Indeno[4,3a-b]furan, decahydro-2,2,7,7,8,9,9-heptamethyl-

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:

8 May 2003 and 18 June 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo 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:

449-360-4

EC Name:

-

Cas Number:

647828-16-8

Molecular formula:

C18H32O

IUPAC Name:

decahydro-2,2,6,6,7,8,8-heptamethyl-2H-Indeno[4,5-b] furan

Test material form:

liquid

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS:
- Source: Charles River (UK) Limited, Margate, Kent
- Age at study initiation: 5-8 weeks old
- Weight at study initiation: 25 to 30g
- Assigned to test groups randomly: Selected at random and given a number unique within the study by ear punching and a number written on a colour coded cage card
- Housing: In groups of up to 7 in solid-floor polypropylene cages with woodflake bedding
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS:
- Temperature (°C): 19 to 25
- Humidity (%):30 to 70
- Air changes (per hr): approximately 15 changes per hour
- Photoperiod (hrs dark / hrs light): controlled by a time switch to give 12 hours light and 12 hours darkness

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

Arachis oil BP

Details on exposure:

Range-Finding Test:
A range-finding toxicity test was performed to determine a suitable dose level and route of administration for the micronucleus test. All animals were dosed once only at the appropriate dose level by gavage using a metal cannula or with a hypodermic needle attached to a graduated syringe. Animals were observed 1-hour after dosing and subsequently once daily for 2 days. Any deaths and evidence of overt toxicity were recorded at each observation. No necropsies were performed.

Micronucleus Test:
Groups, each of 7 mice, were dosed once only via the intraperitoneal route with the test substance at 2000 mg/kg. One group of mice was killed by cervical dislocation 24 hours following treatment and a second group was killed after 48 hours. In addition, 3 further groups of mice were included in the test; 2 groups (7 mice) were dosed via the intraperitoneal route with the vehicle alone (arachis oil, negative control) and a third group (5 mice) was dosed orally with cyclophosphamide (positive control). 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 1-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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Remarks:

Dose level (24 hours)

Doses / concentrations 2

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Remarks:

Dose level (48 hours)

No. of animals per sex per dose:

7

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide

Examinations

Tissues and cell types examined:

Polychromatic and normochromatic erythrocytes in bone marrow.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A range finding toxicity test was performed.

DETAILS OF 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.

METHOD OF ANALYSIS:
Stained bone marrow smears were coded and examined blind using light microscopy at x 1000 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 substance 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 substance 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 (ANOVA).

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Decrease in PCE/NCE ratio

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY:
- Dose range: The Maximum Recommended Dose (MRD) of the test substance was 2000 mg/kg.
- Clinical signs of toxicity in test animals: No marked difference in toxicity to male or female mice observed, both via intraperitoneal and oral route.
- Evidence of cytotoxicity in tissue analysed: No evidence of test substance toxicity demonstrated via either route of administration.
- Rationale for exposure: Based on absence of toxicity, the intraperitoneal route was selected for use in the main test in an attempt to maximise test substance exposure to the bone marrow.

RESULTS OF DEFINITIVE STUDY:
- Types of structural aberrations for significant dose levels: None observed
- Induction of micronuclei: There were no statistically significant increases in the frequency of micronucleated PCEs in any of the test substance dose groups when compared to their concurrent vehicle control groups.
- Ratio of PCE/NCE: 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.
- Appropriateness of dose levels and route: There were no premature deaths seen in any of the dose groups. No clinical signs were observed in animals dosed with the test substance at 2000 mg/kg in either the 24 or 48-hour groups.
- Results of positive control group: 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.

Any other information on results incl. tables

Table 1: Micronucleus Test – Summary of Group Mean Data

		Number PCE with micronuclei per 2000 PCE		PCE/NCE Ratio
	Sampling Time	Group Mean	SD	Group Mean	SD
Vehicle Control	48 hours	1.0	0.8	0.73	0.25
Vehicle Control	24 hours	2.1	2.0	0.90	0.31
Positive Control	24 hours	55.8***	20.4	1.16	0.14
2000 mg/kg	48 hours	1.3	2.1	0.57	0.20
2000 mg/kg	24 hours	2.9	2.5	0.59	0.31

PCE: Polychromatic erythrocytes, NCE: Normochromatic erythrocytes, SD: Standard deviation, ***: P<0.001

 

Applicant's summary and conclusion

Conclusions:

In an in vivo Mammalian Erythrocyte Micronucleus Test which was performed in accordance with OECD TG 474 and under GLP conditions, the test substance was considered to be non-genotoxic under the conditions of the test.

Executive summary:

An in vivo Mammalian Erythrocyte Micronucleus test was performed according to OECD TG 474 and under GLP conditions. The study was performed to assess the potential of the test substance to produce damage to chromosomes or aneuploidy when administered to mice. A range-finding test was performed to find suitable dose levels of the test substance, route of administration and investigate to see if there was a marked difference in toxic response between the sexes. There was no marked difference in test substance toxicity between the sexes; therefore, the main test was performed using only male mice. With no evidence of any toxicity with the test substance via either route of administration the micronucleus test was conducted using the intraperitoneal route to maximise exposure in groups of 7 mice (males) at the maximum recommended dose (2000 mg/kg) only. 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. No statistically significant decreases in the PCE/NCE ratio were observed in the 24 or 48-hour test substance dose groups when compared to their concurrent control groups. However, in both instances marked reductions were observed and 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 substance when compared to the 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. Based on the results of the test, the test substance was considered to be non-genotoxic under the conditions of the test.