methyl 2-hydroxybut-3-enoate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-09-01 to 2021-11-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted with the purified and stabilised form of the test substance. The test item was stored under inert atmosphere (nitrogen).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

1998-08

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2016-07-29

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

methyl 2-hydroxybut-3-enoate

EC Number:

831-109-9

Cas Number:

5837-73-0

Molecular formula:

C5H8O3

IUPAC Name:

methyl 2-hydroxybut-3-enoate

Test animals

Species:

mouse

Strain:

NMRI

Remarks:

Win: NMRI mice

Details on species / strain selection:

The NMRI mouse is one of the standard animals used internationally in this type of mutagenicity testing.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt.
- Age at study initiation: 8 weeks
- Weight at study initiation: 34.2-38.7 g
- Assigned to test groups randomly: Yes, using a randomization scheme. The randomization was checked according to the actual body weights verifying the homogeneity and deviations between the groups.
- Fasting period before study: No
- Housing: 2 animal/cage in the pretest and in the high dose group of main test and 5 animals/cage in the other groups of the main test in type I polypropylene/polycarbonate cages with laboratory bedding
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 40 - 70
- Air changes (per hr): Not indicated
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From day 0 to day 2

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle: Aqua Purificata
- Justification for choice of solvent/vehicle: The suitability of this vehicle was confirmed in a GLP compliant validation study (Analytical report 941-100-5436) Furthermore, suitability of the vehicle is confirmed with the laboratory’s historical control database.
- Concentration of test material in vehicle: The test item was used for treatment in concentrations of 15 mg/mL, 30 mg/mL and 60 mg/mL prepared with the Aqua Purificata.
- Amount of vehicle: 10 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The necessary amount of test item was weighed into a calibrated volumetric flask. A partial volume of Aqua Purificata was added and the formulation was stirred until homogeneity was reached. The formulations were prepared fresh on day of dosing and used within 2 hours.

Duration of treatment / exposure:

48 hours

Frequency of treatment:

Twice at a 24-hour interval (test item concentrations and negative control), once (positive control)

Post exposure period:

24 hours (test groups, negative and positive control)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

150 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

600 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 male animals/group (7 male animals in the high dose group), 5 groups

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Justification for choice of positive control: Commonly used mutagen, good laboratory's HCD available
- Route of administration: Intraperitoneally
- Doses / concentrations: 60 mg/kg bw

Examinations

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: A non GLP Preliminary toxicity test was performed to identify the appropriate maximum dose level for the main test as no toxicity data in mice was available. The preliminary toxicity test determined the MTD and based on death, clinical signs of test item related toxicity and whether there are differences in toxicity between the sexes or not.

DETAILS OF SLIDE PREPARATION: Smears of the vortexed and centriguged bone marrow from two femurs cell were made on standard microscope slides. Slides were then dried at room temperature. They were fixed for a minimum of 5 minutes in methanol and allowed to air-dry and then stained with Giemsa (10%) solution for 25 minutes. Afterwards, slides were rinsed in distilled water. They were dryed at room temperature (at least 12 hours) and coated with EZ-mount.

METHOD OF ANALYSIS: Four thousand mature, polychromatic erythrocytes (PCEs) were scored per animal to assess the micronucleated cells. The frequency of micronucleated cells was expressed as percent of micronucleated cells based on the first 4000 PCEs counted in the optic field. The proportion of immature among total (immature + mature) erythrocytes was determined for each animal by counting a total of at least 500 erythrocytes.

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, the test item is considered clearly positive if:
• At least one of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated immature erythrocytes compared with the concurrent negative control,
• This increase is dose-related at least at one sampling time when evaluated with an appropriate test, and
• Any of these results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits)

Providing that all acceptability criteria are fulfilled, the test item is considered clearly negative if the following criteria had been met:
• None of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated immature erythrocytes compared with the concurrent negative control,
• There is no dose-related increase at any sampling time when evaluated by an appropriate test,
• All results are inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits),
• Bone marrow exposure to the test item occurred.

Statistics:

Statistical analysis was done with SPSS PC+ software for the following data:
- The frequencies of micronucleated polychromatic erythrocytes in animals in the test and positive control groups were compared to the values found in the corresponding negative (vehicle) and historical control groups.
- The proportion of immature among total (immature + mature) erythrocytes in animals in the test and positive control groups were compared to the values found in the corresponding negative (vehicle) and historical control groups.
- The data was checked for a linear trend in mutant frequency with treatment dose using the adequate regression analysis by Microsoft Excel software.

Results and discussion

Test results

Key result

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 RANGE-FINDING STUDY
- Dose range: 125 - 1500 mg/kg bw
- Solubility: A clear solution was obtained up to a concentration of 200 mg/mL.
- Clinical signs of toxicity in test animals: Groups of two males and females mice were treated two times at 24-hour intervals by oral gavage. Clinical signs like decreased activity, narrow palpebra, piloerection, incoordination and decreased grip strength were observed with dose-dependent increase in severity and decreasing onset. At 750 mg/kg bw/day, one female animal died after the first treatment. Tonic and clonic convulsions were observed before her death. At 1000 mg/kg bw/day, one male animal died half an hour after the first treatment. Tonic and clonic convulsions were observed before death. At 1000 mg/kg bw/day, all animals died between 1-4 hours after treatment.
- Evidence of cytotoxicity in tissue analysed: In two females of the 1000 mg/kg bw/day group, red and haemorrhaged mucous membrane was observed in the stomach.
- Rationale for exposure: The doses were based on the effects (massive local tissue damage in the stomach) noted in acute rat studies.
- Harvest times: Animals were treated twice in 24-hour intervals. The pre-test was finished 24 hours after the last treatment. No bone marrow smears were prepared.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: The frequencies of micronucleated polychromatic erythrocytes (MPCEs) in the treated mice in all dose groups were within acceptable ranges and compatible with the historical control data for this laboratory.
- Ratio of PCE/NCE: In the dose groups of 300 and 600 mg/kg body weight a statistically significant decreased number of PCEs was observed compared to the negative and historical control groups. This effect demonstrated exposure of the test item to the bone marrow (see “Attached background material”)
- Appropriateness of dose levels and route: The dose levels were chosen based on the results of a preliminary range-finding test. In this pre-test, mortality was observed at 750 mg/kg bw/day and higher in male and female mice. Clear signs of toxicity (strong decreased activity, piloerection and grip strength, incoordination and narrow palpebra after the first and second treatment) were observed at 500 mg/kg bw /day. Thus, the maximum tolerated dose was chosen as a dose between 500 and 750 mg/kg bw/day /600 mg/kg bw/day). The dose groups were separated by a factor of 2 as suggested in OECD guideline 474. Oral administration was shown to be appropriate as it was shown that the test substance reached the bone marrow (decreased number of PCEs in the middle and high dose groups).
- Statistical evaluation: No statistical significance was observed in the frequency of MPCEs in male mice at 24 hours after the second treatment compared to the concurrent negative (vehicle) control in any of the examined dose groups (see also "Attached background material").

Applicant's summary and conclusion

Conclusions:

In a Mammalian Erythrocyte Micronucleus Test according to OECD guideline 474, no biologically relevant or statistically significant increases in the frequency of MPCEs were seen in the groups of mice treated with the test item compared to the vehicle and historical control groups. Statistically reduced PCEs at the mid and high dose demonstrated that the bone marrow was reached by the test item did not show any genotoxic activity in this Mouse Micronucleus Test.

Executive summary:

The potential mutagenic activity of the test item was examined in bone marrow of male NMRI mice according to OECD guideline 474 and GLP. The doses of the test item for the Micronucleus Test were determined according to a preliminary oral toxicity study. The doses selected were 150, 300 and 600 mg test item per kg body weight.

Study Design

Negative (vehicle) control and a positive control group were included. Treatment was carried in Aqua Purificata with a constant treatment volume (10 mL/kg body weight). The test item and negative (vehicle) control item were administered by gavage two times at 24-hour intervals. Cyclophosphamide dissolved in Aqua ad injectabilia (positive control) was administered once, intraperitoneally with a treatment volume of 10 mL/kg body weight. In the low, mid and high dose groups and vehicle control group the sampling was made once at 24 hours after the second treatment. In animals treated with Cyclophosphamide (60 mg/kg bw), the sampling was performed at 24 hours post-treatment of the single administration. Five animals per dose group were used. Four thousand polychromatic erythrocytes (PCEs) were scored per animal to assess the micronucleated cells. The suitability of the chosen vehicle for the test item was analytically verified. A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. Measured concentrations of formulations applied in the study varied in the acceptable range (between of 96-103% of the nominal concentrations) and all formulations were homogenous, thereby confirming proper dosing.

Results

The two times oral administration of 150 mg/kg body weight, 300 mg/kg body weight and 600 mg/kg body weight of Methyl vinyl glycolate (MVG) did not induce increases in the frequency of micronucleated polychromatic erythrocytes (MPCEs) in male mice 24 hours after the second treatment compared to the negative and historical control groups. The proportion of immature among total (immature + mature) erythrocytes was determined for each animal by counting a total of at least 500 erythrocytes. Compared to the negative and historical control groups the number of polychromatic erythrocytes (PCEs) at 24 hours after the second treatment in the dose group of 150 mg/kg body weight was not affected. In the dose groups of 300 and 600 mg/kg body weight a statistically significant decreased number of PCEs was observed compared to the negative and historical control groups. This effect demonstrated exposure of the test item to the bone marrow. The frequencies of MPCEs for the negative and positive control mice were within acceptable ranges and in line with the historical control data for this laboratory. Cyclophosphamide treated mice (60 mg/kg body weight) showed a large, statistically significant increase in the MPCE number compared to the negative and historical controls. Thus, the study is considered to be fully valid.

Conclusion

No biologically relevant or statistically significant increases in the frequency of MPCEs were seen in the groups of mice treated with Methyl vinyl glycolate (MVG) compared to the vehicle and historical control groups. Statistically reduced PCEs at the mid and high dose demonstrated that the bone marrow was reached by the test item did not show any genotoxic activity in this Mouse Micronucleus Test.