Document KzJBdVV01L6712jDpee5qb8Ko
^on ^yiuiesi ^un neaearcn urnon & l^o. IM-i
AR226-2986
CCR PROJECT 326428
M1CRONUCLEUS ASSAY IN BONE MARROW CELLS OF THE MOUSE
WITH
U
REPORT
Study Completion Date:
/
February 17,1993
CCR - In den Leppsteinswi'essn 19 D-6101 Ropdorf F.R.G. Telephone: 0 61 54 - 80 7-0 Telefax O 61 54 - 8 33 99
HESSISCHES MINISTER1UM FORUWVELT, ENERGIE
UNO BUNDESANCELEGENHEITEN
GLP-Sescheinigung
Beacheinlgung
Hiennit wird bestaligt, daS dia PriHungs8inochtung(an)
Cytotest Cell-ReseaTch 6nbH & Co KG In den Leppsteinswiesen '19 in .i'iyj^PM0'"^
(On. AraeiCT)
der.^. Holding VerwaJtunganbH
(fiBMl
am 3-08-.0^-08-. 05.08 .und 06.08.92
(OiRJm)
von der (ur dia ClbarwachungzustSndigan Bahflfda Obar .die Snhaltung der Gnjndsitza der Guten Laborpraaa inspba'en wordBn ist (sind). 6s wird hiennit bestaBgl, dafi falgenda Priilungen in dieser Prufeinriditung nach den GrundsteBn dar Guten Laborpraxis dunAgeKihrt warden.
^srs^ Toxikologische Eigenschaftfifl-^ .
Certincata
It is hareby eertilied that the lest (adnty(ies)
Cytotest Cell Research GmbH & Co KG
T 3 " ~ " " 'In'den'Tews'teTnsw'iesen 610'1 RoBdorf
01
(toalicn. KIcinra)
. RCC Holding Verwaltung QnbH
or ...... ..i 1.1.1 r........,,,.....n.......i----_^
(c>Mip*n|rauM)
0(1 03.08.. 04.08., 05.08. and.06.08.92
- (All.)
was (were) inspected by the competent authority
ding
compianca
with
the
Principles of
Good
regar Laboratory
PtacHca.
It is hereby certified lhat studies in IKs lest facility are cLoanbdouracltQeidy iPnraccotimcep.Eanca with the Principles of Good
Toxicological.properties
Im Auf-
^ . ^ ^ r (Or..Hec!cer)
Wiesbaden, den a/^-
nin'teec. re
Copy of GLP-Certificate
PREFACE
General
Project Staff
Schedule
Project Staff Signatures Quality Assurance
Guidelines
Archiving
STATEMENT OF COMPLIANCE
QUALITY ASSURANCE UNIT Statement
SUMMARY
Conclusion
OBJECTIVE
Aims of the Study Reasons for the Study
MATERIALS AND METHODS
The Test Article
Controls The Test System Experimental Performance Data Recording Evaluatipn of Results
BIOMETRY
RESULTS CONCLUSIONS
Pre-Experiment for Toxicity
Summary of Results Tables of Results
DISTRIBUTION
REFERENCES .
mnteec.re
GENERAL
Sponsor:
ISEGA Forschungs- und
Untersuchungsgesellschaft mbH D-8750 Aschaffenburg
Study Monitor:
Heike Kramer
Testing Facility:
C CR
CYTOTEST CELL RESEARCH GMBH 5 CO. KG
D-6101 RoBdorf
CCR Project No.:
Test Article; Title:
PROJECT STAFF
326428
&
0
Micronucleus Assay in Bone Marrow Cells of the Mouse wit
Management:
Dr. H.-E. Knoell
Scientific consultant: Prof. Dr. Herbert G. Miltenburger
Study Director:
, Dr. Wolfgang Volkner
Quality Assurance Unit: Dr. Cnristiane Helmrich
SCHEDULE
Date of Protocol: Start of Experiment; End of Experiment;
Date of Draft;
Date of Report:
November 09, 1992 November 11, 1992 January 26, 1993 January 25, 1993 February 17, 1993
ronteec.re
Study Director;
Management;
Dr. Wolfgang Volkner
6).to'<J
Date: February 17, 1993
Dr. Hans
Knoell
1^...... D..a.te..: ..F.e.b.ruary 17, 1993
QUALITY ASSURANCE
"
The study was performed in compliance with:
Chemikaliengesetz ("Chemicals Act") of the Federal Republic of
Germany, Aniage 1 ("Annex I"), dated March 14, 1990 (BGBL. I S.
521).
"The OECD Principles of Good Laboratory Practice", Paris 1981.
GHIDELIHES
/
This study followed the procedures indicated by the following internationally accepted guidelines and recommendations:
First Addendum to the OECD Guideline for Testing of Chemicals,
Section 4, No. 474, adopted May 26, 1983, "Micronucleus Test".
EEC Directive 79/831, Annex V, B 12
Environmental Protection Agency, Code of Federal Regulations,
Title 40, Subpart F-Genetic Toxicity, Revision July 1, 1986
"In vivo mammalian bone marrow cytogenetics tests: Micronucleus assay."
mnteec.re
CompMWSanBired.Doesi
CCR, D-6101 Rofldorf will archive the following data for 30 years;
raw data, protocol and copy of report.
'The following 12 years:
specimen
and
samples
will
be
archieved
for
at
least
sample of test article, microscopic slides.
No raw data without the
or material relating to sponsor's prior consent.
the
study
will
be
discarded
amteec. re
6
not @mtain TSCA CBI
Project Number:
Test Article ;
Study Director:
Title
;
326428
Dr. Wolfgang VolJcner
Micronucleus Assay in Bone Marrow Cells of the Mouse with|)BB------
To the best of my knowledge
the testing facility of CCR
and belief,
this
study performed
in
Laboratory
Practice
was conducted Regulations;
in
compliance
with
Good
Chemikaliengesetz Germany, Aniage 1
("Chemicals Act")
of
the
Federal
Republic
521).
("Annex I"), dated March 14, 1990 (BGBL. I of
S.
"OECD Principles of Good Laboratory Practice", Paris, 1981 iTnhteeregrwiteyreonf othceirc'sutmudsyta. nces that may have effected the quality or
Study Director
CCR
Dr. Wolfgang Volkner
Date: TU^^ ^ A^
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C C R, Cytotest Cell Research GmbH 5 Co. KG, In den Leppsteinswiesen 19, D-6101 RoBdorf
STATEMENT
Project Number;
Test Article :
Study Director:
Title
*
326428
Dr. Wolfgang Volkner Micronucleus Assay in D Cells of the Mouse wi
dsTathutiedssy.reapnodr/ot r wtaesstaiundgitefdacbiylittyheweQrue alinitsypeAcstseudranonce thUenitfoallnodwitnhge
Dates of QAU Inspections/ Audits
November 10, November 20, January 29,
1992 1992 1993
Dates of Reports to the Study Director and to Management
November 10, 1992 November 20, 1992 January 29, 1993
Head of Quality Assurance Unit
Dr. Christiane Helmrich
Date: ^y^y ^ ^yj
mnteec.re
SUMMARY
f^^^^ Th^ study was
BFO induce
performed
to
.
investigate
.
the
potential
of||BH----B
the bone marromwicorfonthueclmeiouisne. polychromatic erythrocytes~RpCE) in
wThase utesesdt aasrtnicegleatiwvaes dissolved in aqua deionized. This
10 ml/kg b.w.. 24
test article the
h
control.
and 48 h
The volume administered
after a single
solvent orally was
application
analysis.
bone
marrow
cells
were
collected
for
of the micronuclei
Ten animals (5 males, 5
for the occurrence of
females)
per
test
group
were
evaluated
cytes (PCE) To describe
per
animal
micronuclei. 1000 polychromatic were scored for micronuclei.
erythro-
article
the
a cytotoxic effect due to the ratio between polychromatic
treatment
with
the
test
tthherocnuymtebser (NCf EN) CEwaps edr e1te0r0m0 inPeCdE. in the sameansdamnpolremaoncdhrorempaotrictederays-
The
following
24 h
dose
levels
of
the
test
article
were
48
h
preparation interval: preparation interval:
200,
2000
666,
and
investigated: 2000 nig/kg b.w..
The highest dose estimated by
recommended
by
mg/kg b.w.. guideline (2000
mg/kg)
pressed
a pre-experiment
slight toxic reactions.
to
be
suitable.
The
animals
was ex
After treatment
increased as
with
the
test
article
the
number
of
NCEs
indicating
thactorm--p--are--d --to--t--he--, c|foiarrdesponding
negative
was not controls thus
^^^^^^^^^^r no cytotoxic effectiveness.
At preparation
the
interval
24
hours
no
substantial
the
frequency of the detected
test article was observed.
micronuclei
after
enhancement in treatment with
In comparison
statistically
to the corresponding significant
negative
control
there
was
a
detected micronuclei treatment with the
at
enhancement in the frequency of preparation interval 48 hours
the
rHeolweveavnerc,e.this finditnegst isartnioctlec. onsidered to be of a biologaifctealr
An appropriate reference mutagen positive control which showed a
micronucleus frequency.
(cyclophosphamide)
distinct
was used as
increase of induced
COKCLPSIOM
In conclusion, and under the
it
can be
stated that
during the
study described
did not induceexmpeircirmoennutcalleicoansditdioetnesrmrienpeodrted, the test article
test with bone marrow cells
Therefore f'WWWSm.s
of
by the NMRI-mouse.
the
micronucleus
micronucleus assay. J considered to be non-mutagenic in this
nmteec. re
9
Cmaswsy
not contain TSCA
OBJECTIVE
AIMS OF THE STUDY
This in vivo experiment was performed to assess the mutagenic
properties of the test article by means of the micronucleus test
in bone marrow cells of the mouse.
REASONS FOR THE STUDY
The occurrence of micronuclei in interphase cells provides an indirect but easy and rapid measure of chromosomal damage. Micronuclei arise from acentric chromosomal fragments or whole chromo somes induced by clastogens or agents affecting the spindle apparatus (1,2,3,4). Polychromatic erythrocytes (PCE) in the bone marrow of the mouse
are the cell population of choice for mammalian cells in vivo.
PCEs are newly formed red blood cells and are easily identifiable by their staining properties. These cells have the advantage that the micronuclei can be readily detected because the nucleus is extruded from the erythroblast after the last cell division .
The first appearance of micronuclei in PCEs is at least 10-12
hours after a clastogenic exposure. This lag is due to the time required for the affected erythroblast to differentiate into a PCE. This differentiation process includes;
1. The time required for the damaged erythroblast to proceed to mitosis.
2. The mitotic delay induced by the treatment. 3. The formation of micronuclei due to acentric fragments or
chromosomes that are not included in the daughter nuclei. 4. The time required for the expulsion of the main nucleus after
the last mitosis to become a micronucleated PCE. This newly formed cell population persists for about 20 hours in the bone marrow of mouse. During this time micronucleated PCEs accumulate in the bone marrow as the production of micronuclei
extends over a considerable period of time.
The time at which the micronucleus frequency is at a maximum va ries from agent to agent (5). Due to mitotic delay or metabolic and pharmacokinetic effects the appearance of micronucleated PCEs
can be considerably delayed. Therefore a single sampling time is not optimal. Results obtained with model mutagens showed that
samples taken at 24 h and 48 h after treatment cover the inter
vals in which maximum frequencies of micronuclei occur.
mnteec.re
10
For the initial assessment of clastogenic activity a single dose
level at the maximum tolerated dose or that producing some indi cation of cytotoxicity (change in the ratio of polychromatic to nonnochromatic erythrocytes) and sampling at 24 h and 48 h after treatment is recommended. For verification two additional dose levels are tested at the central sampling time 24 h after treat ment to establish a dose response effect. To validate the test, a reference mutagen is tested in parallel to the test article.
mnteec.re
11 Company Sanitized. Does not contain TSCA CBj
MATERIALS AND METHODS
THE TEST ARTICLE
The test article and the information concerning the test article
were provided by the sponsor.
Name;
Batch No.
Aggregate State
at RT:
f -- 3 C^----3 liquid
Colour:
-
Purity:
Analysis:
S
t
a
b
i
l
i
t
y
*
not indicated
C^^^------------B 3
C.^----3
Pure:
not indicated
In vehicle: not indicated
Storage:
4 C
Expiration date; not indicated
On the day of the experiment/ the test article was dissolved in aqua deionized. The solvent was chosen to its non-toxicity for the animals. All animals received a single standard volume of
10 ml/kg body weight orally.
mnteec.re
12 . Company Sanitized. Does not conSain TSCA CM
THE COHTROLS
The Negative Control
The vehicle of the test article was used as negative control,
^Hame:
Route and Frequency
of Administration: Volume Administered:
aqua deionized
.orally, once
10 ml/kg b.w.
The Positive Control
Name;
Supplier: Catalogue no.;.
Dissolved in:
Dosing:
Route and Frequency
of Administration: Volume Administered:
CPA; Cyclophosphamide
SERVA, D-6900 Heidelberg
17681
physiological saline
30 mg/kg b.w.
orally, once
10 ml/kg b.w.
Solution prepared on day of administration.
The stability of CPA at room temperature is good. At 20 C only
1 % of CPA is hydrolysed per day in aqueous solution.
mnteec.re
13 Company Sanitized. Does not onrtain TSCA CBE
THE TEST SYSTEM
Reasons for the Choice of the Escperijmental Animal Species
The mouse is an animal which has been used for many years as suitable experimental animal in cytogenetic investigations. There are many data available from such investigations which may be helpful in the interpretation of results from the micronucleus test. In addition, the mouse is an experimental animal in many physiological, pharmacological and toxicological studies. Data from such experiments also may be useful for the design and the performance of the micronucleus test (1,2/3,4,5).
Strain:
Source:
Number of Animals:
Initial Age at Start
of Acclimatization: Acclimatization:
Initial Body Weight
at Start of Treatment:
NMRI
Charles River Wiga GmbH Sandhofer Weg 7, D-8741 Suizfeld 1 84 (42 males/4 2 females)
minimum 10 weeks minimum 5 days
20 - 40 g
According to the suppliers assurance the animals were in healthy condition. The animals were under quarantine in the animal house
of C C R for a minimum of five days after their arrival. During this period the animals did not show any signs of illness or
altered behaviour.
The animals were distributed into the test groups at random and identified by cage number.
mnteec.re
14 .Kpaa^f Sanitized. Does not contain TSCA CBI
Husbandry
The animals were kept conventionally. The experiment was conduct ed under standard laboratory conditions.
Housing; Cage Type: Bedding: Feed: Water; Environment:
single
Makrolon Type I, with wire mesh top
(EHRET GmbH, D-7830 Emmendingen)
granulated soft wood bedding
(ALTROMIM, D-4937 Lage/Lippe)
pelleted standard diet, ad libitum
(ALTROMIN 1324, D-4937 Lage/Lippe)
tap water, ad libitum
(Gemeindewerke, D-6101 Rofidorf)
temperature 21 .fc 3"a
relative humidity 30-70%
artificial light 6.00 a.m. -
6.00 p.m.
mnteec.re
15 Company Sanitized. Does not contain TSCA CBE
EXPERIMEMTflI. PERFORM&HCE
Pre-Experiment for Toxicitv
A preliminary study on acute toxicity was performed with the same strain and under identical conditions as in the mutagenicity
.study.
Dose Selection
It is generally recommended to use the maximum tolerated dose or
the highest dose that can be formulated and administered repro-
ducibly or 2000 mg/kg as the upper limit for non-toxic test articles. The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on
survival within 48 hours. The volume to be administered should be compatible with physio
logical space available.
Three adequate spaced dose levels extending over a
range were applied at the central sampling interval treatment. For the highest dose level an additional taken at 48 h after treatment.
single log
24 h after
sample was
Study Procedure
Test Groups;
Six males and six females were assigned to each test group. The
animals were identified by their cage number as shown below in
the table.
/
Test group
Negative control
Low dose Medium dose High dose
Positive control
hours pos-lt-treatment
24
48
male/female male/female
1- 6/ 7-12 61-66/67-72
13-18/19-24
-
/
-
25-30/31-36
-
/
-
37-42/43-48 73-78/79-84
49-54/55-60
-
/
-
mnteec.re
16
Treatment:
Approximately 18 hours before treatment with the test article the
animals received no food but water ad libitum. At -the beginning of the treatment the animals were weighed and the individual volume to be administered was adjusted to the animal's body
weight. The animals received the test article once. Twelve ani
mals, six males and six females, were treated per dose group. Sampling of the bone marrow was done 24 and 48 hours after treat
ment.
Preparation of the Animals:
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a 5 ml syringe. The cell-suspension was centrifuged at 1,500 rpm for 10 minutes
and the supe~rnatant was discarded. A small drop of the resuspend-
ed cell pellet was spread on a slide. The smear was air-dried and
then stained with May-Griinwald (MERCK, D-6100 Darmstadt)/Giemsa
(Gurr, BDH Limited Poo Ie, Great Britain). Cover slips were mount
ed with EUKITT (KINDLER, D-7800 Freiburg). At least one slide was
made from each bone marrow sample.
Analysis of Cells:
Evaluation of the slides was performed using NIKON microscopes
with lOOx oil immersion objectives. 1000 polychromatic erythro-
cytes (PCE) were analysed per animal for micronuclei. To describe
a cytotoxic effect the ratio between polychromatic and normochro-
matic erythrocytes was determined in the same sample and ex pressed in normochromatic erythrocytes per 1000 the PCEs. The
>
analysis was performed with coded slides.
Five animals per sex and group were evaluated as described. The
remaining animal of each test group was evaluated in case an
animal had died in its test group.
mnteec.re
17
DATA RECORDING
The data generated are recorded in the laboratory protocol. The results are presented in tabular form, including experimental groups, negative and positive control. The micronucleated cells per thousand and the ratio of polychromatic to normochromatic erythrocytes are presented for each animal.
EwaLnATioM OF RESULTS
A test article is classified as mutagenic if it induces either a
statistically significant dose-related increase in the number of
micronucleated polychromatic erythrocytes or a reproducible
statistically significant positive response for at least one of
the test points.
A test article producing neither a statistically significant
dose-related"increase in the number of micronucleated polychro
matic erythrocytes nor a statistically significant and reproduci ble positive response at any of the test points is considered
non-mutagenic in this system.
This can be confirmed by means of the nonparametric Mann-Whitney test (6).
However, both biological and statistical significance should be
considered together.
mnteec.re
18 Company Sanitized. Does i
BIOMETRY
Statistical significance at the five per cent level (p < 0.05)
was evaluated by means of the non-parametric Mann-Whitney test.
Negative control versus
Test group
' 200 mg/kg b.w.; 24 h 666 mg/kg b.w.; 24 h
2000 mg/kg b.w.; 24 h 2000 mg/kg b.w.; 48 h
Significance
-
n.t* n.t*
+
P
0.395
0.014
+ = significant; - = not significant; n.t. = not tested
* = mean micronucleus frequency was not above the mean corresponding negative control value
mnteec.re
19
PRE-EXPERIMENT FOR TOXICITY
In a pre-experiment 4 animals (2 males, 2 females) received
orally a single dose of 2000 ing/kg b.w. JJpBBflHHRf^ solved in
aqua deionized. The volume administered was 10 ml/k^b.w..
The treated animals expressed toxic reactions as shown below in the table:
toxic reactions
reduction o
spontaneous activity
eyelid closure
apathy
h<ours posst-treatmesnt male;/female
1 h
6 h
24 h
48 h
2/2
2/2
2/2
2/2
0/0
1/1
1/1
1/1
0/0
0/0
1/0
1/1
In accordance to the guideline-draft "EEC Directive 79/831, Annex
V, B 12" 2000 mg/kg b.w. of the test article was used as maximum
dose in the micronucleus assay.
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20 ^"ecl.Doesi
Summary of results
:test group dose mg/kg sampling
b.w.
time (h)
PCEs with micronuclei
(Z)
range
PCE/NCS
vehicle
0
24
0.13
0-2 1000/ 639
test article
200
24
0.15
0-3 1000/ 716
test
article
666
24
test
article
2000
24
cyclo-
phosphamide
30
24
vehicle
0
'48
test
article
2000
48
0.10 0.07 1.58 0.03 0.11
0-3 1000/ 663 0-2 1000/ 732
8-24 1000/ 923
0-1 1000/736 0-2 1000/ 666
inirteec. re
21 SaniSIzed; Does not contain TSCA CB?'
Micronuclei in polychromatic erythrocytes (PGE) and relationsjtup PCE/MCE (HCE = nonnochromatic erythrocytes) scoring 24 hours after treatment
Table I; vehicle
animal no.
1 2 3 4 5 7 8 9
10 11
sex test group dose
mg/kg b.w.
m
aqua cie ion.
0
m
m
m
m
f f f f f
micronuclei
in 1000 PCE
per animal
2 2 2 1 2 0 1 1 1 1
PCE/NCE
1000/ 669 1000/ 600 1000/ 594 1000/ 536 1000/ 482
IOLOO/ 418
1000/ 726 1000/ 683 1000/ 846 1000/ 834
sum mean
percent cells with micronuclei
13
1.3 0.13
10000/ 6388 1000/ 639
Table II:
test article
fl-Tijjna-1 -
no-r
sex
13
m
14
m
15
m
16
m
17
m
19
f
20
f
,
21
f
22
f
23
f
test
group
-do,semg /kg
b-Tw.
2 00
sum mean
percent cells with micronuclei
-micronuclei
in 1000 PCE
per animal
1 0 1 2 1 1 3 2 1 3
15
1.5 0.15
PCE/NCE
1000/ 817 1000/ 763 1000/ 629 1000/ 679 1000/ 598 1000/ 805 1000/ 789 1000/ 545 1000/ 766 1000/ 767 10000/ 7158 1000/ 716
Table III: test article
micronuclei
in 1000 PCE
per animal
10
1.0 0.10
PCE/NCE
1000/ 763 1000/ 620 1000/ 675 1000/ 485
1QOO/ 696
1000/ 729 10-OQI 851 1000/ 497 1000/ 602 1000/ 709 10000/ 6627 1000/ 663
mnteec.re
22
Micronuclei in polychromatic erythrocytes (PCS) and relationship PCE/HCE (NCE = nomochromatic erythrocytes) scoring 24 hours after treatment
Table IV; test article
micronuclei in 1000 PCE per animal
7
0.7 0.07
PCE/NCE
1000/ 705 1000/ 835 1000/ 584 1000/ 799 1000/ 810 1000/ 603 1000/ 674 1000/ 708 1000/ 810 1000/ 789 10000/ 7317 1000/ 732
Table V: cyclophosphamide
animal no.
49 50 51 52 53 55 56 57 58 59 60
sex test group dose
mg/kg b.w.
m
C: 'A
3 0
m
m
m
m
f
f
f
f
f
/
f
n
sum
mean
percent cells with micronnclei
micronuclei in 1000 PCE per animal
PCE/NCE
14 12 24 13 21
8
14 17
15 not
20
1000/ 1000/ 1000/ 1000/ 1000/ 1000/ 1000/ 1000/ secarable 1000/ 1000/
1097 983 767
1122 989
1122 788 790
693 881
158
15.8 1.58
10000/ 9232 1000/ 923
nmteec.re
23
Does nS contain TSCA CM
Micronuclei in polychromatic erythrocytes (PCE) and relationship PCE/NCE (NCE = normochromatic erythrocytes) scoring 48 hours after treatment
Table VI; vehicle
animal sex test group dose
no.
nig/kg
b.w.
61
m
aqua cieion.
( 3
62
m
63
a
64
m
65
m
67
f
68
f
69
f
70
f
71
f
sum' mean
percent -cells with micronnclei
inicronuclei
in 1000 PCE
per animal
0 1 0 0 1 0 0 0 0 1
3
0.3
0-. 03
PCE/NCE
1000/ 774 1000/ 605 1000/ 758 1000/ 796 1000/ 624 1000/ 711 1000/ 742 1000/ 814 1000/ 795 1000/ 741 10000/ 7360 1000/ 736
Table VII: test article
animal sex test group dose
no.
nig/kg
b.w.
73
m
74
m
75
m
76
m
77
m
79
f
80
f
81
f
82
f
83
f
sum
mean
percent cells <ri.th micronuclei
micronuclei
in 1000 PCE
per animal
1 2 2 0 1 2 1 1 1 0
11
0I..1I1
PCE/MCE
1000/ 687 1000/ 567 1000/ 492 1000/ 756 1000/ 648 1000/ 733 1000/ 455 1000/ 721 1000/ 771 1000/ 827 10000/ 6657 1000/ 666
nmteec.re
24
f
Does not Gontain T8CA CB|
^uptoi-uoiuno
The test; articlelJBBHBflP^f^5 assessed in the micronucleus assay for its potential to jES&uce micronuclei in polychromatic
erythrocytes (PCE) in the bone marrow of the mouse.
The test article was dissolved in aqua deionized. The solvent was used as negative- control. The volume administered orally was 10 ml/kg b.w.. 24 h and 48 h after a single application of the test article the bone marrow cells were collected for micronuclei
analysis.
Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 1000 polychromatic erythro cytes (PCE) per animal were scored for micronuclei.
To describe, a cytotoxic effect due to the treatment with the test article the"ratio between polychromatic and normochromatic ery throcytes (NCE) was determined in the same sample and reported as
the number of NCE per 1000 PCE.
The following dose levels of the test article were investigated: 24 h preparation interval: 200, 666, and 2000 mg/kg b.w., 48 h preparation interval: 2000 mg/kg b.w..
As determined in a pre-experiment 2000 mg/kg b.w. of the test article were tolerated by the animals. Slight toxic reactions
were observed.
The mean number of normochromatic erythrocytes was not increased
after treatment with the test article as compared to the mean
values of NCEs o^the corresponding negative controls, indicating
that^UIIJBBByad no" cytotoxic properties.
At preparation interval 24 hours no substantial enhancement in the frequency of the detected micronuclei after treatment with the test article was observed. The mean micronucleus values were in the range of the corresponding negative control rate.
In comparison to the corresponding negative control at prepara tion interval 48 hours, however, there was a statistically significant enhancement in--the frequency of the detected micronu clei after treatment withRft----HHHBB^B
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This statistically significant effect is considered to be of
minor importance:
1. The corresponding actual negative control rate at prepa ration interval 48 hours was low in this study. The mean historical negative control value obtained within the last 20 experiments at preparation interval 48 hours
was 0.096 %. The range varied from 0.03 % to 0.18 %.
2. The micronucleus frequency of 0.11 % PCEs with micronu-
clei after treatment with 2000 ing/kg ^wQ------||^------H.s
within the range of the historical controJL data presented.
3. The number of micronuclei per animal did not exceed 2 per
1000 PCEs.
Therefore, the statistically significant response is not consid ered to be an indication for an induced mutagenic effect due to the test article.
30 mg/kg b.w. cyclophosphamide administered per os was used as
positive control which showed a distinct increase in induced
micronucleus frequency.
In conclusion., it can be stated that during the study described
and under the experimental conditions reported, the test article
did not induce micronuciei as determined by the micronucleus test with bone marrow cells of the NMRI-mouse.
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DISTRIBUTION OF THE REPORT
Sponsor;
Study Director:
2x (original, copy) Ix (copy)
REFERENCES
1. Heddle, J.A (1973)
A- rapi-d Jbn-vivo test for chromosomal damage
Mutation Research, 18, 187-190
2. Schmid.W. (1976)
The micronucleus test for cytogenetic analysis In: A. Hollaender (Ed.), Chemical Mutagens, Vol.4, Plenum
Press, New York, pp. 31-53
3. Matter, B.E., and J. Grauwiler (1974) Micronuclei in mouse bone marrow cells, a simple in vivo model for the evaluation of drug induced chromosomal aberrations Mutation Res., 23, 239-249
4. Heddle, J.A. and A.V. Carrano (1977) The DMA content of micronuclei induced in mouse bone marrow by
X-irradiation: evidence that micronuclei arise from acentric
chromosomal fragments. Mutation Research, 44, 63
5. Salomone, M.F., J.A. Heddle, E. Stuart and M. Katz (1980) Towards an improved micronucleus test - studies on three model
agents, mitomycin C, cyclophosphamide and dimethylbenz anthra cene. Mutation Research 74, 347
6. Krauth, J. (1971)
Locally most powerful tied rank test in a Wilcoxon situation Annals of Mathematical Statistics, 42, 1949 - 1956
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