6-glycidyloxynapht-1-yl oxymethyloxirane

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:

03.07.1997 to 22.08.1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

sampling did not occur after 48 hours.

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

429-960-2

EC Name:

-

Cas Number:

27610-48-6

Molecular formula:

C16H16O4

IUPAC Name:

2-[({6-[(oxiran-2-yl)methoxy]naphthalen-1-yl}oxy)methyl]oxirane

Test material form:

other: viscous liquid

Details on test material:

Description: Brown viscous liquid
Storage conditions: At approximately 4°C in the dark

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

Sufficient albino CD-1 Strain mice were supplied by Charles River (UK) Limited, Margate, Kent. At the start of the main study the male mice weighed 24 to 29 g and the female mice weighed 20 to 26 g and were approximately five to seven weeks old. After a minimum acclimatisation period of five days the animals were selected at random and given a number unique within the study by ear punching and a number written on a colour coded cage card.
The animals were housed in groups of up to five in solid-floor polypropylene cages with woodflakes beding. Free access to mains drinking water and food (Rat and mouse Expanded Diet N°1, Special Diets Services Limited, Witham, Essex, UK) was allowed throughout the study.
The animal room was maintened at a temmperature of 20 to 22 °C and relative humidity of 55 to 60%. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours light and twelve hours darkness.

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

The vehicle was supplied by Analytical Supplies Ltd, as follows:
Supplier's identification: Arachis oil
Supplier's batch number: T17
Safepharm serial number: CO/1226
Date received: 13 June 1997
Description: straw coloured viscous liquid
Storage conditions: Room temperature

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

The test item was freshly prepared as required as a dispersion at the appropriate concentration in arachis oil.

Duration of treatment / exposure:

Duration of treatment: 24 hours and 48 hours

Frequency of treatment:

Groups of mice were dosed once only via the intraperitoneal route.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
125 mg/kg
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
250 mg/kg
Basis:
nominal conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
500 mg/kg
Basis:
nominal conc.

No. of animals per sex per dose:

Groups of mice (five males and five females) were dosed with the test material at 125, 250 or 500 mg/kg. One group of mice from each dose level was killed by cervical dislocation 24 hours following treatment and a second group dosed with the test material at 500 mg/kg at 48 hours.

Control animals:

yes

Positive control(s):

A group was dosed orally with cyclophosphamide, a positive control material known to produce micronuclei under the conditions of the test.

Examinations

Tissues and cell types examined:

Femurs were dissected from each animal, aspirated with foetal calf serum and bone marrow smears prepared.

Details of tissue and slide preparation:

Immediately following sacrifice (ie 24 or 48 hours following dosing), both femurs were dissected from each animal, aspirated with foetal calf serum and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol and stained in May-Grünwald/Giesma.

Evaluation criteria:

Stained bone marrow smears were coded and examined blind using light microscopy at 1000x magnification. The incidence of micronucleated cells per 1000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored.
Micronuclei are normally circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes were counted;these cells were also scored of micronuclei.
The ratio of polychromatic to monochromatic erythrocytes was calculated together with appropriate group mean values.

Statistics:

Student's t-test and any significant results were confirmed using the one way analysis of variance.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

positive

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Mortality Data and clinical Signs

There were premature deaths seen in the 24 and 48 hour 500 mg/kg test material dose groups such that in both groups three animals died prematurely. It was considered that the integrity of the study was not compromised by the reduction in group size and that the result obtained was valid. Clinical signs were observed, particulary in animals dosed with the test material at 500 mg/kg, in both the 24 and 48 hour groups; these were as follows: hunched posture, vocalisation, occasional body tremors, decreased respiratory rate, dehydration, tonic convulsions, splayed gait, pilo-erection, clonic convulsions, ptosis, emaciation, lethargy and laboured respiration. It should be noted that the test material induced a greater level of toxicity to that observed in the range-finding toxicity study.

Evaluation of Bone Marrow Slides.

Statistically significant increases in the frequency of micronucleated PCEs were recorded for both the 24 and 48 -hour 500 mg/kg test material dose groups when compared to their concurreent vehicle control groups. In addition, unsually large frequencies of micronucleated NCEs were recorded for the 500 mg/kg 48 -hour group.

There was a statistically significant decrease in the PCE/NCE ratio in the 48 -hour test material group (500 mg/kg) when compared to the concurrent vehicle control group. This, in addition to the presence of clinical signs and premature deaths at 500 mg/kg was taken to indicate that exposure to the target tissue had been achieved. When the slides were scored it was found that one animal had very few monochromatic erythrocytes present, and none were recorded during the scoring of data.

The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

The tet material, Epiclon HP-4032, was found to produce a statistically significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice under the conditions of the test.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): positive
In conclusion, Epiclon HP-4032 was considered to be genotoxic under the conditions of the test.

Executive summary:

The study was performed to assess teh potential of the test material to produce damage to chromosomes or aneuploidy when administred to mice. The study design complies with the OECD Guidelines for testing of chemicals N° 474 "Micronucleus test" and Method B12 of the EEC Commission Directive 84/449/EEC.

A range-finding study was performed to find suitable dose levels adn route of administartion for the test material.The micronucleus study was conducted via the intraperitoneal route in groups of ten mice (5 males and 5 females) at the maximum tolerated dose (MTD) 500mg/kg with 125 and 250 mg/kg as the lower dose levels.Animals were killed 24 or 48 hours later, the bone marrow extracted and smear preparations made and stained. Polychromatic and normochromatic erythrocytes were scored for the presence of micronuclei.

Further groups of mice were given a single intraperitoneal dose of arachis oil or dosed orally with cyclophosphamide, to serve as vehicle and positive controls respectively.

There were statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test material at 500 mg/kg when compared to the concurrent vehicle control groups. A statistically significant decrease in the PCE/NCE ratio was observed in the 48 -hour 500 mg/kg test material dose group when compared to the concurrent control group. Furthermore, clinical signs were observed and prematurate deaths occured in the 24 and 48 -hour 500 mg/kg test material dose group; these observations were taken to indicate that exposure to the target tissue had been achieved.

The positive control material produced a marked increase in the frequency of micronucleated polychromatic erythrocytes.

The test material, EPICLON HP-4032, was considered to be genotoxic under the conditions of the test.