Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
10 August 1998 - 9 September 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to standard protocol and under GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to guideline
Guideline:
other: US EPA (1997) Toxic Substances Control Act Test Guidelines; Title 40 Code of Federal Regulations Part 799.
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Reference substance name:
849705-80-2
Cas Number:
849705-80-2
IUPAC Name:
849705-80-2
Details on test material:
Identity: P-2290
Chemical name: 2-Oxetanone, 3-(C14-16 and C16-unsatd. branched and linear alkyl) 4-(C15-17 and C17-unsatd.branched and linear alkylidene) derivs
Appearance: waxy liquid
Storage conditions: Refridgerated at 4ºC (dark)
Batch number: 290
Purity: assumed to be 100%
Date of receipt at Huntingdon: 8 May 1998
Supplier: Raisio Chemical Oy
Stability of formulations: Stable for 15 days at 4ºC (dark)

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, England.
- Age at study initiation: no data
- Weight at study initiation: Males weighed between 28 and 30 grams and females weighed between 22 and 24 grams
- Assigned to test groups randomly: yes, under following basis: no data
- Fasting period before study: No
- Housing: Each group was kept, with the sexes separated, in plastic cages.
- Diet (e.g. ad libitum): All animals were allowed free access to pelleted expanded rat and mouse No.1 maintenance diet (SQC grade obtained from Special Diets Services Ltd, Witham, Essex, UK) , ad libitum.
- Water (e.g. ad libitum): tap water, ad libitum.
- Acclimation period: minumum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 50
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 10 August 1998 - 9 September 1998

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: solubility
- Concentration of test material in vehicle: depending on the dose of the main study: 25, 50, 100 mg/ml
- Lot/batch no. (if required): F8048
- Purity: no data
Duration of treatment / exposure:
A single dose was given. Following dosing, the animals were examined regularly and any mortalities or clinical signs of reaction were recorded. Five males and five females from the negative control, each of the test substance groups and the positive control group were sacrificed 24 hours after dosing. In addition five male and five female animals in the negative control and high level treatment groups were sacrificed 48 hours after dosing. Additional animals not used in the preparation of bone marrow smears were killed at the 48 hour time point.
Frequency of treatment:
Single dose
Post exposure period:
24 or 48 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1000 and 2000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
vehicle control and high dose group: 10
postive control, mid- and low dose group: 5
Control animals:
yes, concurrent vehicle
Positive control(s):
mitomycin C
- Justification for choice of positive control(s): standard from the guildeine
- Route of administration: administered orally by intragastric gavage
- Doses / concentrations: 12 mg/kg bw/0.6 mg/ml

Examinations

Tissues and cell types examined:
Bone marrow from the femur.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
In a preliminary toxicity test 2 male and 2 females animals were given 250, 500, 1000 mg/kg/bw. Following dosing, the animals were observed regularly during the working day for a period of 48 hours and any mortalities or clinical signs of reaction during the experiment were recorded. At the end of this observation period, surviving animals were sacrificed.Observed toxicty after doing was hunched posture and piloeraction. no mortalities. Results showed that a dose level of 2000 mg/kg (the standard limit), was considered to be the appropriate maximum for use in the micronucleus test. Dose levels of 500, 1000 and 2000 mg/kg bodyweight were chosen for use in the main test.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
No additional information.

DETAILS OF SLIDE PREPARATION:
The animals were killed by cervical dislocation following carbon dioxide inhalation and both femurs dissected out from each animal. The femurs were cleared of tissue and the proximal epiphysis removed from each bone. The bone marrow of both femurs from each animal was flushed out and
pooled in a total volume of 2 ml of pre-filtered foetal calf serum using a 2 ml disposable syringe fitted with a 21 gauge needle. The cells were sedimented by centrifugation, the supernatant was discarded and the cells were resuspended in a small volume of fresh serum. A small drop of the cell
suspension was transferred to a glass microscope slide and a smear was prepared in the conventional manner (Schmid 1976). At least three smears were made from each animal. The prepared smears were fixed in methanol (> 10 minutes). After air-drying the smears were stained for 20 minutes in 5% Giemsa prepared by 1 : 19 dilution of Gun's improved R66 Giemsa (BDH) with purified water. Following rinsing in purified water and differentiation in purified buffered water, the smears were air-dried and mounted with coverslips using DPX.

METHOD OF ANALYSIS:
The stained smears were examined (under code) by light microscopy to determine the incidence of micronucleated cells per 2000 polychromatic erythrocytes per animal. Usually only one smear per animal was examined. The remaining smears were held temporarily in reserve in case of technical problems with the first smear. The proportion of immature erythrocytes for each animal was assessed by examination of at least 1000 erythrocytes. A record of the number of micronucleated mature erythrocytes observed during assessment of this proportion was also kept as recommended by Schmid (1976).

OTHER: None
Evaluation criteria:
A positive response is normally indicated by a statistically significant dose-related increase in the incidence of micronucleated immature erythrocytes for the treatment group compared with the concurrent control group (P<0.01); individual and/or group mean values should exceed the laboratory historical control range (Morrison and Ashby 1995). A negative result is indicated where individual and group mean incidences of micronucleated immature erythrocytes for the group treated with the test substance are not significantly greater than incidences for the concurrent control group (P>0.01) and where these values fall within the historical control range. An equivocal response is obtained when the results do not meet the criteria specified for a positive or negative response. Bone marrow cell toxicity (or depression) is normally indicated by a substantial and statistically
significant dose-related decrease in the proportion of immature erythrocytes (P<0.01). This decrease would normally be evident at the 48 hour sampling time.
Statistics:
For incidences of micronucleated immature erythrocytes, exact one-sided p-values are calculated by permutation (StatXact, CYTEL Software Corporation, Cambridge, Massachussetts). Comparison of several dose levels are made with the concurrent control using the Linear by Linear Association test
for trend in a step-down fashion if significance is detected (Agresti et al. 1990); for individual intergroup comparisons (ie the positive control group) this procedure simplifies to a straight forward permutation test (Gibbons 1985). For assessment of effects on the proportion of immature erythrocytes, equivalent permutation tests based on rank scores are used, ie exact versions of Wilcoxon's sum of ranks test and Jonckheere's test for trend.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Piloerection and hunched posture
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 500, 1000 and 2000 mg/kg
- Solubility: no data
- Clinical signs of toxicity in test animals: piloerection and hunched posture
- Evidence of cytotoxicity in tissue analyzed: none
- Rationale for exposure: more direct exposure
- Harvest times: 48 hours
- High dose with and without activation: 2000 mg/kg bw
- Other: none


RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): None
- Induction of micronuclei (for Micronucleus assay): None
- Ratio of PCE/NCE (for Micronucleus assay): The test substance failed to cause any significant decreases in the proportion of immature erythrocytes.
- Appropriateness of dose levels and route: dosed up to the limit concentration, route of exposure as stated in the guideline
- Statistical evaluation: no significant effects observed

Any other information on results incl. tables

No mortalities occurred in any group in any part of the study.

PRELIMINARY TOXICITY TEST

Results showed that a dose level of 2000 mg/kg (the standard limit), was considered to be the appropriate maximum for use in the micronucleus test. Dose levels of 500, 1000 and 2000 mg/kg bodyweight were chosen for use in the main test.

MICRONUCLEUS TEST

Clinical signs and mortalities

No mortalities were obtained in the micronucleus test. Clinical signs for the high level group were consistent with the maximum tolerated dose having effectively been achieved. No adverse clinical signs were obtained for the vehicle control or positive control treated animals over the duration of the test.

Micronucleated immature erythrocyte counts (mie)

The test substance did not cause any statistically significant increases in the number of micronucleated immature erythrocytes at either sampling time (P>0.01). Mitomycin C caused large, highly significant increases (P<0.001) in the frequency of micronucleated immature erythrocytes.

Micronucleated mature erythrocytes (mme) The test substance did not cause any substantial increases in the incidence of micronucleated mature

erythrocytes at either sampling time.

Proportion of immature erythrocytes (% ie/ie + me)

The test substance failed to cause any significant decreases in the proportion of immature erythrocytes (P>0.0 1 ). Mitomycin C caused statistically significant decreases in the proportion (P<0.001).

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
No statistically significant increases in the frequency of micronucleated immature erythrocytes and no substantial decrease in the proportion of immature erythrocytes were observed in mice treated with P-2290 and killed 24 or 48 hours later, compared to vehicle control values (p>0.01 in each case). It is concluded that P-2290 did not show any evidence of causing chromosome damage or bone marrow cell toxicity when administered by intraperitoneal injection in this in vivo test procedure.
Executive summary:

This study was designed to assess the potential induction of micronuclei by P-2290 in bone marrow cells of mice. Mice were treated with a single intraperitoneal administration of the test substance at dose levels of 500, 1000 and 2000 mg/kg bodyweight. A preliminary toxicity test had previously

shown that a dose of 2000 mg/kg was expected to be approximately the maximum tolerated and standard limit; this level was therefore selected as an appropriate maximum for use in the micronucleus test.

The test substance and negative control were administered by intraperitoneal injection. The negative control group received the vehicle, corn oil. A positive control group was dosed orally, by intragastric gavage, with mitomycin C at 12 mg/kg bodyweight. Bone marrow smears were obtained from five male and five female animals in the negative control, each of the test substance groups and the positive control group 24 hours after dosing. In addition bone marrow smears were obtained from five male and five female animals in the negative control and high level treatment groups 48 hours after dosing. One smear from each animal was examined for the presence of micronuclei in 2000 immature erythrocytes. The proportion of immature

erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal. A record of the incidence of micronucleated mature erythrocytes was also kept. Mice treated with the test substance did not show any significant increase in the frequency of micronucleated immature erythrocytes at either sampling time. There was no significant decrease in the proportion of immature erythrocytes after treatment of the animals with the test substance. No statistically significant increases in the frequency of micronucleated immature erythrocytes or any substantial decrease in the proportion of immature erythrocytes were observed in mice treated with P-2290 and killed 24 or 48 hours later, compared to vehicle control values (p>0.01 in each case). The positive control compound, mitomycin C, produced large, highly significant increases in the frequency of micronucleated immature erythrocytes and a highly significant decrease in the proportion of immature erythrocytes (p<0.001 in each case). It is concluded that P-2290 did not show any evidence of causing chromosome damage or bone marrow cell toxicity when administered by intraperitoneal injection in this in vivo test procedure.