.85	1.47 ± 1.25
500	6.15 ± 2.26	17.30 ± 9.56	0.77 ± 0.45
1000	12.86 ± 4.91	33.33 ± 10.77	1.79 ± 0.88
2000	6.25 ± 1.49	13.90 ± 5.48 * ^DN	0.65 ± 0.18
Positive (EMS) control	31.21 ± 5.62 ** ^DN	60.58 ± 12.58 ** ^DN	7.34 ± 2.61 ** ^U
Negative control ranges	0.00 – 14.96	0.00 – 35.00	0.00 – 2.63
Positive (EMS) control ranges	16.96 – 44.32	23.18 −130.47	0.00 – 23.91

 

Dose (mg/kg body weight/day)	Duodenum
	Measured parameters
	% Tail DNA	Tail length (µm)	OTM
Negative (vehicle) control	4.27 ± 0.70	8.26 ± 1.97	0.40 ± 0.08
500	4.37 ± 0.50	8.76 ± 1.23	0.43 ± 0.05
1000	7.30 ± 3.23	15.19 ± 10.17	0.73 ± 0.47
2000	5.72 ± 2.39	13.29 ± 9.91	0.61 ± 0.31
Positive (EMS) control	25.24 ± 3.74 ** ^U	50.73 ± 7.27 ** ^U	5.00 ± 1.17 ** ^U
Negative control ranges	1.19 – 7.13	4.23 – 11.36	0.14 – 0.63
Positive (EMS) control ranges	11.47 – 46.12	26.21 − 127.89	1.78 – 18.68

 

Dose (mg/kg body weight/day)	Liver
	Measured parameters
	% Tail DNA	Tail length (µm)	OTM
Negative (vehicle) control	3.08 ± 0.27	6.56 ± 0.26	0.32 ± 0.02
500	2.70 ± 0.45	6.80 ± 0.52	0.29 ± 0.04
1000	3.33 ± 0.78	7.10 ± 0.97	0.34 ± 0.08
2000	2.80 ± 0.83	7.03 ± 0.95	0.31 ± 0.07
Positive (EMS) control	27.66 ± 3.40 ** ^U	54.63 ± 10.10 ** ^U	6.30 ± 1.23 ** ^U
Negative control ranges	2.62 – 6.61	5.78 – 10.50	0.27 – 0.69
Positive (EMS) control ranges	11.06 – 34.44	34.96 −99.19	1.13 – 13.80

EMS:Ethyl methanesulfonate

Remark: The table contains the group mean values of each parameter.

             The negative and positive control ranges are the 95 % confidence intervals, C-charts.

Statistically significant:

     **      : p<0.01

     *        : p<0.05

DN: Duncan's multiple range test

U: Mann-Whitney U-test Versus Control

 

Bone Marrow Micronucleus Test Observations: Proportion of PCEs

The proportion of immature among total (immature + mature) erythrocytes was determined for each animal by counting a total of 500 erythrocytes. Compared to the negative control group, the number of polychromatic erythrocytes (PCEs) at 24 hours after the second treatment in all dose groups was not affected, no reduction was detected. The proportion of PCEs among total erythrocytes in CP positive control differed statistically significantly from that of the negative control; however, was in acceptable range, and corresponded with laboratory’s historical control data, C-chart.

Bone Marrow Micronucleus Test

Three times oral administration of 500 mg/kg body weight of Hydroxyacetone did not induce biologically and statistically significant increases in the frequency of MPCEs at 24 hours after the second treatment compared to the concurrent negative (vehicle) control (Table below). Three times oral administration of 1000 mg/kg body weight and 2000 mg/kg body weight of Hydroxyacetone induced biologically and statistically increases in the frequency of MPCEs at 24 hours after the second treatment compared to the concurrent negative (vehicle) control. The increases were dose dependent as confirmed by adequate regression analysis. The number of MPCEs were outside the distribution of the historical negative control data (95 % confidence intervals, C-chart) in the dose groups of 1000 and 2000 mg/kg body weight.

Table. Summarized results of Micronucleus test

Dose (mg/kg body weight/day)	Mean of MPCE	Mean of PCE/PCE+NCE
Negative (vehicle) control	9.80 ± 4.49	0.56 ± 0.07
500	13.80 ± 2.59	0.54 ± 0.06
1000	17.80 ± 4.55 * ^KW	0.59 ± 0.03
2000	21.40 ± 4.04 * ^KW	0.55 ± 0.05
Positive (CP) control	81.80 ± 14.62 ** ^KW	0.47 ± 0.07 * ^DN
Negative control ranges	0.37 – 16.96	0.38 – 0.65
Positive (CP) control ranges	33.59 – 153.61	0.27 – 0.50

CP: Cyclophosphamide

PCE: Polychromatic Erythrocyte

NCE: Normochromatic Erythrocyte

MPCE: Number of Micronucleated Polychromatic Erythrocytes per 4000 PCE

Remark: The negative and positive control ranges are the 95 % confidence intervals, C-charts.

Statistically significant:

     **      : p<0.01

     *       : p<0.05

KW: Kruskall Wallis Non Parametric ANOVA

DN: Duncan's multiple range test

Applicant's summary and conclusion

Conclusions:

The test item did not induce statistically significant increases in DNA strand breaks at any of the tested dose levels in stomach, duodenum and liver cells. No biologically and statistically significant increases in the frequency of MPCEs were seen in the dose groups of 500 mg/kg body weight/day compared to the negative control group, however, a biologically relevant, dose-dependent and statistically significant increase in the frequency of MPCEs were seen at dose levels of 1000 and 2000 mg/kg body weight/day compared to the concurrent vehicle control group. While the investigated test item was not genotoxic in the examined tissues in the comet assay, it showed unequivocally genotoxic activity in the rat bone marrow micronucleus test.

Executive summary:

The test item was investigated by the means of a combined assay of an in vivo comet assay on isolated stomach, duodenum and liver cells under alkaline conditions and in parallel in an in vivo bone marrow micronucleus test in the male HAN:WIST rats. The test was done according to OECD Guideline 489, 474 and EU Method B.12 under GLP compliance. The test item was administered three times via oral gavage at the dose levels 2000, 1000 and 500 mg/kg body weight/day. The applied treatment regime (dose 1 at 0-hour, dose 2 at 24 hours, dose 3 at 45 hours) as well as the samplings (e.g.: 3 hours after the last treatment for comet assay, 24 hours after the second treatment for micronucleus test) were adequate for above method combination approach. Concurrent controls confirmed the sensitivity and validity of both test parts of combined assay. Under the experimental conditions presented in this report, the test item did not induce statistically significant increases in DNA strand breaks at any of the tested dose levels in stomach, duodenum and liver cells. No biologically and statistically significant increases in the frequency of MPCEs were seen in the dose groups of 500 mg/kg body weight/day compared to the negative control group, however, a biologically relevant, dose-dependent and statistically significant increase in the frequency of MPCEs were seen at dose levels of 1000 and 2000 mg/kg body weight/day compared to the concurrent vehicle control group. While the investigated test item was not genotoxic in the examined tissues in the comet assay, it showed unequivocally genotoxic activity in the rat bone marrow micronucleus test.