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:
27 December 1999 to 15 February 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Principles of method if other than guideline:
The study protocol states that it was written to comply with the standardised guideline OECD 474.
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Appearance: Light yellow liquid, Clear Liquid
- Stability: There was no apparent change in the physical state of the test material during administration
- Storage conditions of test material: Room temperature, protected from exposure to light

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 6 to 8 weeks old
- Weights at study initiation: Weights at randomisation were within the following ranges: Males 27.5 - 31.5 g and females 25.2 - 27.5 g (pilot toxicity study); males 26.2 - 33.5 g and females 25.6 - 29.5 g (toxicity study); males 30.3 - 34.5 g and females 26.2 - 28.7 g (supplemental toxicity study); and males 29.1 - 33.5 g and females 25.4 - 30.0 g (micronucleus study)
- Assigned to test groups randomly: Yes. In the micronucleus assay, mice were assigned to experimental groups according to a computer-generated program which is based on distribution according to body weight.
- Fasting period before study: No
- Housing: Housed in an AAALAC-accredited facility with a controlled environment. Mice of the same sex were housed up to five per cage in polycarbonate cages which were maintained on stainless steel racks equipped with automatic watering manifolds and which were covered with filter material. Heat-treated hardwood chips were used for bedding.
- Diet: ad libitum certified laboratory rodent chow
- Water: ad libitum tap water
- Acclimation period: no less than 5 days of quarantine

ENVIRONMENTAL CONDITIONS
- Temperature: 72 ± 3 °F
- Humidity: 50 ± 20 % relative humidity
- Photoperiod: 12 hour light/dark cycle

IN-LIFE DATES
From: 21 December 1999 (pilot study); 04 January 2000 (toxicity study); 18 January 2000 (supplemental toxicity study); and 25 January 2000 (micronucleus study)
To: No data

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: Corn oil
- Justification for choice of solvent/vehicle: Determined to be the solvent of choice based on information provided by the Sponsor and compatibility of the vehicle with the test system animals. The test material was soluble in corn oil at 100 mg/mL.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Test material, vehicle and positive control were administered at a constant volume of 20 mL/kg body weight.
Duration of treatment / exposure:
- Pilot study, toxicity assay and the supplemental toxicity study: 3 days
- Micronucleus study: 24 and 48 hours
Frequency of treatment:
Dosed once through a single intraperitoneal injection
Post exposure period:
In the pilot toxicity study, toxicity assay and the supplemental toxicity study, mice were observed after dose administration and daily thereafter for 3 days for clinical signs of chemical effect. Body weights were recorded prior to dose administration and 1 and 3 days after dose administration.
For the micronucleus assay, bone marrow was collected from 5 animals per sex per dose group for all dose levels after 24 hours post dose and from 5 animals per sex from the vehicle control and high dose groups after 48 hours post dose.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
Pilot toxicity study: male mice were dosed with 1, 10, 100 or 1000 mg test material/kg body weight and male and female mice were dosed with 2000 mg/kg.
Basis:
other: actual administered
Remarks:
Doses / Concentrations:
Toxicity assay: male and female mice were dosed with 1200, 1400, 1600 or 1800 mg test material/kg body weight.
Basis:
other: actual administered
Remarks:
Doses / Concentrations:
Supplemental toxicity study: male and female mice were dosed with 1000 mg test material/kg body weight.
Basis:
other: actual administered
Remarks:
Doses / Concentrations:
Micronucleus assay: male and female mice were dosed with 181, 362 or 725 mg test material/kg body weight.
Basis:
other: actual administered
No. of animals per sex per dose:
5 animals per sex per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
- Cyclophosphamide dissolved in sterile distilled water at a concentration of 2.5 mg/mL.
- Route of administration: Administered in a constant volume of 20 mL/kg body weight by a single intraperitoneal injection.
- Doses / concentrations: 50 mg/kg

Examinations

Tissues and cell types examined:
Bone marrow erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION
The LD50/3 in the supplemental toxicity study was calculated by probit analysis to be approximately 1034.5 mg/kg for male and female mice. The high dose for the micronucleus test was set at 725 mg/kg for male and female mice which was estimated to beapproximately70 % of the LD50/3.

DETAILS OF SLIDE PREPARATION
At the scheduled sacrifice times, five mice per sex per treatment were sacrificed by CO2 asphyxiation. Immediately following sacrifice, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing foetal bovine serum. The bone marrow cells were transferred to a capped centrifuge tube containing approximately 1 mL foetal bovine serum. The bone marrow cells were pelleted by centrifugation at approximately 100 x g for five minutes and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were re-suspended by aspiration with a capillary pipette and a small drop of bone marrow suspension was spread onto a clean glass slide. Two to four slides were prepared from each mouse. The slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted.

METHOD OF ANALYSIS - SCORING FOR MICRONUCLEI
Using medium magnification, an area of acceptable quality was selected such that the cells were well spread and stained. Using oil immersion, 2000 polychromatic erythrocytes were scored for the presence of micronuclei which are defined as round, darkly staining nuclear fragments, having a sharp contour with diameters usually from 1/20 to 1/5 of the erythrocyte. The number of micronucleated normochromatic erythrocytes in the field of 2000 polychromatic erythrocytes was enumerated. The proportion of polychromatic erythrocytes to total erythrocytes was also recorded per 1000 erythrocytes.
Evaluation criteria:
EVALUATION OF TEST RESULTS
The incidence of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes was determined for each mouse and treatment group. All analyses were performed separately for each sex and sampling time.
In order to quantify the proliferation state of the bone marrow as an indicator of bone marrow toxicity, the proportion of polychromatic erythrocytes to total erythrocytes was determined for each animal and treatment group.
All conclusions were based on sound scientific judgement; however, as a guide to interpretation of the data, the test material was considered to induce a positive response if a dose-responsive increase in micronucleated polychromatic erythrocytes was observed and one or more doses were statistically elevated relative to the vehicle control (p≤0.05, Kastenbaum-Bowman Tables) at any sampling time. If a single treatment group was significantly elevated at one sacrifice time with no evidence of a dose-response, the assay was considered a suspect or unconfirmed positive and a repeat assay recommended. The test material was considered negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control was observed at any sampling time.

CRITERIA FOR A VALID TEST
The mean incidence of micronucleated polychromatic erythrocytes must not exceed 5/1000 polychromatic erythrocytes (0.5 %) in the vehicle control. The incidence of micronucleated polychromatic erythrocytes in the positive control group must be significantly increased relative to the vehicle control group (p≤0.05, Kastenbaum-Bowman Tables).
Statistics:
Statistical significance of the incidence of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes was determined using the Kastenbaum-Bowman tables which are based on the binomial distribution (Kastenbaum and Bowman, 1970).

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
PILOT STUDY
Mortality occurred after dose administration in 5/5 males and 5/5 females at 2000 mg/kg. Clinical signs which were noted following dose administration included lethargy, piloerection, irregular breathing and crusty eyes in male mice at 1000 mg/kg and convulsions in males and females at 2000 mg/kg. All other animals appeared normal throughout the observation period.

TOXICITY ASSAY
Mortality occurred within two days of dose administration in 4/5 males and 5/5 females at 1200 mg/kg and all males and females at 1400, 1600 and 1800 mg/kg. Clinical signs which were noted after dose administration included convulsions in all animals at all dose levels and prostration, irregular breathing and crusty eyes in males and females at 1200, 1400, 1600 and 1800 mg/kg. Lethargy and piloerection were observed in one surviving male mouse at 1200 mg/kg.

SUPPLEMENTAL TOXICITY STUDY
Mortality was observed in 2/5 female mice. Clinical signs following dose administration included prostration, irregular breathing, crusty eyes, tremors, lethargy and piloerection in males and females at 1000 mg/kg.

MICRONUCLEUS ASSAY
One female mouse receiving 725 mg/kg was found dead on the day following dose administration and was replaced at the time of bone marrow collection with an animal from a replacement group which had also been dosed with 725 mg/kg. Mortality in the other test material-treated groups was not observed. Clinical signs which were noted after dose administration included lethargy and piloerection in males and females at 362 and 725 mg/kg and prostration and irregular breathing in animals at 725 mg/kg. All other mice treated with the test and control materials appeared normal during the study.
Slight reductions of 3 to 12 % in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test material-treated groups relative to their respective vehicle controls. Reductions were observed in female dose groups 24 hours after treatment with 181, 362 and 725 mg/kg and in the male dose group 48 hours after treatment with 725 mg/kg. The number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes in test material-treated groups was not statistically increased relative to their respective vehicle controls in either male or female mice, regardless of dose level or bone marrow collection time (p>0.05, Kastenbaum-Bowman Tables). Cyclophosphamide (positive control) induced a significant increase in micronucleated polychromatic erythrocytes in both male and female mice (p≤0.05, Kastenbaum-Bowman Tables).

Any other information on results incl. tables

Table 1: Summary of Bone Marrow Micronucleus Study

Treatment (mg/kg)

Sex

Time (hours)

No. of Mice

PCE/Total Erythrocytes (Mean ± SD)

Change from Control (%)

Micronucleated Polychromatic Erythrocytes

No./1000 PCEs (Mean ± SD)

No./PCEs Scored

Vehicle

M

24

5

0.483 ± 0.04

-

0.3 ± 0.27

3/10 000

F

5

0.568 ± 0.03

-

0.9 ± 0.55

9/10 000

181

M

5

0.487 ± 0.11

1

0.1 ± 0.22

1/10 000

F

5

0.549 ± 0.04

-3

0.6 ± 0.22

6/10 000

362

M

5

0.535 ± 0.04

11

0.5 ± 0.00

5/10 000

F

5

0.529 ± 0.04

-7

0.4 ± 0.42

4/10 000

725

M

5

0.546 ± 0.06

13

0.3 ± 0.27

3/10 000

F

5

0.536 ± 0.05

-6

0.5 ± 0.35

5/10 000

CP 50

M

5

0.571 ± 0.05

18

25.6 ± 5.24

256/10 000*

F

5

0.549 ± 0.09

-3

18.3 ± 6.47

183/10 000*

Vehicle

M

48

5

0.615 ± 0.04

-

0.2 ± 0.27

2/10 000

F

5

0.553 ± 0.03

-

0.3 ± 0.45

3/10 000

725

M

5

0.540 ± 0.07

-12

0.0 ± 0.00

0/10 000

F

5

0.554 ± 0.04

0

0.5 ± 0.50

5/10 000

*p≤0.05 (Kastenbaum-Bowman Tables)

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study, the test material was concluded to be negative in the micronucleus test using male and female mice.
Executive summary:

A mammalian erythrocyte micronucleus test in ICR mice was conducted to assess the toxicity of the test material using a protocol written to comply with the standardised guideline OECD 474 under GLP conditions.

The assay was performed in two phases. The first phase, designed to set dose levels for the definitive study, consisted of a pilot toxicity study followed by a toxicity study and supplemental toxicity study. The second phase, the micronucleus study, evaluated the potential of the test material to increase the incidence of micronucleated polychromatic erythrocytes in bone marrow of male and female mice. In both phases of the study, test and control materials were administered in a constant volume of 20 mL/kg body weight through single intraperitoneal injection. Corn oil was determined to be the solvent of choice based on compatibility of the vehicle with the test system animals..

In the pilot toxicity study, male mice were dosed with 1, 10, 100 or 1000 mg test material/kg body weight and male and female mice were dosed with 2000 mg/kg (5 animals per sex per dose level). Mortality was observed in 5/5 male mice and 5/5 female mice at 2000 mg/kg. Clinical signs following dose administration included lethargy, piloerection, crusty eyes and irregular breathing in all males at 1000 mg/kg and convulsions in all males and females at 2000 mg/kg.

In the toxicity assay, male and female mice were dosed with 1200, 1400, 1600 or 1800 mg test material/kg body weight (5 animals per sex per dose level). Mortality was observed in 4/5 male mice and 5/5 female mice at 1200 mg/kg and in all males and females at 1400, 1600 and 1800 mg/kg. Clinical signs following dose administration included convulsions in all animals at all dose levels and prostration, irregular breathing and crusty eyes in males and females at 1200, 1400, 1600 and 1800 mg/kg. Lethargy and piloerection were observed in one surviving animal at 1200 mg/kg. Due to high mortality at 1200 mg/kg and clinical signs at 1000 mg/kg (pilot study), a supplemental toxicity study was performed using the test material at a dose level of 1000 mg/kg.

In the supplemental toxicity study, male and female mice were dosed with 1000 mg test material/kg body weight (5 animals per sex per dose level). Mortality was observed in 2/5 female mice. Clinical signs following dose administration included prostration, irregular breathing, crusty eyes, tremors, lethargy and piloerection in males and females at 1000 mg/kg. The high dose for the micronucleus test was set at 725 mg/kg which was estimated to be approximately 70 % of the LD50/3.

In the micronucleus assay, male and female mice were dosed with 0, 181, 362 or 725 mg test material/kg body weight (15 animals per sex per dose). A concurrent positive control group was dosed with cyclophosphamide. Mortality was observed in 1/15 female mice receiving 725 mg/kg. Clinical signs following dose administration included lethargy and piloerection in male and female mice at 362 and 725 mg/kg and prostration and irregular breathing in males and females at 725 mg/kg. Bone marrow cells, collected 24 and 48 hours after treatment (5 animals per sex per dose per time interval), were examined microscopically for micronucleated polychromatic erythrocytes. Slight reductions (up to 12 %) in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test material-treated groups relative to the respective vehicle controls. These reductions suggest that the test material did not inhibit erythropoiesis. No significant increase in micronucleated polychromatic erythrocytes in test material-treated groups relative to the respective vehicle control groups was observed in male or female mice at 24 or 48 hours after dose administration.

CP induced a significant increase in micronucleated polychromatic erythrocytes in both male and female mice.

Under the conditions of the study, the test material did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow and was concluded to be negative in the micronucleus test using male and female mice.