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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Modern guideline and GLP-compliant study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EPA OPP 84-2
Principles of method if other than guideline:
The experimental procedures described in this study comply with OECD Guideline No. 474 adopted 21 July 1997), ICH Guidelines, EC Guideline B.12, US EPA Pesticide Assessment Guidelines Subdivision F Series 84-2, and Japanese MHLW Guidelines on Genotoxicity Testing (Notification No. 1604 of the Pharmaceutical Affair Bureau of MHW, November 1st, 1999). The described test also follows recommendations published by the US Environmental Protection Agency Gene-Tox Program and the Japanese Collaborative Study Group for Micronucleus Testing (Mavournin et al 1990; CSGMT/JEMS.MMS, 1990).
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid
Details on test material:
Polyol-PX, Batch No. 391191549(B) was received at Charles River on 28 July 2009. The test item, a dark amber, waxy solid, was stored at room temperature and protected from light when not in use. The test item had an expiry date of 15 May 2014. A copy of the certificate of analysis was supplied by the sponsor.

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 Charles River UK, (Kent, England). All mice were housed in suspended polycarbonate cages (36.5 x 20.7 x 14.0 cm) with stainless steel tops. Sterilised white wood shavings and nestlets provided the bedding in the cages. Cages were changed on a weekly basis. The room was designed to provide at least 15 air changes per hour and had automatic control of temperature, humidity and lighting, with
a 12 h light/dark cycle. 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 0.5% Tego 2000 and water bottles changed on a weekly basis during the animal housing period. The mice were individually identified soon after arriving from the suppliers using an ear punching system routinely used at Charles River.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.

Food was freely available to the mice, except for ca 2-4 h prior to dosing and 1-2 h after dosing. The diet used was SDS Rat and Mouse Maintenance Diet No. 1 which was obtained from Special Diet Services Limited, Witham, Essex, England.

Tap water was available ad libitum. The water used at Charles River Laboratories is analysed by the local water authority at 6 month intervals for dissolved materials, heavy metals, pesticide residues, pH, nitrates and nitrites.

All the animals were under the care of Charles Rivers’ clinical veterinary surgeons who were available at all times to provide advice and assistance. Any treatment used to prevent or control intercurrent diseases was implemented at the discretion of the Study Director. Records of any affected individual animals were maintained and include date of first observation and duration of the condition, the nature and dates of the treatment administered
and the outcome of the treatment in relation to the disease and to the test results.

For the micronucleus test, cages were arranged in sequences, each sequence containing one cage of each treatment group. Computer-generated random numbers were used to determine the order of these sequences. The male mice were individually housed and the female mice were group housed in sets of two or three.The high dose group of mice consisted of an increased group size of 7 males and 7 females of
which 5 males and 5 females provided the regular assessment base. The additional mice were processed in normal fashion and the slides labelled with the original animal number. The slides from this spare group were kept as a contingency in case of unscheduled deaths or potential sex differences. In the event of death, the first available animal in the relevant contingency group replaced the missing animal. Preparations were made from remaining
contingency group animals and the slides were kept as spares. For the main micronucleus test, the coding procedure was completed by giving test tubes random numbers from a computer-generated numerical sequence, 201-225. From this stage onwards, the operators were therefore unaware of the dose group and treated animal from which the sample had been taken. The randomisation code was only broken when the last slide had been assessed at which point the generated data were computer-sorted and then tabulated as relevant.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
The test item was dissolved in water to give the required dosing concentrations. Due to the availability of formulation stability, dose formulations were prepared in advance and stored in the fridge when not in use. The dose volume used for both the control and test item treated animals was a constant 10 mL/kg body weight.

Cyclophosphamide was prepared fresh as a 5 mg/mL solution in distilled water. It was administered to the positive control animals in dose volumes of 10 mL/kg to give the required target dose of 50 mg/kg.
Details on exposure:
Groups of CD-1 mice were dosed orally at 0 h and 24 h with test or control materials, then marrow samples taken 24 h later
Duration of treatment / exposure:
Oral exposure (gavage)
Frequency of treatment:
Twice (0h, 24h)
Post exposure period:
24 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 2000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5/sex (7/sex in the 2000 mg/kg bw group)
Control animals:
yes, concurrent vehicle
Positive control(s):
50 mg Cyclophosphamide/kg (males only)

Examinations

Tissues and cell types examined:
Femoral bone marrow PCEs
Details of tissue and slide preparation:
Mice were killed by cervical dislocation. One femur of each mouse was quickly dissected out and freed of adherent tissue. A small hole was made in the neck of one femur 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 calf serum and 0.8% trisodium citrate in Sorensen's buffer, pH 6.8 (Sorensen's buffer, pH 6.8 = 2.84 g Na2HPO4/L plus 2.72 g KH2PO4/L distilled water). Routine tissue culture antibiotics were included to prevent microbial growth. 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 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/animal. The smear was left to air dry, fixed in methanol for ca 5 min and then immersed for 15 min in 15% Giemsa stain, prepared in tap water, to give optimum erythrocyte discrimination. The stained smears were finally rinsed in water for ca 1 min and left to air dry overnight. Permanent slide preparations were made by sealing coverslips onto the glass slides using DPX mounting medium. The better of the 2 prepared slides was selected for examination and the coded slides assessed blind by the same operator. At least two thousand (2000) 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 numbers of micronucleated normochromatic erythrocytes (MNNCE) in mature red blood corpuscles were also recorded (Maier and Schmid, 1976; Hamoud et a , 1989). In addition, scored micronuclei were assigned on the basis of size into small or large categories, historically defined as micronuclei occupying less or more than 25% of the visible cellular area. This classification provided a non-specific measure of compound induced spindle dysfunction, as large micronuclei appear to derive from lagging chromosomes caused by damage to the mitotic apparatus during bone marrow erythropoiesis (Yamamoto and Kikuchi, 1980; Vanderkerken et al, 1989). The PCE/NCE ratio, a measure of any induced systemic toxicity, was determined by counting a minimum total of 1000 erythrocytes (PCE + NCE) per marrow preparation.A suitable binocular microscope was used for the assessment. The scoring was done under a nominal magnification of x 1250 using x 12.5 magnification eye pieces and a x 100 oil immersion objective.
Evaluation criteria:
The average micronucleus incidence in vehicle control dosed and untreated CD-1 mice, has in this laboratory been determined as 0.05±0.06% , a range of 0.00-0.23% per group of 5 animals and 0.00-0.18% per group of 10 animals (Appendix 3). This frequency is in published data for micronucleus tests with CD-1 mice.

The test would be judged negative if no biologically relevant increases in the numbers of MNPCE were observed, relative to the concurrent and established historical control frequencies for MN-PCE induction.

The test would be judged positive if an increase in the number of micronucleated polychromatic erythrocytes (MN-PCE) was obtained for one or more of the test item treated dose groups. That is, an increase greater than 10% over the expected historical control ranges for a group of animals. The increase observed should be biologically relevant and statistically significant relative to concurrent and historical control frequencies for MN-PCE and/or MNNCE induction.

The test would be considered inconclusive if the levels of MN-PCE within any one dose group were increased above the established historical control frequencies for MN-PCE induction, but not high enough to meet the criteria for a positive response. That is an increase up to 10% over the maximum negative control frequency for a group of animals.
Statistics:
No statistical analysis was performed as the levels of MN-PCE induction fell within the determined historical control frequencies.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The prepared slides had uniform staining properties and sufficient number of PCE cells present to allow accurate micronucleus determination. The assay was considered acceptable as the MN-PCE frequencies for the vehicle control dosed mice were within the expected historical range. The ranges are defined in accordance with Charles Rivers experience of the bone marrow micronucleus test using CD-1 mice. The third requirement for an acceptable assay is an adequate positive control response for at least 2 animals and the dose group as a whole. This criterion was also met in this micronucleus test.

Any other information on results incl. tables

No animal deaths occurred in the vehicle control and test item group. There were also no clinical signs in these groups. In the positive control group, animal 127M displayed an extreme response after dosing on day 2. Due to the severity and unexpected nature of the response (positive control animals typically show no adverse reactions throughout the study) the animal was humanely killed and sent for post mortem examination. Necropsy findings indicated that there was a rupture at the thoracic inlet, suggesting that the animal had been inadvertently damaged during the dosing procedure.

The numbers of micronucleated bone marrow polychromatic erythrocytes (MN-PCE) in mice dosed with the vehicle, 10 mL water/kg, averaged 0.02%. This MN-PCE frequency conformed to the established in-house control range for vehicle treated mice of the CD-1 strain (0.00-0.18% per 10 mice).

Exposure of mice to the positive control agent, 50 mg cyclophosphamide/kg, induced large increases in bone marrow micronuclei. The mean MN-PCE frequency for the mice was 0.89%. An evident increase in the number of MN-NCE was also observed. Bone marrow toxicity accompanied these findings as shown by a suppression of the PCE/NCE ratios.

There was no indication that Polyol-PX induced bone marrow micronuclei in the treated mice. The highest MN-PCE frequency recorded for the test item was for males where an incidence of 0.04% was observed.

Summary of findings

Group

Micronucleated PCEs (%)

M

F

M+F

-Control

0.04

0.01

0.02

Polyol PX 2000 mg/kg bw/d

0.04

0.02

0.03

Cyclophosphamide

 

 

0.89

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
It is concluded that Polyol-PX did not induce micronuclei in bone marrow cells when tested to the maximum recommended dose of 2000 mg/kg/day in male and female CD-1 mice using a 0 h + 24 h oral dosing and 48 h sampling regimen.
Executive summary:

The in vivo genotoxic potential of Polyol-PX was evaluated in a micronucleus test in bone marrow erythrocytes of young male and female CD-1 mice following a 0 h + 24 h oral dosing and 48 h sampling regimen. A toxicity study was undertaken to establish a suitable dose range for the micronucleus experiment. Based on the findings of the toxicity study, the maximum recommended dose of 2000 mg Polyol-PX/kg bw/day was judged to be non-toxic and was selected for the main micronucleus test. In the micronucleus test, a group of CD-1 mice were therefore dosed at 0 h and 24 h via the oral route with the test item at a concentration of 2000 mg/kg/day. Bone marrow samples were taken 48 h after the initial 0 h dose. Two control groups of CD-1 mice were dosed orally with the vehicle, 10 mL water/kg/day, or the positive control agent, 50 mg cyclophosphamide/kg/day. The experimental schedule for the control groups followed that of the test item treated mice. No micronucleus induction was detected in bone marrow erythrocytes of mice dosed with Polyol-PX. Animals treated with the vehicle alone showed normal background levels of micronuclei, while animals dosed with cyclophosphamide responded with substantial increases in the numbers of bone marrow micronuclei. It was concluded that Polyol-PX did not induce micronuclei in bone marrow cells when tested to the maximum recommended dose of 2000 mg/kg/day in male and female CD-1 mice using a 0 h + 24 h oral dosing and 48 h sampling regimen.