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:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Remarks:
Not specified in report
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
AMP-95 (lot 35662)

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
ICR mice from Harlan Sprague-Dawley, INC, were 6-8 weeks old at study initiation. For the pilot, toxicity, and MNT assay, males ranged in weight from 27.4g to 31.9g, and females ranged from 23.0g to 28.0g. After arrival to the lab, the animals were monitored for parasites and infections, and were quarantined a minimum of 5 days. They were observed daily for general health, food and water consumption patterns, and other conditions. The animals were deemed healthy prior to placement on study.

Mice were housed in an AALAC-accredited facility with appropriate temperature, humidity, and photocycle for the species. They were housed up to 5 per cage, and given food and water ad libitum. The feed and water contained no contaminants that influenced the study.

The mice were randomized and placed into assigned dose groups, and were identified by a uniquely numbered ear tag.

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
water
Details on exposure:
In each the Pilot & Toxicity Studies (designed to set appropriate dose levels for the micronucleus assay), and the Micronucleus Assay, mice were weighed immediately prior to dose administration and the dose volume was based on individual body weights. Mice were given a single IP injection of the test article in water, the vehicle alone, or the positive control substance.

Pilot Study: 2 male mice were dosed with 1, 10, 100, or 1000 mg test material /kg body weight, and 5 males and 5 females were dosed with 2000 mg/kg.
Toxicity Study:
5 male and 5 female mice were dosed with 200, 400, 600, 800 mg/kg
Duration of treatment / exposure:
Single IP injection
Frequency of treatment:
Once
Post exposure period:
24 and 48 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
200, 400, 600, 800 mg AMP/kg
Basis:
nominal conc.
No. of animals per sex per dose:
5/sex/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Yes, cyclophosphamide

Examinations

Tissues and cell types examined:
bone marrow cells
Details of tissue and slide preparation:
At the scheduled sacrifice times (24 and 48 hours), up to 5 mice / sex / dose were sacrificed, the femurs exposed, and bone marrow aspirated into a syringe of fetal bovine serum. The bone marrow cells were pelleted by centrifugation and the supernatant drawn off. The cells were resuspended and a small amount of suspension spread onto a glass slide. Two to four slides were prepared per animal. The slides were fixed in methanol, stained, and permanently mounted. Slides were coded and scored blindly.

Using oil immersion, 2000 polychromatic erythrocytes were scored for the presence of micronuclei. 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:
The test article was considered to induce a positive response if a dose-responsive increase in mocronucleated polychromatic erythrocytes was observed and one or more doses were statistically-elevated relative to the vehicle control (p>0.05) at any sampling time.  If a single dose group was significantly elevated without a dose-response trend, the assay was considered a suspect or unconfirmed positive and a repeat assay recommended.  The test article would be considered negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control was observed at any sampling time.
Statistics:
Statistical significance was determined using the Kastenbaum-Bowman tables which are based on the binomial distribution.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Clinical signs noted on the day of dosing included: lethargy in males and females at 60 mg/kg (highest dose).
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Pilot:
Mortality occurred within 4 hours following dosing: 5/5 males and 5/5 females at 2000 mg/kg, and 2/2 males at 1000 mg/kg. There were no clinical observations noted.

Toxicity:
Mortality occurred within 3 days of dosing: 5/5 males and 5/5 females at 400, 600, and 800 mg/kg, and 3/5 males and 5/5 females at 200 mg/kg. Clinical signs included: lethargy in males and females at all dose levels, piloerection in males and females, and crusty eyes in males at 200 mg/kg. The LD50/3 was calculated by probit analysis to be approximately 73.7 mg/kg for male and female mice.

Micronucleus Assay:
There was no treatment-related mortality at any dose level. Clinical signs noted on the day of dosing included: lethargy in males and females at 60 mg/kg (highest dose). All other mice treated with test material and control articles appeared normal during the study. The number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes in test article-treated groups was not statistically increased relative to their respective vehicle control in either male of female mice, regardless of dose level or bone marrow collection time.

Applicant's summary and conclusion

Conclusions:
Under the conditions of the assay described in this report, 2-amino-2-methyl-1-propanol did not induce a sugnificant 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.