Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 January 2001 and 4 March 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report date:
2001

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
EPA OPP 84-2
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Iodomethane
EC Number:
200-819-5
EC Name:
Iodomethane
Cas Number:
74-88-4
Molecular formula:
CH3I
IUPAC Name:
iodomethane
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Iodomethane
- Physical state: liquid
- Analytical purity: 99.7%
- Impurities (identity and concentrations): 0.2 % water and <0.1 % methanol
- Composition of test material, percentage of components: not stated
- Isomers composition: not stated

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 6 to 8 weeks
- Weight at study initiation: Toxicity study: males: 27.7 to 35.1 g and female: 24.6 to 31.0 g and Micronucleus assay: males: 28.4 to 33.7 g and female: 25.8 to 29.3 g.
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: 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
- Water: ad libitum
- Acclimation period: 5 days

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

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle used: sterile distilled water
- Justification for choice of solvent/vehicle: Water was determined to be the solvent of choice based on a request by the Sponsor and compatibility of the vehicle with the test system animals. The test material was soluble in water at 14 mg/mL, the maximum concentration tested in the study.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Dosing solutions were prepared in an appropriate sized, sealed amber glass vials.
- Half of the needed amount of solvent was added and the vials were sealed. In each vial, the appropriate amount of the test material was injected through the sealed stopper. To achieve the proper concentration of dosing solutions, the amount of additional solvent was adjusted according to the measured amount of the test material.
- Dose administration was conducted within, approximately, 30 minutes of preparation.


Duration of treatment / exposure:
Single administration
Frequency of treatment:
Single treatment
Post exposure period:
Bone marrow harvested at 24 and 48 hrs post dosng
Doses / concentrationsopen allclose all
Dose / conc.:
25 mg/kg bw (total dose)
Dose / conc.:
50 mg/kg bw (total dose)
Dose / conc.:
100 mg/kg bw (total dose)
No. of animals per sex per dose:
5 animals per sex per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Route of administration: intraperitoneal injection
- Doses / concentrations: 50 mg/kg
- Cyclophosphamide was dissolved in sterile distilled water at a concentration of 2.5 mg/mL.

Examinations

Tissues and cell types examined:
Bone marrow; 2000 polychromatic erythrocytes (PCEs) examined/animal for the presence of micronuclei. For toxicity assessment, the proportion of PCE to total erythrocytes was recorded (number of PCE in 1000 erythrocytes scored).
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
- Toxicity Study doses: 50, 100, 200 or 280 mg/kg body weight.
- Based on the results of the toxicity study the high dose for the micronucleus test was set at 100 mg/kg for male and female mice which was estimated to be the maximum tolerated dose.
- Micronucleus Assay doses: 25, 50, 100 mg/kg body weight. An additional group of five males and five females were designated as replacement animals in the event of mortality prior to the scheduled sacrifice time and was dosed with the test material high dose level.

TREATMENT AND SAMPLING TIMES:
- Bone Marrow Collection was performed at 24 and 48 hours after dose administration.
- Vehicle: 5 animals at 24 hours and 5 animals at 48 hours
- 25 mg/kg test material: 5 animals at 24 hours
- 50 mg/kg test material: 5 animals at 24 hours
- 100 mg/kg test material: 5 animals at 24 hours and 5 animals at 48 hours
- Positive control: 5 animals at 24 hours


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 resuspended by aspiration with a capillary pipet and a small drop of bone marrow suspension was spread onto a clean glass slide. Two slides were prepared from each mouse. The slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted.

METHOD OF ANALYSIS:
- Slides were coded using a random number table by an individual not involved with the scoring process.
- 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 normochrornatic 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 RESULTS
- The incidence of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes was determined for each mouse and treatment group.
- 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:
The incidence of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes was determined for each mouse and treatment group. Statistical significance was determined using the Kastenbaum-Bowman tables which are based on the binomial distribution. All analyses were performed separately for each sex and sampling time.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TOXICITY ASSAY
- Mortality occurred after dose administration as follows: 3/5 male mice and 4/5 female mice at 200 mg/kg and 4/5 males and 4/5 females at 280 mg/kg.
- Clinical signs, which were noted on the days following dose administration included: lethargy and piloerection in all males and all females at 200 mg/kg, but only in 3/5 males and 4/5 females at 280 mg/kg. Tremors were observed in 2/5 males and 1/5 females at 280 mg/kg. In addition, crusty eyes and/or a crusty nose were observed in some mice at 200 and 280 mg/kg. All other mice appeared normal throughout the observation period.
- A loss in animal body weight from approximately 3 to 18 % was recorded in all of the test material treated groups. The high dose for the micronucleus test was set at 100 mg/kg for male and female mice which was estimated to be the maximum tolerated dose.

MICRONUCLEUS ASSAY
- No mortality occurred at any dose level during the course of the micronucleus study.
- All mice treated with test or control materials appeared normal throughout the observation period.
- The incidence of micronucleated polychromatic erythrocytes per 10000 polychromatic erythrocytes were scored (2000 PCEs/animal). Reductions of 2 to 20 % 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. These reductions suggest that the test material did not inhibit erythropoiesis. The number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes in test material-treated groups was not statistically increased relative to their respective vehicle control in either male or female mice, regardless of dose level or bone marrow collection time (p>0.05, Kastenbaum-Bowman Tables).
- The positive control, cyclophosphamide, induced a significant increase in micronucleated polychromatic erythrocytes in both male and female mice (p ≤ 0.05, Kastenbaum-Bowman Tables). All criteria for a valid test were met.

Any other information on results incl. tables

Table 1: Summary of Micronucleus test

Treatment (mg/kg)

Sex

Time (hours)

PCE/Total erythrocytes (Mean ± SD)

Change from control (%)

Micronucleated Polychromatic Erythrocytes

Number per 1000 PCEs (Mean ± SD)

Number per PCE’s scored

Vehicle

M

24

0.451 ± 0.11

-

0.3 ± 0.27

3/ 1000

F

24

0.470 ± 0.10

-

0.3 ± 0.27

3/ 1000

25

M

24

0.518 ± 0.05

15

0.3 ± 0.27

3/ 1000

F

24

0.484 ± 0.04

3

0.3 ± 0.27

3/ 1000

50

M

24

0.443 ± 0.06

-2

0.4 ± 0.22

4/ 1000

F

24

0.406 ± 0.05

-14

0.4 ± 0.22

4/ 1000

100

M

24

0.384 ± 0.09

-15

0.0 ± 0.00

0/ 1000

F

24

0.374 ± 0.07

-20

0.5 ± 0.35

5/ 1000

Cyclophosphamide 50

M

24

0.323 ± 0.04

-28

24.7 ± 3.68

247/ 1000*

F

24

0.313 ± 0.03

-33

23.2 ± 5.66

232/ 1000*

Vehicle

M

48

0.491 ± 0.05

-

0.1 ± 0.22

1/ 1000

F

48

0.501 ± 0.05

-

0.4 ± 0.22

4/ 1000

100

M

48

0.425 ± 0.02

-13

0.5 ± 0.35

5/ 1000

F

48

0.441 ± 0.02

-12

0.3 ± 0.27

3/ 1000

*= p0.05 (Kastenbaum-Bowman Tables)

Applicant's summary and conclusion

Conclusions:
Under the conditions of this 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 ICR mice .
Executive summary:

The genetic toxicity of the test material was investigated in accordance with the standardised guideline 84-2, under GLP conditions.

The test material was tested in the mouse micronucleus assay. 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. 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 by a single intraperitoneal injection.

Water was determined to be the solvent of choice based on a request by the Sponsor and compatibility of the vehicle with the test system animals. The test material was soluble in water at 14 mg/mL, the maximum concentration tested. Dosing concentrations were delivered to the test system as clear, colourless solutions.

In the pilot toxicity study, male mice were dosed with 100, 120, 180 or 225 mg/kg body weight and male and female mice were dosed with 280 mg/kg. No mortality was observed in any male or female test material treated group. Clinical signs, observed three days after dose administrations, included: lethargy in males at 180 and 225 mg/kg and lethargy and piloerection in males and females at 280 mg/kg . In this phase of the study, the maximum tolerated dose was not established and the toxicity study was conducted. In the toxicity study, the test material dosing solutions were prepared following the Sponsor's instructions. Male and female mice were dosed with 50, 100, 200 or 280 mg/kg body weight. Mortality was observed in 3/5 male mice and 4/5 female mice at 200 mg/kg and in 4/5 males and 4/5 females at 280 mg/kg. Clinical signs following dose administration included: lethargy and piloerection in males and females at 200 and 280 mg/kg, and tremors in males and females at 280 mg/kg. In addition, crusty eyes were noted in males at 200 and 280 mg/kg and crusty nose in females at 280 mg/kg. The high dose for the micronucleus test was set at mg/kg which was estimated to be the maximum tolerated dose.

In the micronucleus assay, male and female mice were dosed with 25, 50 or 100 mg/kg body weight. No mortality or clinical signs were observed in any male or female mice in the micronucleus study. All animals treated with the test or control materials appeared normal following dose administration. Bone marrow cells, collected 24 and 48 hours after treatment, were examined microscopically for micronucleated polychromatic erythrocytes. Reductions (up to 20 %) 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. The positive control, cyclophosphamide, induced a significant increase in micronucleated polychromatic erythrocytes in both male and female mice, all criteria for a valid test were met.

Under the conditions of this 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 ICR mice.

Categories Display