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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Dec 2002 - Mar 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Unnamed
Year:
2003
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report date:
2011

Materials and methods

Principles of method if other than guideline:
Peripheral blood samples were obtained from male and female B6C3F1 mice at the end of a 14-week toxicity study. Smears were immediately prepared and fixed in absolute methanol, stained with a chromatin-specific fluorescent dye mixture of Hoechst 33258/pyronin Y and coded. Slides were scanned at 630 or 1000 times magnification using a semi-automated image analysis system to determine the frequency of micronuclei in 10000 normochromatic erythrocytes (NCEs) in each of 10 animals per dose group. A detailed discussion of this assay can be found in MacGregor et al. (1990).
GLP compliance:
no
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Diethylamine
EC Number:
203-716-3
EC Name:
Diethylamine
Cas Number:
109-89-7
Molecular formula:
C4H11N
IUPAC Name:
diethylamine
Specific details on test material used for the study:
- Physical state: colorless liquid with a strong ammonia odor
- Analytical purity: approximately 99.9%
- Lot/batch No.: BE/07/01
- Stability under test conditions: no degradation of the bulk chemical was detected
- Storage condition of test material: at controlled room temperature

Test animals

Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Taconic Farms, Inc. (Germantown, NY)
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: males: 23.3 g (mean), female: 19.8 g (mean)
- Housing: individually in stainless steel wire bottom (Lab Products, Inc., Seaford, DE), changed weekly and rotated daily
- Diet: NTP-2000 irradiated wafers (Zeigler Brothers, Inc., Gardners, PA), available ad libitum, except during exposure periods
- Water: Tap water (Richland municipal supply) via automatic watering system (Edstrom Industries, Waterford, WI), available ad libitum
- Acclimation period: 12 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3 °C (72°±3 °F)
- Humidity (%): 50% ± 15%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: vapour
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Inhalation chamber (Harvord Systems Division of Lab Products, Inc. Aberdeen, MD)
- Method of conditioning air: glass beads in a heated glass coloum for vapourization
- Air flow rate: 15 air changes /h
Duration of treatment / exposure:
14 weeks (93 days exposure)
Frequency of treatment:
6 hours per day, 5 days per week
Doses / concentrationsopen allclose all
Dose / conc.:
8 ppm (nominal)
Remarks:
analytical concentration 8 ± 0.3 ppm
Dose / conc.:
16 ppm (nominal)
Remarks:
analytical concentration 15.9 ± 0.6 ppm
Dose / conc.:
32 ppm (nominal)
Remarks:
analytical concentration 32 ± 1.3 ppm
Dose / conc.:
62 ppm (nominal)
Remarks:
analytical concentration 62.2 ± 2.3 ppm
Dose / conc.:
125 ppm (nominal)
Remarks:
analytical concentration 126 ± 5 ppm
No. of animals per sex per dose:
5
Control animals:
yes

Examinations

Tissues and cell types examined:
Peripheral blood
Justification: According to the OECD 474, the use of peripheral blood is a valid alternative in mice, if the treatment time exceeds 4 weeks. Since the animals were treated for 90 days, peripheral blood can be used instead of bone marrow.
Statistics:
The frequency of micronucleated cells among normochromatic cells was analysed by statistical software package that tested for increasing trend over exposure groups using a one-tailed Cochran-Armitage trend test followed by pairwise comparisons between each exposure group and the control group.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
not specified

Any other information on results incl. tables

Tab. 1 Frequency of Micronuclei in Mouse Peripheral Blood Erythrocytes













































Dose (ppm)



Micronucleated Normochromatic Erythrocytes / 1000 cells



 



Male



Female



0



2.80 ± 0.30



2.60 ± 0.29



8



4.60 ± 0.60



2.50 ± 0.61



16



4.10 ± 0.48



2.20 ± 0.25



32



3.30 ± 0.34



3.50 ± 0.57



62



4.00 ± 0.52



3.80 ± 0.60



125



2.60 ± 0.33



2.20 ± 0.25



 


No significant increase in micronucleated NCEs was observed in males or females and all tested dose groups.


 


The results from the vehicle control group as well as all treated groups were within the negative control range of micronucleated cells obtained by the lab (1.8 - 5.3 for males, 0.7 - 5.1 for females). The data have been compiled in the table below.


 


Tab. 2: Results for the vehicle control groups obtained by ILS Inc.













































































Year



Report



Male



Female



RoE



2011



TR564



2 +/- 0.61



1.8 +/- 0.25



Inhalation



2014



TR581



2.4 +/-0.33



2.5 +/-0.35



Inhalation



2008



TR552



3.4 +/-0.54



4 +/- 0.47



Inhalation



2009



TR 542



2.4 +/- 0.69



2.3 +/- 0.4



Inhalation



2007



TR 543



5.3 +/- 0.5



5.1 +/- 0.46



Inhalation



2011



TR 567



2.8 +/- 0.2



1.7 +/- 0.2



gavage



2011



TR 570



1.9 +/-0.19



0.7 +/- 0.2



gavage



2011



TR565



4.6 +/- 0.58



3.7 +/- 0.46



feed



2011



tox076



1.8 +/- 0.29



2 +/- 0.26



gavage



 


No concurrent positive control was included, but positive results have been reported by the laboratory. E.g, inhalation exposure for 90-days with α-Methylstyrene results in 9.1 micronucleated NCEs / 1000 NCEs in peripheral blood (TR 543). 

Applicant's summary and conclusion

Conclusions:
There was no increase in the percentage of micronucleated cells compared to the vehicle control group. Additionally, all results were within the negative control range. Consequently, the substance did not cause chromosome damage in this study.
Executive summary:

In a micronucleus assay with B6C3F1 mice, peripheral blood samples were obtained from 5 male/female animals at the end of a 93 days inhalation toxicity study (6 hours per day, 5 days per week; doses of 0, 8, 16, 32, 62, 125 ppm). No significant increase in micronucleated normochromatic erythrocytes was observed in males or females and all tested dose groups.