2-Butyne-1,4-diol, polymer with 2-(chloromethyl)oxirane, brominated, dehydrochlorinated, methoxylated

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, near-guideline study, no restrictions, fully adequate for assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1984

Report date:

1984

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

2-Butyne-1,4-diol, polymer with 2-(chloromethyl)oxirane, brominated, dehydrochlorinated, methoxylated

EC Number:

614-503-3

Cas Number:

68441-62-3

Molecular formula:

(C3H7O2)xC4H4O2Br2(C4H9O2)y with x + y = 2.5

IUPAC Name:

2-Butyne-1,4-diol, polymer with 2-(chloromethyl)oxirane, brominated, dehydrochlorinated, methoxylated

Constituent 2

Reference substance name:

Triethyl phosphate

EC Number:

201-114-5

EC Name:

Triethyl phosphate

Cas Number:

78-40-0

Molecular formula:

C6H15O4P

IUPAC Name:

triethyl phosphate

Test material form:

liquid: viscous

Details on test material:

- Name of test material (as cited in study report): B251, suspensions in 1% tragacanth mucilage.
- Lot/batch No.: Batch No. 1106
- Physical state: liquid
- Analytical purity: not specified

Test animals

Species:

mouse

Strain:

Swiss

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: TNO, Zeist, the Netherlands
- Age at study initiation: sex weeks
- Diet: free access
- Water: ad libitum
- Acclimation period: five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-24
- Humidity (%): 60-90
- Air changes (per hr): 16
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

1% tragacanth

Details on exposure:

The total dosages (3000, 1500 and 750 mg/kg bw) were given as two equal administrations separated by an interval of 24 hours. The mice were fasted overnight only prior to the first dosing. The test compound and vehicle controL were dosed by oral gavage in a volume of 0.1 ml/10 g body weight. Mitomycin C, the positive reference compound, was dosed by intraperitoneal injection in a volume of 0.1 ml/10 g body weight.

Total dosages of 3000, 1500 and 750 mg/kg bw were chosen for the micronucleus test. The top dose of 3000 mg/kg bw would be expected to
cause one or two deaths out of ten, within the 30 hours of the duration of the experiment.

Duration of treatment / exposure:

two equal administrations separated by an interval of 24 hours

Frequency of treatment:

twice

Post exposure period:

6 hours after the second dose (in total 30 hours after the first dose)

Doses / concentrations

Remarks:

Doses / Concentrations:
750, 1500 and 3000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

5 male and 5 female animals

Control animals:

yes, concurrent vehicle

Positive control(s):

Mitomycin C

Examinations

Tissues and cell types examined:

bone marrow, erythrocytes

Details of tissue and slide preparation:

After each administration, the animals were observed. All mortalities during the experiment were recorded.

The animals were killed six hours after the second administration by cervical dislocation and one femur was dissected from each animal. By injection of New Born calf serum in the femur the bone marrow was removed. The suspension of serum and bone marrow was centrifuged for 5 min at 800 rpm. The supernatant was taken off, a bone marrow smear was made of the pellet. After air-drying overnight the smears were fixed in methanol for 5 minutes. The smears were succesively placed in May-Grunwald (1.25%) for 15 minutes and in Giemsa (3%) for 20 minutes. After rinsing in buffered distilled water, the slides were air-dried and mounted in Depex. The smears were examined by light microscopy to determine the incidence of micronucleated cells per 1000 polychromatic erythrocytes per animal and the ratio of normochromatic to polychromatic erythrocytes.

Evaluation criteria:

Incidence of micronucleated cells per 1000 polychromatic erythrocytes per animal and the ratio of normochromatic to polychromatic erythrocytes.

Statistics:

Significance levels of the ratios of normochromatic to-polychromatic erythrocytes were determined by the Mann-Whitney formulation of the Wilcoxon rank sum procedure using pairwise comparison with the control.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Mortalities
After administration of 1%-tragacanth (the vehicle control) and Mitomycin C (the positive reference compound) no mortalities were observed.
In the top dose of B251 (the test compound) two male and two female mice died. In the middle dose one female mouse died.

Micronucleus counts
After administration of B251 at all dosages, the group mean micronucleated cell counts were in the same range as the concurrent control value (vehicle; range 0-3 per 1000 polychromatic erythrocytes)). The positive reference group, administered intraperitoneally with Mitomycin C, gave a mean count of 25.2 micronucleated cells per 1000 polychromatic erythrocytes (range 13-26).

Normochromatic to polychromatic erythrocyte ratios
In this experiment the vehicle control group gave a mean ratio normochromatic to polychromatic erythrocytes of 0.63 (range 0.31-1.53). The positive reference group, dosed with Mitomycin C intraperitoneally, gave a mean ratio of 0.87 normochromatic to polychromatic erythrocytes (range 0.46-1.70). The mean ratio of normochromatic to polychromatic erythrocytes of the top dose level of B251 was 0.78 (range 0.59-1.17). Both were statistically significantly different from the mean ratio of the control compound, indicating toxicity of the test compound and the positive reference compound.

Applicant's summary and conclusion

Conclusions:

Polyol IXOL B251 was negative in a micronucleus study (clastogenicity) in which mice were exposed to doses of 3000, 1500 and 750 mg/kg bw by oral gavage, in two equal dosages, separated by an interval of 24 hours. Toxicity (mortality) was observed at the mid and high dose.

Executive summary:

In a study performed according to a protocol similar to OECD guideline 474 and under GLP, the effect of Polyol IXOL B251 on the incidence of micronucleated polychromatic erythrocytes in mice was assessed (Duphar B.V., 1984). Doses of 3000, 1500 and 750 mg/kg bw were administered by oral gavage, in two equal dosages, separated by an interval of 24 hours. A control group was dosed in an identical manner with the vehicle, 1%-tragacanth. A positive control group was dosed by intraperitoneal injection with Mitomycin C, at a total dosage of 6.6 mg/kg bw. Each of these groups of mice consisted of five females and five males. All the mice were killed six hours after the second treatment, bone marrow smears were examined for the presence of micronuclei in 1000 polychromatic erythrocytes per mouse. At all doses of Polyol IXOL B251, the mean micronucleated cell counts were in the same range as the counts obtained for the negative control group. The positive control compound, Mitomycin C, produced a large increase in the group mean micronucleated cell count. It was concluded that Polyol IXOL B251 did not show any evidence of mutagenic potential for polychromatic erythrocytes of mice in this test.

Regarding the occurrence of toxicity, in the top dose of Polyol IXOL B251 two male and two female mice died. In the middle dose one female mouse died.