Indeno[4,5-d]-1,3-dioxin, 4,4a,5,6,7,8,9,9b-octahydro-7,7,8,9,9-pentamethyl-

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:

The experimental phases of the study were performed between 2 April 2003 and 28 May 2003.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted according to OECD TG 474 and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

446-220-4

EC Name:

-

Cas Number:

365411-50-3

Molecular formula:

C16 H26 O2

IUPAC Name:

7,7,8,9,9-pentamethyl-2H,4H,4aH,5H,6H,7H,8H,9H,9bH-indeno[4,5-d][1,3]dioxine

Test material form:

other: solid

Details on test material:

Sponsor's identification: 2-Eyed Musk
Date received: 5 February 2003
Storage conditions: Room temperature, in the dark

For the purpose of this study the test material was freshly prepared as required as a solution at the appropriate concentration in arachis oil.

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

Sufficient albino Crl:CD-1 TM(ICR)BR strain mice were supplied by Charles River (UK) Limited, Margate, Kent. At the start of the main test the male mice weighed 24 to 29g and were approximately five to eight weeks old. After a minimum acclimatisation period of seven 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 seven in solid-floor polypropylene cages with woodflake bedding. Free access to mains drinking water and food (Certified Rat and Mouse Diet 5LF2, International Product Supplies, Wellingborough, Northants, UK) was allowed throughout the study.

The temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to 70 % respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. 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 Limited, as follows:

Supplier's identification: Arachis oil BP
Supplier's lot number: T63
Safepharm serial number: V-2508
Date received: 16 May 2002
Description: Straw coloured, slightly viscous liquid
Storage conditions: Room temperature

Details on exposure:

Groups, each of seven mice, were dosed once only via the intraperitoneal route with the test material at 250, 500 or 1000 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 1000 mg/kg was killed after 48 hours. In addition, three further groups of mice were included in the test; two groups (seven mice) were dosed via the intraperitoneal route with the vehicle alone (arachis oil) and a third group (five mice) was dosed orally with cyclophosphamide. Cyclophosphamide is a positive control material known to produce micronuclei under the conditions of the test. The vehicle controls were killed 24 or 48 hours following dosing and positive control group animals were killed 24 hours following dosing.

All animals were observed for signs of overt toxicity and death one hour after dosing and then once daily as applicable and immediately prior to termination.

Duration of treatment / exposure:

24 or 48 hours.

Frequency of treatment:

Once only.

Post exposure period:

Not applicable.

Doses / concentrations

Remarks:

Doses / Concentrations:
250, 500, 1000 mg/kg
Basis:
nominal conc.

No. of animals per sex per dose:

7 male mice per test material dose group.

Control animals:

yes, concurrent vehicle

Positive control(s):

The positive control material was supplied by Sigma Chemicals, as follows:

Supplier's identification: Cyclophosphamide
Supplier's lot number: 91K1176
Safepharm serial number: R-2670
Date received: 5 November 2002
Storage conditions: 4°C in the dark

For the purpose of this study the positive control material was freshly prepared as required as a solution at the appropriate concentration in distilled water (IV AX batch no. A3018).

Examinations

Tissues and cell types examined:

Polychromatic and normochromatic erythrocytes in bone marrow.

Details of tissue and slide preparation:

Slide Preparation:
Immediately following termination (i.e. 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, stained in May-Griinwald/Giemsa, allowed to air-dry and coverslipped using mounting medium.

Slide Evaluation:
Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification. The incidence of micronucleated cells per 2000 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 (NCE-pink stained mature cells) associated with 1000 erythrocytes were counted; these cells were also scored for incidence of micronuclei.

The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations.

Evaluation criteria:

A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test material groups and the number occurring in the corresponding vehicle control group.

A positive mutagenic response was demonstrated when a statistically significant, dose-responsive, toxicologically relevant increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48-hour kill times when compared to their corresponding control group.

If these criteria were not fulfilled, then the test material was considered to be non-genotoxic under the conditions of the test.

A positive response for bone marrow toxicity was demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the concurrent vehicle control group.

Statistics:

All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989). The data was analysed using Student's t-test (two tailed) and any significant results were confirmed using the one way analysis of variance.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Decrease in PCE/NCE ratio

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Range-finding Toxicity Test:
In animals dosed with test material via the intraperitoneal route there were no premature deaths, however the clinical signs observed were so severe that the animals were killed in extremis in the interests of animal welfare. Clinical signs were only observed at 2000 mg/kg, and they were as follows: loss of righting reflex, decreased respiratory rate, laboured respiration, ptosis, hypothermia, dehydration, occasional body tremors and increased salivation.

In animals dosed with the test material via the oral route no premature deaths or clinical signs were observed.

The test material showed no marked difference in its toxicity to male or female mice, it was therefore considered to be acceptable to use males only for the main test. Adequate evidence of test material toxicity was demonstrated via the intraperitoneal route of administration, therefore, this was selected for use in the main test. A Maximum Tolerated Dose (MTD) for the test material of 1000 mg/kg, was selected for use in the main test, with 250 and 500 mg/kg as the lower dose levels.

Micronucleus Test:
Mortality Data and Clinical Observations
There were no premature deaths or clinical signs seen in any of the dose groups.

Evaluation of Bone Marrow Slides
A summary of the results of the micronucleus test is given in attached Table 1. Individual and group mean data are presented in attached Tables 2 to 8.

Whilst there were no statistically significant decreases in the PCE/NCE ratio in the 24 or 48-hour test material groups when compared to their concurrent vehicle control groups, it should be noted that a marked decrease was observed in the 48-hour test material group. This was taken to indicate that systemic absorption had occurred and exposure to the target tissue, bone marrow, was achieved.

There were no statistically significant increases in the frequency of micronucleated PCEs in any of the test material dose groups when compared to their concurrent vehicle control groups.

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 test material was found not to produce a significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice under the conditions of the test.

Any other information on results incl. tables

Range-finding Toxicity Test

In animals dosed with test material via the intraperitoneal route there were no premature deaths, however the clinical signs observed were so severe that the animals were killed in extremis in the interests of animal welfare. Clinical signs were only observed at 2000 mg/kg, and they were as follows: loss of righting reflex, decreased respiratory rate, laboured respiration, ptosis, hypothermia, dehydration, occasional body tremors and increased salivation.

In animals dosed with the test material via the oral route no premature deaths or clinical signs were observed. The test material showed no marked difference in its toxicity to male or female mice, it was therefore considered to be acceptable to use males only for the main test. Adequate evidence of test material toxicity was demonstrated via the intraperitoneal route of administration, therefore, this was selected for use in the main test.

A Maximum Tolerated Dose (MTD) for the test material of 1000 mg/kg, was selected for use in the main test, with 250 and 500 mg/kg as the lower dose levels.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
The mutagenicty potential of the test material was assessed according to OECD Guideline 474. The test material was considered to be non-genotoxic under the conditions of the test.

Executive summary:

The study was performed to assess the potential of the test material to produce damage to chromosomes or aneuploidy when administered to mice according to OECD TG 474. The micronucleus test was conducted using the intraperitoneal route in groups of seven mice (males) using the maximum tolerated dose (MID) 1000 mg/kg with 250 and 500 mg/kg as the two lower dose levels. Animals were killed 24 or 48 hours later, the bone marrow was extracted, and smear preparations made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei. Further groups of mice were given a single intraperitoneal dose of arachis oil (7 mice) or dosed orally with cyclophosphamide (5 mice), to serve as vehicle and positive controls respectively. Vehicle control animals were killed 24 or 48 hours later, and positive control animals were killed after 24 hours. Whilst no statistically significant decreases in the PCE/NCE ratio were observed in the 24 or 48-hour test material dose groups when compared to their concurrent control groups, it should be noted that a marked reduction was observed in the 48-hour 1000 mg/kg test material group. With no premature deaths or clinical signs being observed this was taken to indicate that systemic absorption had occurred. There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test material when compared to the concurrent vehicle control groups. The test material was considered to be non-genotoxic under the conditions of the test.