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:
disregarded due to major methodological deficiencies
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: scientifically acceptable study (TS presumably contained unspecified impurities, only one post-treatment sample (after 48 hours), 2 treatments at 48 hours intervals, limited documentation)

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
limited documentation, 2 treatments at 48 hours intervals, only one post-treatment sample (after 48 hours)
GLP compliance:
yes
Type of assay:
chromosome aberration assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): N-BAPED
- Physical state: yellow liquid
- Analytical purity: technical grade; 95%
- Impurities (identity and concentrations): not specified
- Lot/batch No.: 21955-60-1 (Aldrich Chem Co Inc catalog number: 23,939-9)

Test animals

Species:
mouse
Strain:
Balb/c
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: animals from a randomly bred closed colony purchased from Charles River Breeding Laboratories, Inc., Wilmington, MA
- Age at study initiation: not specified; adult male and female mice were used
- Weight at study initiation:
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Fasting period before study: not specified
- Housing: group-housed, 7 mice/cage
- Diet: not specified (a commercial diet was used); ad libitum
- Water: not specified (“water”); ad libitum
No additional detail provided

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: A set of mice was dosed with the solvent (filter sterilized 0.99% saline; intraperitoneal [IP] injection), which served as a negative control.
Details on exposure:
Dosing consisted of 2 intraperitoneal (IP) administrations of the test substance / positive control substances / vehicle.
Duration of treatment / exposure:
48 hours
Frequency of treatment:
The initial dose was given at time-0 (i .e. immediately following the initial blood sampling). A second dose was given 48 hours thereafter.
Post exposure period:
24 hoours; the final blood sample was obtained at 96 hours after the first injection.
Doses / concentrations
Remarks:
Doses / Concentrations:
ca. 119, 238 and 475 mg/kg bw (calculated from 0.125, 0.25 and 0.5 µl/g bw assuming test substance density of 0.95 g/cm³)
Basis:
other: actual injected
No. of animals per sex per dose:
7
Control animals:
yes, concurrent vehicle
other: An initial (0 hour; before dosing began) blood sample from each mouse provides an internal control for comparison with dosed blood (96 hour samples). These initial blood samples provided a mouse-specific control blood sample.
Positive control(s):
methylmethanesulfonate (MMS)
- Route of administration: IP
- Doses / concentrations: 100 mg/kg bw

Examinations

Tissues and cell types examined:
polychromatic cell (PCE) of peripheral blood
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The high dose was detetmined in a preliminary lethal dose study. In order to evaluate the lethal dose range, mice were dosed with the test material diluted in 0.9% sterile saline to the following concentrations: ca. 238, 594, 1188, 1782, and 2375 mg/kg bw (converted from 2.5, 1.875, 1.25, 0.625, and 0.25 μl/g bw). The mice were injected with 25 ml test substance/kg bw. Those animals which were injected with the 1.25 μl/g bw solution and higher did not survive, while all the others survived 96 hours. Therefore, the high dose was set at 0.5 μl/g bw (ca. 475 mg/kg bw), the medium dose at 0.25 μl/g bw (ca. 238 mg/kg bw) and the low dose at 0.125 μl/g bw (ca. 119 mg/kg bw).
A preliminary analysis of the PCE number was obtained in order to insure that stem cell toxicity was not a factor in the micronucleus assay.

DETAILS OF SLIDE PREPARATION:
Blood cells were obtained from the tail vein of each mouse and collected in heparin. The blood samples were fixed with alcohol and were stained with dye solutions of Hoechst 33258 (DNA of the micronucleus) and propidium iodide (RNA of PCEs). With this dye combination, it was possible to provide a quantitative analysis of the number of RBCs, PCEs and micronucleated cells that are present in each sample. A fixed and stained blood sample from a malaria infected mouse was used during the instrument setup in order to evaluate the resolution of the signals.

METHOD OF ANALYSIS:
- The 0 and 96 hour blood samples were quantitatively analyzed by high speed flow cytometry (FCM; DNA of a micronucleus emit blue fluorescence, and RNA of polychromatic cells red). The stained cells were then excited with a 363 nm beam from a 5 watt Argon Ion laser focused to 20 microns at the sample stream. The fluorescence from each cell was collected by photomultiplier tubes (PMTs) and the signal levels were stored in computer memory for later processing. In addition to obtaining fluorescence profiles of each cell, FCM simultaneously provided cell size information by determining the light scatter properties of each cell or combination of cells. By setting hardware or software gates, it was possible to exclude ("gate out") cells and/or debris from analysis and thus focus on those cells of specific interest (e.g. the number of micronucleated cells, PCEs, or RBCs present in a sample).
- For each mouse, the number of micronucleated cells was determined in 2000000 total blood cells 1000000 cells from the t-0 blood and 1000000 cells from the final blood [t-96] for each mouse. The MN cell count of each initial (0 hour) blood sample was compared with the corresponding 96 hour blood sample.
Evaluation criteria:
A mean and standard deviation (SD) of the micronucleated cell counts (t=0 and t=96 hours) was provided for each set (i .e. each treatment group) of mice. In addition, the change between the t=0 and the t=96 hours values were provided for the micronucleated cells and the PCEs. A comparison of the means was done by statistical analysis.
Statistics:
The test substance induced effects were analysed by ANOVA, and a t-test was used to highlight the source of the variation.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
positive
Remarks:
a significant increase in the number of micronucleated cells was observed with all doses for the females and with the high dose for the males, but the variations within the test groups were very high.
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid

Any other information on results incl. tables

Table 1: Summary of the results

 

Treatment group

 

 

Sex

Means of micronucleus (MNs) / polychromatic erythrocytes (PCEs)

MN and changes (MN96 hours– MN0 hour)

PCEs

0 hours value

96 hours value

Change

0 hours value

96 hours value

 

Negative control

Females

1929

1982

53

2340

5606

Males

2665

2699

34

2828

5826

Mean variation in both sexes

44

 

 

 

Low dose (119 mg/kg)

Females

2076

2356

280*

2660

5700

Males

2714

2812

99

2960

5700

Mean variation in both sexes

189

 

 

 

Medium dose (238 mg/kg)

Females

1997

2234

236*

3052

6171

Males

2621

2777

156

2764

5681

Mean variation in both sexes

196

 

 

 

High dose (475 mg/kg)

Females

2080

2338

258*

2914

6173

Males

2670

2905

234

2925

5783

Mean variation in both sexes

246*

 

 

 

Positive control

Females

2013

5225

3212*

2285

5298

Males

2689

570

3071*

2510

4554

Mean variation in both sexes

3142

 

 

 *: significant different from the negative control

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): positive