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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:
16 January - 26 January 1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report date:
1990

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
N-isopropylhydroxylamine
EC Number:
225-791-1
EC Name:
N-isopropylhydroxylamine
Cas Number:
5080-22-8
Molecular formula:
C3H9NO
IUPAC Name:
N-(propan-2-yl)hydroxylamine
Details on test material:
The test article, Nipha Crystals, was supplied in the form of white crystals. No purity information reported.

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals or test system and environmental conditions:
Adult male and female mice, strain ICR, were purchased from Harlan Sprague-Dawley, Inc., Frederick, MD. This healthy, random bred strain was selected to maximize genetic heterogeneity and at the same time assure access to a common source.

Animals were group-housed by sex up to five per cage. The temperature and humidity were maintained at 72±6°F and 50±20%, respectively. A 12-hour light/12-hour dark cycle was maintained. A commercial diet (Purina Certified Laboratory Chow· #5002) and water were available ad libitum for the duration of the study. The feed is analyzed by the manufacturer for concentrations of specified heavy metals, aflatoxin, chlorinated hydrocarbons, organophosphates, and specified nutrients. The water is analyzed on a retrospective basis for specified microorganisms, pesticides, heavy metals, alkalinity, and halogens. Sanitary polycarbonate cages and hardwood bedding were used. Personnel handling animals or working within the animal facilities were required to wear suitable protective garments and equipment.

Animals were quarantined for seven days before being placed on study. Animals were randomly assigned to study groups and were individually weighed prior to the initiation of the study. All animals were dosed based upon the individual body weights. Animals were uniquely identified by ear tag. Dose or treatment groups were identified by cage card.

At the termination of the study all surviving animals were euthanatized by CO2 inhalation. Any extra animals not used for the study were euthanatized at the end of the study.

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
The test article, Nipha Crystals, was supplied in the form of white crystals. The solubility of the test article was evaluated in a previously conducted dose range finding assay (HLA Study No.: 11200-0-459). Based upon the solubility data from the dose range finding assay, the vehicle used to solubilize the test article for the bone marrow micronucleus assay was 0.9% sodium chloride (Abbott, Loti 15-117-DM-03). The stability of the test article under the preparation and dosing conditions of the assay is the responsibility of the sponsor.
Details on exposure:
Trial 1
The animals used in the trial 1 micronucleus assay were dosed on Tuesday, January 16, 1990. The weight range of the animals used in the trial 1 micronucleus assay was 30.0 - 39.9 grams and 21.5 - 27.5 grams for the males and females. respectively. The dosing solutions for the assay were prepared by making a 140 mg/ml stock for the high dose (1400 mg/kg). Dilutions of this stock were prepared for the 467 and 140 mg/kg dose levels.

Trial 2
The animals used in the trial 2 micronucleus assay were dosed on Tuesday, January 30, 1990. The weight range of the animals used in the trial 2 micronucleus assay was 30.3 - 40.5 grams and 20.0 - 25.0 grams for the males and females, respectively. The dosing solution for the assay was prepared by making a 120 mg/ml stock for the 1200 mg/kg dose level.

In both trials, cyclophosphamide (CP, Sigma, Lot #67F-0155). the positive control, was solubilized in sterile deionized water and was administered by oral gavage at 80 mg/kg. The vehicle controls consisted of 0.9% sodium chloride and were administered concurrently with the test article at a volume of 10 ml/kg. A second group of animals (designated Secondary Dose Group) was also assigned to each study and was dosed with the high dose of the test article. These animals were only used in the assays as replacements for any which died in the primary dose groups.

The test article dosed animals were euthanatized 24, 48 and 72 hours after administration of the test article. The positive and vehicle control animals were euthanatized 24 hours after the administration of the control articles.
Duration of treatment / exposure:
Mice received a single ip dose.
Frequency of treatment:
Mice received a single ip dose.
Post exposure period:
The test article dosed animals were euthanatized 24, 48 and 72 hours after administration of the test article. The positive and vehicle control animals were euthanatized 24 hours after the administration of the control articles.
Doses / concentrations
Remarks:
Doses / Concentrations:
140, 467, 1200 and 1400 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
See Table below
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (CP, Sigma, Lot #67F-0155) was used as positive control.

Examinations

Tissues and cell types examined:
The frequency of PCEs versus NCEs was determined by scoring the number of NCEs observed in the optic fields while scoring the 1000 PCEs for micronuclei.
Details of tissue and slide preparation:
The coded slides were then scored for micronuclei and the polychromatic (PCE) to normochromatic (NCE) cell ratio. Standard forms were used to record these data. One thousand PCEs per animal were scored. The frequency of micronucleated cells was expressed as percent micronucleated cells based on the total PCEs present in the scored optic field. The normal frequency of micronuclei in this mouse strain is about 0.0-0.4%.
Evaluation criteria:
General
The criteria for the identification of micronuclei were those of Schmid (1976). Micronuclei were darkly stained and generally round, although almond and ring-shaped micronuclei occasionally occur. Micronuclei had sharp borders and were generally between 1/20 and 1/5 the size of the PCE. The unit of scoring was the micronucleated cell, not the micronucleus; thus the occasional cell with more than one micronucleus was counted as one micronucleated PCE, not two (or more) micronuclei. The staining procedure permitted the differentiation by color of PCEs and NCEs (bluish-grey and red, respectively).

Reference
Schmid (1976).
Statistics:
Data were summarized to include tables indicating the individual animal results and in tables with animal results summarized by sex and dose groups at the different time points. The analysis of the data was performed using an Analysis of Variance (p
The criteria for determining a positive response involved a statistically significant dose-related increase in micronucleated PCEs, or the detection of a reproducible and statistically significant positive response for at least one dose level. A test article that induced neither a statistically significant dose response nor a statistically significant and reproducible increase at one dose level was considered negative. In either case, the final decision was based on scientific judgment.

References
Sokal, R.R. and Rohlf, J.J.: Biometry, Freeman, 1981.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
All animals were observed immediately after dosing and periodically throughout the duration of the assay for toxic symptoms and/or mortalities. In both trials, all animals in the vehicle and positive control groups appeared normal after dosing and remained healthy until the appropriate harvest times.

Trial 1
Within one hour of dosing 35 animals in the 1400 mg/kg dose groups had expired. Due to this toxicity, the 1400 mg/kg dose level was eliminated from the study. All animals surviving at this dose level were euthanized the following morning. Within 15 minutes of dosing the animals in the 467 mg/kg dose group became languid. The animals dosed at 140 mg/kg were slightly ataxic. The following morning, approximately 16 hours after dosing, all animals in the 140 and 467 mg/kg dose levels appeared normal and they remained healthy until the appropriate harvest times. The test article, Nipha Crystals, induced no significant increases in micronucleated polychromatic erythrocytes over the levels observed in the vehicle controls in either sex or at any of the harvest times. The positive control, CP, induced significant increases in micronucleated PCEs in both sexes, with means and standard errors of 1.72% ± 0.41% and 1.90% ± 0.23% for the males and females, respectively.

Trial 2
Within 30 minutes of dosing, the animals dosed with the test article (1200 mg/kg) had become prostrate with dyspnea. A few animals exhibited convulsions. Approximately 30 minutes after dosing two females (#'s 5660 and 5682) expired. Two additional females (#'s 5662 and 5663) died within 3 hours of dosing. All other animals dosed with the test article were languid and several had squinted eyes. The following morning, approximately 22 hours after dosing, all animals appeared normal and they remained healthy until the appropriate harvest times. The test article, Nipha Crystals, induced no significant increases in micronucleated polychromatic erythrocytes over the levels observed in the vehicle controls in either sex or at any of the harvest times. The positive control, CP, induced significant increases in micronucleated PCEs in both sexes, with means and standard errors of 2.42% ± 0.61% and 1.42% ± 0.39% for the males and females, respectively.

Any other information on results incl. tables

TABLE 1. MICRONUCLEUS DATA SUMMARY TABLE

     

       % Micronucleated PCEs

Mean of 1000 per animal + SE

    Ratio PCE:NCE

Mean + SE

 Treatment  Dose  Harvest Time (hr)  Males  Females  Total  Males  Females

 Vehicle Control

(0.9% sodium chloride)

 10 ml/kg  24  0.02 + 0.02  0.04 + 0.04  0.03 + 0.02  0.80 + 0.18  0.87 + 0.27

 Positive Control

CP

 80 mg/kg  24  1.72 + 0.41*  1.90 + 0.23*  1.81 + 0.23*  0.46 + 0.13  0.56 + 0.09
 NIPHA  140 mg/kg  24  0.12 + 0.06  0.04 + 0.02  0.08 + 0.03  0.50 + 0.12  0.71 + 0.11
     48  0.14 + 0.07  0.02 + 0.02  0.08 + 0.04  0.46 + 0.07  0.74 + 0.10
     72  0.12 + 0.06  0.08 + 0.02  0.10 + 0.03  0.60 + 0.13  0.75 + 0.10
   467 mg/kg  24  0.08 + 0.04  0.12 + 0.04  0.10 + 0.03  0.66 + 0.24  0.82 + 0.15
     48  0.08 + 0.04  0.10 + 0.03  0.09 + 0.02  0.61 + 0.07

 0.65 + 0.07

     72  0.04 + 0.02  0.04 + 0.02  0.04 + 0.02  0.59 + 0.08  0.53 + 0.05

* Significantly greater than the corresponding vehicle control, p<0.05.

TABLE 2 MICRONUCLEUS DATA SUMMARY TABLE

     

       % Micronucleated PCEs

Mean of 1000 per animal + SE

    Ratio PCE:NCE

Mean + SE

 Treatment  Dose  Harvest Time (hr)  Males  Females  Total  Males  Females

 Vehicle Control

0.9% Sodium Chloride

 10 ml/kg  24  0.10 + 0.03  0.04 + 0.02  0.07 + 0.02  0.62 + 0.09  0.93 + 0.25

 Positive Control

CP

 80 mg/kg  24  2.42 + 0.61*

 1.42 + 0.39*

 1.92 + 0.38*  0.85 + 0.16  1.30 + 0.35
 Test Article  1200 mg/kg  24  0.08 + 0.04  0.08 + 0.04  0.08 + 0.02  0.47 + 0.03  1.10 + 0.17
     48  0.12 + 0.08  0.16 + 0.07  0.14 + 0.05  0.69 + 0.07  0.69 + 0.16
     72  0.14 + 0.05  0.08 + 0.02  0.11 + 0.03  0.50 + 0.03  0.73 + 0.15

* Significantly greater than the corresponding vehicle control, p<0.05.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test material, Nipha Crystals, did not induce a significant increase in micronuclei in bone marrow polychromatic erythrocytes under the conditions of this assay and is considered negative in the mouse bone marrow micronucleus test.
Executive summary:

The objective of this in vivo assay was to evaluate the ability of the test article, Nipha Crystals, to induce micronuclei in bone marrow polychromatic erythrocytes of ICR mice. The test article was solubilized in 0.9% sodium chloride and dosed by intraperitoneal injection at 140, 467, and 1400 mg/kg based upon the results of a previously conducted dose range finding assay (HLA Study No.: 11200-0-459). The 1400 mg/kg dose group was eliminated from the initial study due to excessive toxicity. A subsequent study was performed at 1200 mg/kg (one dose level only) to replace the high dose eliminated from the initial study. Ten animals (five males and five females) were randomly assigned to each dose/harvest time group. Vehicle and positive control groups euthanatized 24 hours after dosing were included in both studies. The animals were dosed with the test article and were euthanatized 24, 48 and 72 hours after dosing for extraction of the bone marrow. The test material, Nipha Crystals, did not induce a significant increase in micronuclei in bone marrow polychromatic erythrocytes under the conditions of this assay and is considered negative in the mouse bone marrow micronucleus test.

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