(±)-dihydro-3-hydroxy-4,4-dimethylfuran-2(3H)-one

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002-07-15 to 2002-11-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline compliant GLP compliant

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

as at 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

as at 2002

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

(±)-dihydro-3-hydroxy-4,4-dimethylfuran-2(3H)-one

EC Number:

201-210-7

EC Name:

(±)-dihydro-3-hydroxy-4,4-dimethylfuran-2(3H)-one

Cas Number:

79-50-5

Molecular formula:

C6H10O3

IUPAC Name:

(±)-dihydro-3-hydroxy-4,4-dimethylfuran-2(3H)-one

Details on test material:

Physiological saline: not indicated
- Name of test material (as cited in study report): dl-Lactone
- Physical state: white crystalline mass
- Analytical purity: 99.6 %
- Purity test date: not reported
- Lot/batch No.: BX226
- Expiration date of the lot/batch: 31 May 2003
- Stability under test conditions: Stable under storage conditions, Stability in vehicle (physiological saline) not indicated
- Storage condition of test material: In refrigerator in the dark

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: NMRI BR mice (SPF)
- Source: Charles River, Sulzfeld, Germany.
- Age at study initiation: 6-8 weeks old
- Weight at study initiation: males, average = 31.5 g; females, average =
- Assigned to test groups randomly: allocated to treatment groups as they came to hand from delivery boxes.
- Fasting period before study: 3-4 h
- Housing: group housing of 5 animals per sex per cage in labelled polycarbonate cages containing purified sawdust as bedding material (Sawi, Jelu Werk, Rosenberg, Germany).; paper bedding as nest material (supplied by B.M.I. Helmond, The Netherlands).
- Diet (e.g. ad libitum): ad libitum, standard pelleted laboratory animal diet (Altromin (code VRF 1), Lage, Germany).
- Water (e.g. ad libitum): ad libitum, tap-water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30-70
- Air changes (per hr): ca. 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: physiol. saline
- Justification for choice of solvent/vehicle: no
- Concentration of test material in vehicle: 0, 37.5, 75 and 150 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
- Lot/batch no. (if required): not given
- Provider: NPBI, Emmer Compascuum, The Netherlands

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was dissolved in physiological saline at the respective concentration and concentrations were dosed within 4 hours after preparation.

Negative Control:
Negative control animals, treated with physiological saline.
Positive Control:
The positive control used in the micronucleus test was cyclophosphamide (CP; CAS no. 50-18-0; Endoxan, Asta-Werke, F.R.G.) dissolved in physiological saline (NPBI, Emmer Compascuum, The Netherlands) dosed at a single oral intubation of 50 mg salt/kg body weight.

Duration of treatment / exposure:

single oral dose

Frequency of treatment:

single oral dose

Post exposure period:

animals were sacrificed 24 or 48 h post dosing

Doses / concentrations

Remarks:

Doses / Concentrations:
50, 375, 750, 1500 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

5 males and 5 females

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide (CP; CAS no. 50-18-0; Endoxan, Asta-Werke, Germany) dissolved in physiological saline (NPBI, Emmer Compascuum, The Netherlands)
- Justification for choice of positive control(s): accepted positive control substance according to OECD TG 474
- Route of administration: single oral intubation
- Doses / concentrations: 50 mg salt/kg body weight

Examinations

Tissues and cell types examined:

- systemic toxic signs were recorded at least once a day
- animals were weighed at the day of dosing, just prior to dosing
- time of death was recorded as precisely as possible

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
- based on a dose range finding study:
Two dose groups, one comprising 4 males and 4 females and one comprising 3 males and 3 females received a single dose of DL-LACTONE. The study duration per dosing was one to three days. During this period mortality and physical condition were recorded at least once daily. see Table 2

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
see Table 2

DETAILS OF SLIDE PREPARATION:
- bones shortened until a small opening to the marrow canal became visible and flushed with approximately 2 ml of foetal calf serum
- cell suspension centrifuged at 1000 rpm (approximately 100 g) for 5 min
- cells were harvested, resuspended in serum, and a drop of the mixture carefully spread on a slide and fixed for 5 min in 100% methanol and air-dried overnight.
- slides were automatically stained using the "Wright-stain-procedure" in an "Ames" HEMA-tek slide stainer (Miles, Bayer Nederland B.V.). The dry slides were dipped in xylene before they were embedded in MicroMount and mounted with a coverslip.
- 2 slides per animal were prepared

METHOD OF ANALYSIS:
- slides were randomly coded before examination.
- slides were screened at a magnification of 100 x for regions of suitable technical quality
- slides were scored at a magnification of 1000 x; No. of micronucleated polychromatic erythrocytes in 2000 polychromatic erythrocytes.
- ratio polychromatic to normochromatic erythrocytes = counting and differentiating the first 1000 erythrocytes
- micronuclei only counted in polychromatic erythrocytes

OTHER:

Evaluation criteria:

- The positive control substance induced a statistically significant (Wilcoxon Rank Sum Test, two-sided test at P < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes.
- The incidence of micronucleated polychromatic erythrocytes in the control animals should reasonably be within the laboratory historical control data range (mean ± three times the standard deviation):
Males: 1.2%0 ± 3.6%0 indicated are means for n=154. Females: 1.2%0 ± 3.8%0 indicated are means for n=95).

Statistics:

A test substance is considered positive in the micronucleus test if:
It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test; two-sided test at P < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) in the combined data for both sexes or in the data for male or female groups separately.
A test substance is considered negative in the micronucleus test if:
None of the tested concentrations or sampling times showed a statistically significant (P < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes neither in the combined data for both sexes nor in the data for male or female groups separately.
The preceding criteria are not absolute and other modifying factors may enter into the final evaluation
decision.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

at high doses effects of systemic toxicity were seen (convulsions, tremors, lethargy, rough coat, ventro-lateral recumbency, hunched posture), nevertheless no decrease in the ratio of polychromatic to normochromatic erythrocytes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 1.5 and 2.0 g/kg/bw
- Solubility: soluble at all doses
- Clinical signs of toxicity in test animals: convulsions, tremors, lethargy, rough coat, ventro-lateral recumbency, hunched posture
- Evidence of cytotoxicity in tissue analyzed: not analyzed
- Rationale for exposure: 2.0 g/kg bw is the highest dose allowed for animal testing to date in the EU


RESULTS OF DEFINITIVE STUDY

- Induction of micronuclei (for Micronucleus assay): No increase in the frequency of micronucleated polychromatic erythrocytes at any dose level (see Table 4
- Ratio of PCE/NCE (for Micronucleus assay): fluctuating values for all doses, no dose response effect
- Appropriateness of dose levels and route: doses were high enough to cause signs of toxicity (convulsions, tremors, lethargy, rough coat, ventro-lateral recumbency, hunched posture). Based on the high water solubility and the low molecular weight a significant oral bioavailability can be assumed for the test item.

Any other information on results incl. tables

- Table 4: Mean Number of Micronucleated Polychromatic Erythrocytes per 2000 Polychromatic Erythrocytes and Ratio of Polychromatic/Normochromatic Erythrocytes.

Group	Treatment	Dose (mg/kg body weight)	Sampling time (hours	Number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes (mean ± S.D.) (1)	Ratio polychromatic/normochromatic erythrocytes (mean ± S.D.) (1)
	MALES
A	Solvent control	0	24	1.0 ±   1.0	1.21 ± 0.12
B	DL-LACTONE	1500	24	0.4 ±   0.5	0.89 ± 0.10
C	DL-LACTONE	1500	48	0.4 ±   0.5	0.95 ± 0.21
D	DL-LACTONE	750	24	1.2 ±   1.3	1.08 ± 0.15
E	DL-LACTONE	375	24	1.4 ±   0.5	1.48 ± 0.12
F	CP	50	48	45.6 ±24.6 (2)	0.32 ± 0.10
	FEMALES
A	Solvent control	0	24	0.2 ±   0.4	1.31 ± 0.17
B	DL-LACTONE	1500	24	0.8 ±   1.1	1.17 ± 0.09
C	DL-LACTONE	1500	48	1.0 ±   1.4	1.17 ± 0.11
D	DL-LACTONE	750	24	0.6 ±   0.9	1.24 ± 0.23
E	DL-LACTONE	375	24	0.8 ±   0.8	1.13 ± 0.10
F	CP	50	48	20.4 ± 2.6 (2)	0.36 ± 0.10

Solvent control = Physiological saline

CP = Cyclophosphamide

(1) Five animals per treatment group

(2) Significantly different from corresponding control group (Wilcoxon Rank Sum Test, P < 0.01).

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
RS-Pantolactone was tested in an in-vivo micronucleus assay in bone marrow cells of mice according to OECD TG 474 up to maximal tolerated dose of 1500 mg/kg bw (single oral dose). It is concluded that this test is valid and that RS-Pantolactone is not mutagenic in the micronucleus test under the experimental conditions described in this report.

Executive summary:

RS-Pantolactone (named DL-LACTONE in the study report) was tested in the Micronucleus Test in mice, to evaluate its genotoxic effect on erythrocytes in bone marrow. The study was conducted according to OECD TG 474 and is compliant to GLP.

Six groups each comprising 5 males and 5 females, received a single oral intubation. Two groups were dosed with 1500 mg/kg body weight, one group was dosed with 750 mg/kg body weight and one group was dosed with 375 mg/kg body weight. After dosing seven out of twenty animals of the dose level of 1500 mg/kg body weight were lethargic. The other animals of the dose level of 1500 mg/kg body weight showed no abnormalities. The animals of the dose levels of 750 and 375 mg/kg body weight showed no abnormalities after dosing.

A vehicle treated group served as negative control, a group treated with a single oral intubation of cyclophosphamide (CP) at 50 mg/kg body weight served as positive control.

Bone marrow of the groups treated with RS-PANTOLACTONE was sampled 24 or 48 hours after dosing. Bone marrow from the negative control group was harvested at 24 hours after dosing only and bone marrow from the positive control group was harvested at 48 hours after dosing only.

No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with RS-PANTOLACTONE.

The groups that were treated with RS-PANTOLACTONE showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, which reflects a lack of toxic effects of this compound on the erythropoiesis.

The groups that were treated with cyclophosphamide, the positive control substance, showed a decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls. Cyclophosphamide induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes. Thereby the test system was validated for its sensitivity.

It is concluded that RS-PANTOLACTONE is not mutagenic in the micronucleus test under the experimental conditions described in this report.