Registration Dossier

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:
1993-11-11 till 1994-02-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant; guideline study; available as unpublished report; ne restrictions, fully adequate for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Order No. 31-11862-00 (99.3% purity) from Kymi Paper Mills

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
Male and female CD-1 mice were supplied by Harlan Olac Limited, Shaw's Farm, Blackthorn, Bicester, Oxfordshire. On arrival from the supplier, all mice were examined for signs of disease or ill health. Any animal judged unsuitable for testing was automatically excluded from the study. The mice were housed individually in non-SPF conditions in a room with a 12 h light -dark cycle. Animals were kept individually in polypropylene and stainless steel cages measuring 48 cm x 15 cm x 13 cm . White wood shavings provided the bedding in the cages. Benches were washed and floors were swept and disinfected with a mop impregnated with 0.5% Tego 2000 (Th Goldschmidt & Company Limited, Middlesex, England), an ampholytic detergent, at least once each day during the experiments . Walls were washed with Tego and cage racks and water bottles changed on a weekl y basis during the animal housing period .

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Immediately prior to dosing, the test compound was dissolved in corn oil to give the required test concentration . The dose volume used for both the control and test compound treated animals was a constant 10 ml.kg-1 body weight .
Food and water were freely available to the mice at all times except for a brief 2-3 h period prior to dosing when food was withheld . The diet used was SDS Rat and Mouse Maintenance Diet No. 1 obtained from Special Diet Services Limited, Witham, Essex , England.
Frequency of treatment:
Dosing 0 h + 24 h
Post exposure period:
DRF: 6 day
main test: 2 day
micronucleus test: 0 h
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
400 mg.kg-1.day-1
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
800 mg.kg-1.day-1
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
1600 mg.kg-1.day-1
Basis:
nominal in diet
No. of animals per sex per dose:
DRF:
6 groups of 1 male and 1 female; 50 - 125 - 350 - 800 - 2000 - 5000 mg.kg-1.day-1
Main test:
3 groups of 3 male and 3 female; 2000 - 3000 - 4000 mg.kg-1.day-1
Micronucleus test:
400 mg.kg-1.day-1: 5 male and 5 female
800 mg.kg-1.day-1: 5 male and 5 female
1600 mg.kg-1.day-1: 8 male and 8 female
Control animals:
yes, concurrent vehicle
Positive control(s):
One control group of male and female CD-1 mice received the positive control agent, 50 mg cyclophosphamide.kg-1.day-1

Examinations

Tissues and cell types examined:
polychromatic erythrocytes (PCE)
Details of tissue and slide preparation:
Mice were killed by cervical dislocation . The iemora were quickly dissected out and freed of adherent tissue . A small hole was made in the neck of onefemur and the marrow flushed, using a 1 ml syringe fitted with a gauge 25 needle, into a centrifuge tube containing 3 ml of a 1:1 mixture of foetal calserum and 0.8% trisodium citrate in Sorensen's buffer, pH 6 .8 (Sorensen's buffer, pH 6 .8 = 2 .84 g Na2HPO4.L-1 plus 2 .72 g KH2P04.L-1 distilled water). This mildly hypotonic treatment served to make the micronuclei clearly visible and to distinguish them from surrounding artefacts. Following completion of the sampling procedure the contents of the tubes were briefly agitated on a vortex mixer to allow separation of the cells. The tubes were centrifuged for 5 min at 1000 r.p.m. to pellet the cells . All but a few drops of supernatant fluid were discarded. The cells were then resuspended on a vortex mixer in this residual amount of supernatant liquid. Clean slides were assigned numbers corresponding to the tube numbers. A drop of the suspension was placed at one end of the slide and a smear made by drawing the top of a Pasteur pipette horizontally along the slide. Two slides were prepared from each tube and animal. The smear was left to air dry, fixed in methanol for ca 5 min and immersed for 30 min in modified Wright stain (0.3 % w/v buffered at pH 6.9 in methanol) to give optimum erythrocyte discrimination. The stained smears were rinsed in 2-3 changes of distilled water and air dried. Permanent slide preparations were made by sealing glass coverslips onto the microscopic slides using DPX mounting medium.
Evaluation criteria:
1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei and the frequency of micronucleated cells (MN-PCE) determined .As a control against inclusion of artefacts, or action of a mutagen on the G2 and/or mitotic phase of the cell cycle, the number of micronucleated normochromatic erythrocytes (MN-NCE) in mature red blood corpuscles were also recorded.a mean frequency of MN-PCE of 0.128% per mouse and 0.122% in CD-1 mice for both sexes combined.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
However, in the high dose group males a slight increase in MN-PCE to 0.28% was noted. This micronucleus frequency was marginally outside the established control range of 0.00-0.25% for a random group of 8 mice . The response was therefore classified as equivocal in accordance with protocol defined criteria. To clarify whether the obtained increase was due to a biological chance event, it was recommended to the Sponsor that a repeat analysis of the high dose males should be performed using a greater sample size of cells.The additional assessment was performed on re-randomised slides from the high, vehicle and positive control group. Both analyses were performed by the same assessor and a total of 2000 polychromatic erythrocytes per mouse examined for micronuclei. The observed frequency of MN-PCE (0. 10%) in the high dose from this additional assessment was well within the normal negative response range. The vehicle (0.09%) and positive control (1.17 % ) groups showed MN-PCE frequencies which were similar to
the first assessment. No bone marrow toxicity was detected in the 2-ethylhexanoic acid exposed mice, as the PCE/NCE ratios were of a normal order for all 3 test material groups. It was concluded that 2-ethvlhexanoic acid did not induce micronuclei in bone marrow when given as maximum tolerated oral doses to male and female mice.

Applicant's summary and conclusion