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:
1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study similar to OECD Guideline 474; Study under FDA GLP Regulations 40 CFR 792;

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1987
Report Date:
1987

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
See information in the field "Confidential details on test material"

Test animals

Species:
mouse
Strain:
other: Crl:DC-1 (ICR)BR
Sex:
male/female
Details on test animals and environmental conditions:
Male and female Cr1:Cne-l(ICR)BR mice (43 days old) were from Charles River, Kingston, NY. They were quarantined and acclimated to laboratory conditions for at least 6 days prior to initiation of the study. Except during exposure, the animals were housed in standard 4" x 7'0 x 5" wire mesh cages, with Purina Certified Rodent and tap water supplied ad libidum. Animal rooms were maintained on a timer controlled 12-hr-light/dark cycle. Environmental conditions of the rooms were targeted for a temperature of 23 + 2°C and relative humidity of 50 + 10%. Any excursions outside these ranges were of small magnitude and/or brief duration and did not adversely affect the validity of the study. During the quarantine period, the mice were housed three per cage; during testing, they were housed individually. All animals were uniquely identified by individual cage numbers and colored markings on their tails

Administration / exposure

Route of administration:
inhalation: aerosol
Vehicle:
- Physical State: liquid
- Vehicle(s)/solvent(s) used: air;
- Justification for choice of solvent/vehicle: used for aerosol generation
- Concentration of test material in vehicle: 5.5, 11.0, and 19.0 mg/L DBE in aerosol air



Details on exposure:
TYPE OF INHALATION EXPOSURE: nose only

In the rangefinding studies aerosol atmospheres of DBE were generated with either a Spraying Systems or Solosphere8 nebulizer.

All atmospheres were generated for the micronucleus assay using the Solosphere8 nebulizer. With the Spraying Systems nebulizer, liquid DBE was pumped into the nebulizer with a Harvard Model 975 compact infusion pump. Air introduced at the nebulizer aerosolized the liquid DBE. and swept the aerosol stream into the exposure chamber. With the Solophere8 nebulizer. liquid DBE was drawn from a reservoir by capillary action into a high pressure air stream and was aerosolized off a spherical glass bead. For both methods, the resulting aerosol stream was dispersed with a baffle as it entered the chamber.

The chamber exhausts were drawn through scrubbers containing acetone, dry-ice cold traps and MSA cartridge filters prior to being discharged into the exhaust hoods. For the control chamber, air only was pumped into a similar exposure chamber.

Atmosphere Analyses: The atmospheric concentration of DBE in each exposure chamber was monitored at approximately 30 minute intervals. Known volumes of the chamber atmosphere were drawn from the animals' breathing zones through 2 fritted glass midget impingers containing acetone as a with a Hewlett Packard 5710A gas chromatograph equipped with a flame ion trapping solvent. The resulting solutions were analyzed in duplicate. The injection port and detector temperatures were 250°C. Nitrogen was used as the carrier gas. Samples were chromatographed isothermally at 125°C on a 3 ft. x 2 mm 10 glass column packed with 10% SP-I000 on 100/120 mesh Chromosorb W-AW.

Atmospheric concentrations of DBE were determined by comparing the detector responses against standard curves calculated by regression. Standards were prepared as needed by diluting known amounts of liquid DBE in acetone. Particle size (mass median aerodynamic diameter and percent respirable) was determined during each exposure with a Sierra Series 210 Cascade Impactor. During each exposure, chamber temperatures were monitored with mercury thermometers. Relative humidities were measured with a Vaisala Model HMI 31F Temperature and Humidity Indicator. Chamber oxygen concentrations were determined with a Biosystems Model 3100R oxygen monitor.
Duration of treatment / exposure:
One day prior to exposure, mice (approximately 7 weeks old) were assigned to exposure groups with equivalent mean body weights (sexes separate) using a computer-based algorithm. The mean pre-treatment weight of the males was 34 g (range 32-38 g) and the females was 25 g (range 20-28 g). Two groups of 15 male and 15 female mice (low and intermediate dose groups) and one group of 18 male and 18 female mice (high dose group) were restrained and exposed as described for the range-finding study to concentrations of DBE for 6 hrs. One control group of 15 male and 15 female mice were similarly exposed to air only for 6 hrs. Concurrent with the initiation of these exposures, 5 male and 5 female mice were dosed by intraperitoneal injection with 20 mg/kg CP, the positive indicator. The mice were observed for clinical signs of toxicity during and immediately following exposure. Surviving mice were weighed and observed daily until sacrifice. Approximately 24, 48 and 72 hrs after initiation of exposure. mice were sacrificed by CO2 asphyxiation.



Frequency of treatment:
1 time, 6 hours;
Post exposure period:
10 to 13 days;
Doses / concentrations
Remarks:
Doses / Concentrations:
range-finding study: 6.9, 11.0, 13.0, and 19 mg/L in aerosol; in micrunucleus test: 5.0, 10.0, and 23 mg/L in aerosol;
Basis:
nominal conc.
No. of animals per sex per dose:
In range finding: 15 males, 15 females per group;
In micronucleus test: 5 males, 5 females per group:
Control animals:
yes
Positive control(s):
cyclophosphamide;
- Route of administration: intraperitoneal (i.p.);
- Doses / concentrations:Stock solution: 2.9 mg/L in 0.9% sterile saline; effective dose applied = 20 mg/kg.

Examinations

Details of tissue and slide preparation:
Immediately following sacrifice, the marrow from both femurs of each animal was aspirated and flushed into 5 mL prewarmed (37°C) fetal bovine serum (Hyc1one Laboratories, Lot 1111536 or Gibco Laboratories, Lot 56K6455). The marrow button was collected by centrifugation (IEC Centrifuge Model HNS-II) at approximately 1000 rpm for 5 min. Most of the supernatant was removed and the cells were resuspended in the remaining 1-2 drops of serum. A Miniprep. Automatic Blood Smearing Instrument was used to make the bone marrow smears. Three slides per animal were prepared and fixed in absolute methanol for 5 min. The slides were stained for 2.5 min in acridine orange (Sigma Chemical Co., Lot 73F-3673) at a concentration of 0.042 mg/mL in phosphate buffer (pH 7.4), followed by phosphate buffer rinses. Immediately prior to scoring, a coverslip was floated on each slide using phosphate buffer.
Evaluation criteria:
Representative slides from each animal were examined in a blind manner using incident light fluorescence microscopy (Leitz Dialux 22 microscope with Ploempak and H-2 filter cube). Only cells showing good morphology and staining were selected for scoring. PCEs were identified by their characteristic reddish staining; NCEs appeared dark green. One thousand PCEs per animal were scored for the presence of micronuclei, which are typically round, bright yellow-green staining bodies.
Inclusions in PCEs which were irregularly shaped or stained, or were not
in the focal plane of the cell were judged to be artifacts and were not
scored. Cells containing more than one micronucleus were counted as
having a single micronucleus; the unit of scoring was the micronucleated
PCE, not the micronucleus. The number of micronucleated NCEs seen in the
optic fields scored to obtain 1000 PCEs was also recorded. The ratio of
PCEs to NCEs was determined for each animal based on 1000 total
erythrocytes.

Results and discussion

Test resultsopen allclose all
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Sex:
female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

During exposure, no adverse clinical signs were observed in the 5.5 mg/L-treated mice. Mice exposed to 11 mg/L had no response to sound. At 19 mg/L, observations could not be made due to the dense aerosol cloud. When released from restrainers after exposure, mice in all groups (including controls) were wet and stained by feces. One control mouse had bloody hindquarters, presumably caused by struggling in the restrainer. In addition, 5 male mice (1 low dose and 4 high dose) and 2 female high dose mice were found dead upon unloading the animals from the exposure chambers. These deaths were believed to be restrainer-related.

At 24 hrs post-exposure, 25 of the surviving animals had ruffled fur, with the greatest number of animals (10 males and 4 females) exhibiting this condition in the high DBE exposure group. Additionally, the following clinical signs were observed at 24 hrs post-exposure: labored breathing in 3 high dose males, tremors, slow movement, and diarrhea each in 1 high dose male, yellow stained fur in 1 low dose male and hypersensitivity in 1 low dose female. Ruffled fur was present among fewer animals at the 48- and 72-hr observation periods; the only additional clinical sign exhibited was
hunched posture by a high dose male.

 

When compared to the concurrent negative controls, significant weight loss was seen in all of the DBE exposed males at the 24-hr sacrifice time, the high dose 48- and 72-hr males, the high dose 24-hr females and the low dose 72-hr females. No significant weight loss or decreased weight gain was observed in any other test group.

 

Table 2 summarizes the data for micronucleated PCEs and PCE:NCE ratios. Individual animal data for the 24, 48, and 72-hr sacrifice groups are shown in Appendices A, B, and C, respectively. Significant depression of the PCE:NCE ratios was seen in the 24-hr 11 mg/L- and 19 mg/L-treated females. There were no statistically significant differences in the proportion of micronucleated PCEs between DBE-treated mice and the concurrent negative control groups at any sampling interval, and no statistically significant concentration-related trends were present. In comparison, both the CP-treated males and females showed significant increases in micronucleated PCEs compared to the concurrent air controls.

Table 2: Micronucleaus Data Summary

DBE (mg/L

Mean MN-PCEs +/- S.E.

(1000 PCEs Scored)

Mean Ratio PCEs / NCEs

+/- S.E.

Males

Sacrifice time

Sacrifice time

N

24h

N

48h

N

72h

24h

48h

72h

0

5

1.6 ± 0.2

5

1.6 ± 0.6

5

1.6 ± 0.6

0.9 ± 0.2

0.8 ± 0.2

0.9 ± 0.1

5.5

5

1.8 ± 0.8

4

1.0 ± 0.4

5

1.8 ± 0.7

0.8 ± 0.1

0.7 ± 0.1

0.8 ± 0.1

11.0

5

2.0 ± 0.3

5

1.4 ± 0.9

5

1.4 ± 0.5

0.6 ± 0.1

0.8 ± 0.1

0.7 ± 0.1

19

5a

3.0 ±-0.8

5a

1.8 ± 0.6

4b

1.5 ± 0.3

0.7 ± 0.1

0.8 ± 0.1

0.5 ± 0.1

Trend

N.S.

N.S.

N.S.

CP

9.6 ±1.5***

0.8 ±0.1

Females

0

5

1.4 ± 0.4

5

3.2 ± 0.7

5

1.6 ± 0.8

1.2 ± 0.1

0.8 ± 0.2

1.0 ± 0.2

5.5

5

1.8 ± 0.4

5

1.4 ± 0.5

5

2.2 ± 1.5

0.9 ± 0.1

0.9 ± 0.1

0.7 ± 0.1

11.0

5

1.0 ± 0.5

5

1.0 ± 0.4

5

1.6 ± 0.5

0.7 ± 0.1**

0.9 ± 0.2

0.6 ± 0.0

19.0

5a

1.6 ± 0.7

6

2.3 ± 0.7

5a

1.8 ± 0.6

0.7 ±0.0*

0.8 ± 0.1

.07 ± 0.1

Trend

N.S.

N.S.

N.S.

CP

15.4 ± 2.0***

1.0 ± 0.1

MN-PCEs, micronucleated polychromatic erythrocytes; NCEs, normochromatic erythrocyte; CP,  cyclophospamide, 20 mg/kg, i.p.

a, one mouse found dead before scheduled sacrifice time;

a, two mice fond dead before scheduled sacrifice time.

*, p 005; **,  p 0.01; ***, p 0.001; N.S., not significant;

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of this assay, DBE did not induce micronuclei in bone marrow cells of mice.
Executive summary:

A mouse bone marrow micronucleus test similar to the OECD Guideline 474 was performed under GLP regulation. While significant depression of the micronucleated polychromatic erythrocytes (PCE) to normochromatic erythrocytes (NCE) ratios was seen in the 24-hr 11 mg/L- and 19 mg/L DBE-treated females, there were no statistically significant differences in the proportion of micronucleated PCEs between DBE-treated mice and the concurrent negative control groups at any sampling interval, and no statistically significant concentration-related trends were present. In comparison, both the cyclophosphamide-treated males and females showed significant increases in micronucleated PCEs compared to the concurrent air controls. Under the conditions of this assay, DBE did not induce micronuclei in bone marrow of mice.