Reaction products of alkenyl dihydrofuran-2,5-dione and polyisoalkenyl dihydro-2,5-furandione with alkylene glycol

Genetic toxicity: in vivo

Some information in this page has been claimed confidential.

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:

25 July 1995 and 17 August 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

read-across: supporting information

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Safepharm Standard Method Number EEC 29A

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

liquid

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

- Sufficient male and female albino CD-1 Strain mice were supplied by Charles River (UK) Limited, Manston, Kent.
- At the start of the main study the males weighed 23 to 30g and the females 20 to 24g, and were approximately 5 to 7 weeks old.
- Minimum acclimatisation period of 5 days.
- The animals were housed in groups of up to 5 in solid-floor polypropylene cages with woodflake bedding.
- Free access to mains drinking water and food.
- The animal room was maintained at a temperature of 19 to 25°C and relative humidity of 50 to 65%.
- The rate of air exchange was approximately 15 changes per hour.
- Lighting was controlled by a time switch to give 12 hours light and 12 hours darkness.

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

Supplier's identification : Arachis oil
Supplier's batch number : 158
Saf-epharm serial number : CO/926
Date received : 7 June 1995
Description : straw coloured slightly viscous liquid
Storage conditions : room temperature

Details on exposure:

Groups, each of ten mice (five males and five females), were dosed once only via the intraperitoneal route with test material at 37.5, 75 or 150 mg/kg. One group of mice from each dose group was killed by cervical dislocation 24 hours following treatment and a second at 48 hours. In addition three further groups of ten mice (five males and five females) were included in the study; two groups were given a single intraperitoneal dose of the vehicle (arachis oil) and the third group was dosed orally with cyclophosphamide, a positive control material known to produce micronuclei under the conditions of the test. The vehicle control groups were killed 24 and 48 hours following dosing and positive control group animals were killed 24 hours following dosing.

Duration of treatment / exposure:

24 and 48 hours

Frequency of treatment:

once

Post exposure period:

No

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 37.5, 75, 150
Basis:
nominal conc.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Supplier's identification : Cyclophosphamide
Supplier's lot number : 73HO846
Safepharm serial number : CO/911
Date received : 17 May 1995
Description : white powder
Storage conditions : 4°C
The concentration, homogeneity and stability of the positive control material and its preparation were not determined by analysis.

Examinations

Tissues and cell types examined:

Bone marrow polychromatic erythrocytes were examined for the presence or absence of micronuclei.

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/Giemsa, dried and coverslipped using mounting medium.

Evaluation criteria:

Stained bone marrow smears were coded and examined ’blind’ using light microscopy at x1000 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 (NCE-pink stained mature cells) associated with 1000 polychromatic erythrocytes was 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 for males and females
separately and combined.

Statistics:

A positive response for bone marrow toxicity is demonstrated when the dose group mean polychromatic to normochromatic ratio is shown to be statistically significantly lower than the concurrent vehicle control group.
All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part lll (1989). The data was analysed following a*racine (x+1) transformation 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/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

clinical signs were observed in the RF study

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

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 groups.
A positive mutagenic response is demonstrated when a statistically significant increase in the number of micronucleated polychromatic erythrocytes is observed for either the 24 or 48-hour kill times when compared to their corresponding control group.
If these criteria are not demonstrated, then the test material is considered to be non-genotoxic under the conditions of the test.

Any other information on results incl. tables

There was no significant increase in the frequency of micronucleated PCEs in any of the test material dose groups (37.5, 75 and 150 mg/kg) when compared to their concurrent vehicle control groups.

There was no statistically significant change in the PCE/NCE ratio in any of the test material dose groups when compared to their concurrent vehicle control groups. However, the presence of clinical signs and premature deaths in the range-finding toxicity study indicated that systemic absorption did occur. The steep toxicity curve seen in the range-finding study resulted in the selection of a maximum tolerated dose level that was expected to produce clinical signs in the main study. The absence of clinical signs in the main study was considered not to affect its integrity or validity.

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.

Applicant's summary and conclusion

Conclusions:

The genetic toxicity of the test material was assessed in accordance with OECD Guideline 474, Mammalian Erythrocyte Micronucleus Test. The test item was considered to be non-genotoxic under the conditions of the test.

Executive summary:

A study was performed to assess the potential of the test material to produce damage to chromosomes or aneuploidy when administered via the intraperitoneal route to mice. The method followed was designed to comply with Method B12 of the EEC Commission Directive 92/69/EEC which constitutes Annex V of Council Directive 57/548/EEC.

Following a preliminary range-finding study to find a suitable dose level for the test material, the micronucleus study was conducted using the test material at the maximum tolerated dose level of 150 mg/kg with 75 and 37.5 mg/kg as the lower dose levels.

In the micronucleus study, groups of ten mice (five males and five females) were given a single intraperitoneal dose of the test material at 37.5, 75 or 150 mg/kg. 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 dosed via the intraperitoneal route with arachis oil or orally with cyclophosphamide, to serve as vehicle and positive controls respectively.

There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with test material when compared to the concurrent vehicle control groups. No significant change in the PCE/NCE ratio was observed after dosing with the test material. Although, the presence of clinical signs and premature cleaths during the preliminary range-finding study would indicate systemic absorption had occurred.

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

The test material was considered to be non-genotoxic under the conditions of the test.