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CONFIDENTIAL
DPT 435/984104
IN VITRO MAMMALIAN CHROMOSOME ABERRATION TEST IN HUMAN LYMPHOCYTES
Sponsor
DuPont SpecialityChemicals, Jackson Laboratory, Chambers Works, Deepwater, NJ 08023, USA.
Research Laboratory
Huntingdon Life Sciences Ltd.,
P.O.Box!,
Huntingdon, Cambridgeshire, PE18 6ES,
ENGLAND.
Paee 1 of 27
Report issued 27 October 1998 Comoanv SanKh-crf n^oo---s- -.
DPT 435/984104
CONTENTS
Page
COMPLIANCEWTTH GOOD LABORATORY PRACTICE STANDARDS.........................
3
QUALITY ASSURANCE STATEMENT..................................................L...;..........................
~4
5 SUMMARY................................................................................................................................
6 INTRODUCTION.......................................................................................................................
TEST SUBSTANCE................................................................................................................... 7
EXPERIMENTAL PROCEDURE............................................................................................. 8
ASSESSMENT OF RESULTS................................................................................................... 12
MAINTENANCE OFRECORDS..............................................................................................
12
14 RESULTS....................................................................................................................................
15 CONCLUSION...........................................................................................................................
16 REFERENCES..................................................................................................................
TABLES
/
1. Summary of Results....................----.........----.--..--.--..----.--.--..--.--...........
17
2. Mitotic index data-first test................................. ...............................,,..............--.
18.
3. Metaphase analysis data - first test.--.................--......--.....--.............................
20
4. Mitotic index data - second test................................................................................
22
5. Metaphase analysis data - second test..................................:.........--...........--........
24
APPENDIX
Historical control data.......................................................................................................
26
Comply San.Si.ad. Doss not c^ain TSCA Cm
DPT 435/984104 COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS
The study described in this report was conducted in compliance with the following Good Laboratory Practice standards and I consider the date generated to be valid.
The United Kingdom Good Laboratory Practice Regulations 1997, Statutory Instalment No.654. EC Council Directive, 87/18 EEC of 18 December 1986, (No. L15/29).
OECD Principles of Good Laboratory Practice (as revised in 1997), ENV/MC/CHEM(98)17.
Leslie C. Akhurst B.Sc.(Hons.), C.Biol. M.I.Biol., Study Director, Department of Genetic Toxicology, Huntingdon Life Sciences Ltd.
3 -
Company SgsiEEized. Does iiot contain TSCA CBS
QUALITY ASSURANCE STATEMENT
DPT 435/984104
The following have been inspected or audited in relation to this study
Study Phases Inspected Protocol
Date of Inspection 20 August 1998
Date of Reporting 20 August 1998
Process Based Inspections Cell culture preparation Test material preparation Dosing Harvesting cells Slide preparation Slide reading Media change Record keeping
Report
} 11 June 1998 } }
5 October 1998
12 June 1998 9 October 1998
Protocol: An audit of the protocol for this study was conducted and reported to the Study Director and Company Management as indicated above.
Process based inspections: At or about the time this study was in progress inspections and audits of routine and repetitive procedures employed on this type of study were carried out. These were
conducted and reported to appropriate Company Management as indicated above
Report Audit: This report has been audited by the Quality Assurance Department. This audit was conducted and reported to the Study Director and Company Management as indicated above.
The methods, procedures and observations were found to be accurately described and the reported results to reflect the raw data.
Kevin P. de-Salis, B.A. (Hons.), C.BioL, M.I.BioL, Dip R.Q.A., Quality-Assurance Unit Head,
Department Of Quality Assurance,
Huntingdon Life Sciences Ltd.
Date
r ^ 4: Company Sanitized. Does no? enn^.n <
SUMMARY
DPT 435/984104
A study was performed to assess the ability ofjjHHHBHRto induce chromosomal aberrations in
human lymphocytes cultured in vitro.
Human lymphocytes, in whole blood culture, were stimulated to divide by addition; of phytohaemagglutinin, and exposed to the test substance both in the presence and absence of S9 mix
derived from rat livers. Solvent and positive control cultures were also prepared. Two hours before the end of the incubation period, cell division was arrested using Colcemid,the cells harvested and slides
prepared, so that metaphase cells could be examined for chromosomal damage.
otUBBfq10 In order to assess the toxicity
^ra^--__^^^^^^_^^^J--
-
cultured
human
lymphocytes,
_
the
mi--toti.c-.i.n-d_e-x-w,,as
calculated for all cultures treated with the test substance and the solvent control. On the basis of these
data, the following concentrations were selected for metaphase analysis:
First test Without S9 mix - 3 hours treatment, 18 hours recovery: With S9 mix - 3 hours treatment, 18 hours recovery:
312.5, 625 and 1250 pg/ml. 156.3, 312.5 and 625 pg/ml.
Second test
Without S9 mix - 3 hour treatment, 18 hours recovery:
312.5.450 and 550 pg/ml.
With S9 mix - 3 hours treatment, 18 hours recovery:
312.5,450 and 625 ug/ml.
In the absence of S9 niixij,^|^f^lH^^U^^B^^|^H^^c^aBused a statistically significant increase (P0.001) in the
proportion of metaphase figures containu^chromosomal aberrations, at the two highest dose levels
analysed when compared with the solvent control, in the first test. In the second test a statistically
significant increase (P0.01) in the proportion of metaphase figures containing chromosomal
aberrations was observed at the highest dose level analysed.
In me presence ofS9 mixi|^|fBBI|causeda statistically significant increase (P< 0.001) in the
proportion of metaphase figures containing chromosomal aberrations, at the highest dose level analysed when compared with the solvent control, in the first test. In the second test, statistically significant
increases were seen at me intermediate (P0.001) and highest (P0.01) dose levels analysed.
A quantitative analysis for polyploidy was made in cultures treated with the negative control and highest dose level. Statistically significant increases in the proportion of polyploid cells were seen in both tests in the absence and presence ofS9 mix.
All positive control compounds caused large, statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix.
It is concluded thatnIBBHBRias shown evidence of clastogenic and polyploidy-inducing activity
in both the presence and absence ofS9 mix in this in vitro cytogenetic test system.
:5:
company san{feed- D- ^ contain T.^ ..
INTRODUCTION
DPT 435/984104
This report describes a study designed to assess the ability o^fi^p^B^B^H^^H^W1f0 cause chromosomal
aberrations in human lymphocytes cultured in vitro.
The study was conducted in compliance with the foUowmgjpndeline:
the Testing of Chemicals. (1997) Genetic Toxicology: In Vitro
OECD Guideline for
Guideline 473.
Mammalian Chromosome Aberration Test,
been
used
in this
type
of study
for
a
number of years
(Evans
and O'Riordan divide unless
H1u97m5a, nSclyomttp, hDoecaynte, sDhaanvfeord and Kirldand 1990).
They are cultured in vitro but do not
phytohaemagglutini(nPHA) to the culture which
stimulated to do so. This is achieved by adding
results in a high mitotic yield (Nowell 1960).
healthy male donors was pooled and diluted with tissue culture medium.
In this study, blood taken from incubated in
the
presence
of
PHA
before
being
treated
with
the
test substance. Colcemid
The cultures were Following treatment
the
cells
were
arrested
at
metaphase
using
the
mitotic
inhibitor,
.
of chromosome
Chromosomes
in
these
metaphase
cells
were
then
examined for the presence the first cell division after initiation of
of the aberration frequency sisubastequent cell divisions, hi this laboratory
tarbeeartrmateionnt ss.incTehceerbteasint etysptiemsaotef damage may be lost during
is approximately 15 hours. On this
the cell cycle time for human lymphocytes in whole blood culture
basis, the cells were examined after 21 hours.
The study was performedon two separate occasions. both the presence and the absence of S9 mix.
In both tests, a three hour treatment was used in
according to the classification of the ISCN (1985).
Traditionally gaps have been
Aberrations were scored quanthation of chromosome aberrations.
Some gaps, however, have Pafhak
been
excluded
from the discontinuities
in
DNA
(Heddle and Bodycote
1970,
Satya-Prakash, Hsu
and gaps
has
shown to be real
number of cells containing aberrations both with and without
1981). In this study the total
been calculated.
mutagenic effect until they have been metabolised by enzyme
Many substances do not exert a not found in cultured cells.
Therefore the cultures an(Sd 9temsitx)supbrsetpaanrceed wfreorme
systems that are
presence of a supplementedliver fraction high level of enzymic
incubparteevdioinusblyoutrHehateedabwseitnhcea asnudbstance (Aroelor 1254) known to induce a
rats
activity (Maron and Ames
1983,
Natarajan et al.
1976).
;^
Huntingdon Life Sciences Management on 7 July 1998, by the Sponsor
The protocol was approved by
19 August 1998.
on 17 July 1998 and by the Study Director on
conducted between 19 August'and 21 September 1998.
The experimentalphase of the "study was
:6:
Company
Sanitized.
Doos
not
contain
TSCA
CM
Identity: Chemical name:
Intended use: Appearance: Storage conditions:
Lot number: Expiry: Purity: Date received:
TEST SUBSTANCE
DPT 435/984104
23 June 1998
7:
<.,..,,,,,, <Sflna?7pd- DreES not contain TSCA CBB
EXPERIMENTAL PROCEDURE
DPT 435/984104
CULTURE OF LYMPHOCYTES Human blood was collected aseptically from healfev mate-doBOEs,^>oiriiBdfinddilnted^aath RPMI 1640
tissue culture medium (Life Technologies) supplemented with 10% foetal calf serum (Life Technologies), 201.U./mI peniciUin/20 ug/ml streptomycin (Imperial) and 2 mM glutamine<Imperial). Aliquots (0.4 ml blood : 4.5 ml medium : 0.1 ml phytohaemagglutinin(Life Technologies)) of the cell suspension were placed in sterile universal containers and incubated at 37C for approximately 48 hours. The cultures were gently shaken once daily to resuspendthe cells.
POSITIVE CONTROLS
In the absence of S9 mis.
Identity: Supplier Appearance: Batch number Solvent: Final concentration:
In the presence ofS9 mix
Identity: Supplier Appearance: Batch number: Solvent: Final concentration:
Mitoniycin C
BDH
Blue powder B37741 Sterile deionised water
0.4 and 0.8 ug/ml, one dose level of which was selected for
the metaphase analysis
Cyclophosphamide Sigma Chemical Co Ltd White powder 87H0207 Sterile deionised water 20 and 30 |ig/ml, one dose level of which was selected for the metaphase analysis
PREPARATION OF S9 FRACTION
Species:
Sex: Strain: Source: Age: Weight: Diet:
Rat
Male Sprague-Dawley derived
Harlan Olac Ltd
7-8 weeks
<300g SDS R+M no. 1 (modified) nuts
:8:
Company Sanifae'd. Dsss siot conta'n TSCA CBE
DPT 435/984104
S9 fraction was prepared from a group ofca. 20 animals. Mixed function oxidase systems in the rat livers were stimulated by Aroclor 1254, administered as a single intraperitoneal injection in Arachis oil at a dosage of 500 ing/kg bodyweight. On the fifth day after injection, following an overnight starvation, the rats were killed and their livers aseptically removed.
The following steps were carried out at 0 - 4C under aseptic conditions. The livers were placed in
0.15 M
KC1 (3 ml KC1:
1 gliver)--before being transferred to an homogeniser. Following preparation,
supernatant fraction (S9 fractions-
the homogenates were centrifiiged at 9000 g for 10 minuutnetsi.l reTqhueired. The efficacy of each batch of
wS9as dfirsapcetinosned iwntaos alitqeusotetsd anidn staoredbaact t-e8r0ialC omrubtaetlioown assay with the mutagenic precursors
7,12-dimethylben2anthracene and 2-aminoanfhracene. The sterility was also checked.
PREPARATION OF S9 MIX
S9 mix contained: S9 fraction (10% v/v), MgClz (8 mM), KC1 (33 mM), sodium orthophosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADP (4 mM). All fhe cofectors were filter-
sterilised before use.
SELECTION OF SOLVENT
Prior to commencing testing^the. solubilityproofvtidheedteasst HsuBbs^t^anccteivineingsorelvdeienntst icnowmaptaetri,blie.e.w2it5h0 tmheg/tmesl.t
flHBBHqwas system was assessed.
cleared on warming (37C) to a clear solution. On
aflBBHBHHI^^^ The appearance was
dosing at 1% v/v into aqueous tissue culmre medium,
giving
a
final
concentration
of 2500
[ig/ml,
a
thick precipitate and a colour change to yellow was observed which remained after two hours
incubation at 37C. Further dilutions to 125, 62.5 and 31.3 (ig/ml were made and on dosing at 1% v/v
in aqueous tissue culture medium, gave final concenatlrsaotiopnrsesoefnt12a5f0te, r62tw5 oanhdo3u1rs3 uingc/umbla. tiTohnisatres3u7ltCed.
iCnondceecnretraastiinognslewvietlhs hoigfhpiorenciicpsittarteen,gwthhaicnhd owsemreolality may cause chromosomal aberrations (Galloway
et al. 1987).
Therefore, concentrations greater than 5000 p.g/ml or 10 mM are not used in this test highest final concentration used for subsequent testing was 1250 (ig/ml, in
system. In this case, fhe order to ensure that several dose levels would be in the insoluble range.
TREATMENT OF CELLS WITH TEST SUBSTANCE - FIRST TEST if
ofwUBUlRwere After approximately 48 hours, 50 ul aliquots
added to one set of duplicate
156.3, 312.5, 625 and 1250 ug/ml.
cultures to give final concentrations of 9,8. 19.5, 39.1, 78.1, the solvent control, in 50 ul aliquots, was added to two cultures.
Mitomycin C, at final
Water, concentrations
of 0.4
and
0.8
u.g/ml,
was
added
to
duplicate
cultures.
:9 : f^,ww,, %anEt;7(5rf. D&es not contain TSCA CB5
DPT 435/984104
Immediately before treatment of the second set of cultures, 1 ml of medium was removed from each
culture and discarded. This was replaced with 1 ml of S9 mix, followed by 50 [d aliquots of the
ofM^^^^^^I various dilutions
JB^^JI--------^^^^^^^^^JB giving the same series of final concentrations as above.
i^^^^^^^^^^^^^^^^^^^^
Water
(50 pi) was added to two cultures. Cyclophosphamide was added to duplicate cultures at final
concentrations of 20 and 30 ug/nri.
Three hours after dosing, the cultures were centrifuged at 500 g for 10 minutes and the cell pellets resuspended in fresh medium. They were then incubated for a further 18 hours.
HARVESTING AND FIXATION
Two hours before the cells were harvested, mitotic activity was arrested by addition of Colcemid (Sigrna) to each culture at a final concentration of 0.1 ug/ml. After 2 hours incubation, each cell suspension was transferred to a conical centrifuge tube and centrifuged for 5 minutes at 500 g. The cell pellets were treated with 4 ml ofahypotonic solution (0.075M KC1 prewarmed at 37C). After a 10 minute period ofhypotonic incubation at 37C, 4 ml of ice-cold fresh fixative (3 parts methanol: 1 part glacial acetic acid) was added with gentle agitation. The cultures were then centrifuged at 200 g for 10 minutes, the supernatant removed, and the cell pellets resuspended in 4 ml fixative. The fixative was
replaced further times until it became colourless.
SLIDE PREPARATION
The pellets were resuspended, then centrifuged at 200 g for 10 minutes and finally resuspended in a
small volume of fresh fixative. A few drops of the cell suspensions were dropped onto pre-cleaned microscope slides which were then allowed to air-dry. The slides were then stained in 10% Giemsa, prepared in buffered water (pH 6.8). After rinsing in buffered water the slides were left to air-dry and then mounted in DPX.
MICROSCOPIC EXAMINATION
The
prepared
,"
slides
were
examined
by
light
microscopy
using
a
low power objective.
The proportion
of mitotic cells per 1000 cells in each culture was recorded except for positive control treated cultures.
From these results the dose level causing a decrease in mitotic index of at least 50% of the solvent
control value or, if there was no decrease, the maximum achievable concentration was used 'as the
highest dose level for the metaphase analysis. The intermediate and low dose levels were also selected.
The concentration of each positive control compound selected for analysis was the lowest concentration dosed unless a preliminary scan of metaphase figures indicated an insufficient level of aberrant cells.
The selected slides were then coded. Metaphase cells were identified using a low power objective and examined at a magnification of xlOOO using an oil immersion objective. One hundred metaphase figures were examined, where possible, from each culture. Chromosome aberrations were scored according to the classification of the ISCN (1985). Only cells with 44 - 48 chromosomes were
in
DPT 435/984104
analysed. Polyploid and endoreduplicated cells were noted when seen. aberrant metaphase figures were recorded.
The vernier readings of all
The incidence ofpolyploid metaphase cells, out of 500 metaphase cells, where possible, was determined quantitatively for negative control cultures and cultures treated with the highest dose level of the test
substance used in the analysis for chromosomal aberrations.
The number of aberrant metaphase cells in each treatment group was compared with the solvent control value using Fisher's test (Fisher 1973).
SECOND TEST
Cultures were initiated and maintained as previously described, m this second test a repeat of the first
test was carried out. Concentrations ovfl^H^^H^H^^n^wWfere as follows:
Without S9 mix:
156.3, 312.5,450, 550, 625, 750, 1000 and 1250 ug/ml.
With S9 mix:
78.1. 156.3, 312.5,450, 550, 625, 750 and 1250 ug/ml.
Duplicate cultures were used for each treatment and two cultures were treated with the solvent control. Positive control cultures were treated as in the first test.
Three hours after dosing, all cultures were centrifuged and the cell pellets resuspended in fresh medium. They were then incubated for a further 18 hours.
All cultures were treated with Colcemid,at a final concentration of 0.1 ug/ml, two hours before the end of the incubation period. They were then harvested, fixed and the slides prepared as previously
described. The slides were then examined microscopically as previously described.
STABILITY AND FORMULATION ANALYSIS
The stability of the test substance and of the test substance in the solvent were not determined as part of this study. Analysis of achieved concentration was not performed as part of this study.
: 11 :
ASSESSMENT OF RESULTS
DPT 435/984104
An assay is considered to be acceptable if the negative and positive control values lie within the
current historical control range.
The test substance is considered to cause a positiver^ponssTf'A^MluwingTTOiditiims-areTnet:
Statistically significant increases (P<0.01) in the frequency ofmetaphases with aberrant
chromosomes (excluding gaps) are observed at one or more test concentration.
The increases exceed the negative control range of this laboratory, taken at the 95% confidence limit.
The increases are reproducible between replicate cultures.
The increases are not associated with large changes in osmolality of the treatment medium or extreme toxicfty.
Evidence of a dose-relationship is considered to support the conclusion.
A negative response is claimed if no statistically significant increases in the number of aberrant
cells above concurrent control frequencies are observed, at any dose level.
A further evaluation may be carried out if the above criteria for a positive or a negative response are not met.
MAINTENANCE OF RECORDS
All experimental data arising from the study (including documentary raw data, specimens, records and other materials; collectively defined as the "materials") will remain the property of me
Sponsor. i!
Huntingdon Life Sciences shall retain the materials in its archive for a period of five years from the date of issue of the final report. After such time, the Sponsor will be contacted and their advice
sought on the return, disposal or further retention of the materials; If requested, Huntingdon Life
Sciences will continue to retain' the materials, subject to a reasonable fee being agreed with the
Sponsor.
^
Huntingdon Life Sciences shall also retain a copy of the final report in its archive at Huntingdon indefinitely.
: 12: Company Sanitized. Does r-oi cositain TSCA CB6
RESULTS
DPT 435/984104
FIRST TEST
Toricity data
Mitotic indices of cultured human lymphocytes treated witl^fBHR^re shown in Table 2.
In die absence of S9 mix^M^^f^J^f&^^c^akused a reduction in the mitotic index to 43% of the solvent
control value at 1250 ngTml.This concentration was selected as the highest dose level for the metaphase analysis. Treatment concentrations of 625 and 312.5 pg/ml were chosen as the intermediate
and lowest concentrations for metaphase analysis.
m the presence of S9 mix, no live cells were seen in cultures treated with the highest dose level,
flUfiBR^11^ 1250 pg/ml.
at 625 ug/ml.
a reduction in the mitotic index to 42% of the solvent control value
This concentration was selected as the highest dose level for the metaphase analysis.
Treatment concentrations of 312.5 and 156.3 pg/ml were chosen as the intermediate and lowest
concentrations for metaphase analysis.
The quantitative analysis for polyploidy showed statistically significant increases in the number of polyploid metaphase figures, when compared to the solvent control, in both tests.
The concentrations of the positive control compounds selected for analysis were mitomycin C at 0.8 (ig/ml and cyclophosphamide at 30 u.g/ml.
Metaphase analysis
The effects oq|UBMI^)n the chromosomes of cultured human lymphocytes are shown in Table 3
and summarised in Table 1.
m the absence of S9 inixiu^^^B^B^B^B^B^^Acaused statistically significant increases in the proportion of
cells with chromosomal aberrations (P<0.001) at the highest two dose levels analysed, 625 and 1250 pg/ml. These increases were to 5.5 and 7.5% aberrant cells, respectively, which lie well above the upper 95% limit of the historical control range (1.5%),
In the presence of $9 mix, treatment witjlNi^H^^H^^^H^^B^^^I^^^^n^^prfksulted in a statistically significant increase in the proportion of metaphase figures with chromosomal aberrations (P0.001) at the highest dose level
analysed, 625 pg/ml. This increase was to 9.0% aberrant cells (excluding gaps), which lies well above the upper 95% limit of the historical control range (1.5%).
Both positive control compounds, mitomycin C and cyclophosphamide, caused large, statistically
significant increases (PO.OOl). in the proportion of aberrant cells. This demonstrated the efficacy of the S9 mix and the sensitivity of the test system.
: 13 : Compaq Sanifeed. Does not contain TSCA C3I
DPT 435/984104
SECOND TEST
Toricity data
Mitotic indices of cultured human lymphocytes treated ^itbA^^f^^^^^H^^jjaF shown in Table 4. v^^^^ss--^^"^'
In the absence ofS9 mixj,^n^^^H^^^|^B^R^<caused a reduction in the mitotic index to 43% of the solvent
control value at 550 Hg/mi'^nusconcentrationwas selecled as^lne^ highest dose level for fhe-metaphase--analysis. Treatment concentrations of 450 and 312.5 jig/mi were chosen as the intermediate and lowest
concentrations for metaphase analysis.
In the presence ofS9 mix^Sflfifflfcaased a reduction in the mitotic index to 49% of the solvent
control value at 625 (Jig/mi. This concentration was selected as the highest dose level for the metaphase analysis. Treatment concentrations of 450 and 312.5 ug/ml were chosen as the intermediate and lowest concentrations for metaphase analysis. The quantitative analysis for polyploidy showed statistically significant increases in the number of polyploid metaphase figures, when compared to the solvent control, in both tests. The concentrations of the positive control compounds selected for analysis were mitomycin C at 0.8 ug/ml and cyclophosphamideat 30 ug/ml. Metaphase analysis
The effects ^fM^^f^^S^^W^S^Ay^ the chromosomes of cultured human lymphocytes are shown in Table 5
and summarised in Table 1.
In the absence of S9 niixff^U^--^^--^--^Scakused a statistically significant increase in the proportion of
cells with chromosomal aberrations (P0.01) at the highest dose level analysed, 550 ug/ml. This increase was to 6.5% aberrant cells, which lies well above the upper 95% limit of the historical control
range (1.5%).
In the presence ofS9 mix, treatment witiQBi||BHJ|fesuiteidn statistically significant increases in
the proportion of metaphase figures with chromosomal aberrations (P<0.001 and P0.01) at the two
highest dose levels analysed, 450 and 625 ug/ml, respectively. These increases were to 7.0 and 5.5% aberrant cells, respectively, which lie well above the upper 95% limit of the historical control range (1.5%). Bom positive control compounds, mitomycin C and cyclophosphamide, caused large, statistically
significant increases (P0.001) in the proportion of aberrant cells.
14 <-.-"------,. --;*;'-.).> n-,-t. ,<- 4mi>:n Tisr^K /ot
CONCLUSION
DPT 435/984104
It is concluded that|BlHBIB|Rhas shown evidence of clastogenic and polyploidy-inducing activity
in both the presence ancraieabsence ofS9 mix in this in vitro cytogenetic test system.
15 : Company s^Wz^, ^ ^ ,._. .
REFERENCES
DPT 435/984104
EVANS, HJ., and O'RIORDAN, M.L., (1975) Human peripheral blood lymphocytes for the analysis of chromosome aberrations in mutagen tests. Mutation Research, 31, 135.
FISHER, R.A. (1973) The Exact; Treatment of 2 x 2 Table in: Statistical Methods/or Research Workers. Hafher Publishing Company, New York.
GALLOWAY, S.M-, DEASY, D.A, BEAN, C.L., KRAGNAK, A.R, ARMSTRONG, M.J. and BRADLEY, M.0.-(1987) Effects of high osmotic strength on chromosome aberrations, sister chromatid exchanges and DNA strand breaks, and the relation to toxicity. Mutation Research, 189,
15.
HEDDLE, J.A. and BODYCOTE, D.J. (1970) On the formation of chromosomal aberrations.
Mutation Research, 9, 117.
ISCN (1985) An International System for Human Cytogenetic Nomenclature, HARNDEN, D.G. and KLINGER, H.P. (Eds). S. Karger AG, Basel.
MARON, D.M. and AMES, B.N. (1983). Revised methods for the Salmonella mutagenicity test.
Mutation Research, 113, 173.
NATARAJAN, A.T., TATES, A.D., van BUUL, P.P.W., MEUERS, M. and de VOGEL, N. (1976) Cytogenetic effects of mutagens/carcinogens after activation in a microsomal system in vitro. Mutation Research, 37, 83.
NOWELL, P.C. (1960) Phytohaemagglutinin: an initiator of mitosis in cultures of normal human leukocytes. Cancer Research 20,462.
SATYA-PRAKASH, K.L., HSU, T.C. and PATHAK, S. (1981) Chromatid lesions and chromatid core morphology. Cytogenetics and Cell Genetics, 30, 248.
SCOTT, D-, DEAN, B., DANFORD, N.D. and KIRKLAND, D.J. (1990) Metaphase chromosome abenation assays in vitro in KIRKLAND, D.J. (Ed.) Basic Mutagenicity Tests: UKEMS Recommended procedures. Report of the UKEMS sub-committee on guidelines for mutagenicity
testing. Report, Part I revised p. 62. Cambridge University Press, Cambridge.
^ : Company Sanitized. Does not contain TSCA CBS
TABLE 1
DPT 435/984104
Summary of Results
Testi
Exposure S9
period mix
(hours)
3
-
Concentration of
(ug/ml)
0 (Water) 312.5 625
1250 0.8 (Mitomycin C)
. Cells with aberrations
Excluding gaps
Individual values (%)
Mean (%)
0
0
0.0
0
0
0.0
4
7 - 5.5***
8
7
7.5***
10
10
20.0***
-. - Cells Mth-abenationsL Including gaps
Individual values (%)
Mean (%)
0
0
0
0
4
7
8
7
10
10
0.0 0.0
5.5*** 7.5*** 20.0***
Relative Mitotic Index
(%)
-
95 64 43
-
3
+
0 (Water) 156.3
00 0
0
0.0
0.0
-
0
3
1.5
1
3
2.0
88
312.5 625
0
1
6
12
0.5
9.0***
12 8
12
1.5
10.0***
72 42
30 (Cyclophosphamide)
11
9
20.0*** 12 10
22.0***
-
Test 2
Exposure S9 period mix
(hours)
Concentration of
CtHBBHI.1
(u.g/nu)
3
-
0 (Water)
312.5 450
550
0.8 (Mitomycin C)
3
+
0 (Water)
- 312.5 450
625
;'
J'.-'
30 (Cyclophosphamide)
*** **
Otherwise
P0.001 P0.01
P>0.01
Cells with aberrations Excluding gaps
Individual values (%)
Mean (%)
1
I
1.0
0
1
5
3
8
5
12
12
0.5 4.0
6.5** 24.0***
Cells with aberrations Including gaps
Individual values (%)
Mean (%)
1
1
0
1
5
4
8
5
12
12
1.0 0.5 4.5
6.5** 24.0***
1
1
1
3
6
8
6
5
14
17
1.0
1
1
2.0
1
3
7.0*** 6
8
5.5**
6
5
15.5*** 15 17
1.0 2.0
7.0*** 5.5** 16.0***
Relative Mitotic Index
(%)
-
84 55 43
-
.
85 67 49
-
: 17 : Comnatw Sanitized. Doss noS contain TSCA CBS
DPT 435/984104
TABLE 2
Mitotic index data - first test
Without S9 mix, 3 hours treatment and 18 hours recovery
Concentration o^
^Uig/nu)
0 (Water)
MitotieiHdex-
Incidence
124/1000 152/1000
%Mean
13.8
----Relative mitotic index (%)
100
Eolxploidx
..
...
Incidence
0/500 1/500
% Mean 0.1
9.8 19.5 39.1 78.1 156.3 312.5
625^
1250
147/1000
15.6
113
165/1000
169/1000
17.2
125
175/1000
159/1000
15.7
114
155/1000
154/1000
14.6
106
137/1000
118/1000
11.6
84
114/1000
139/1000
13.1
95
122/1000
87/1000
8.9
64
90/1000
68/1000
6.0
43
52/1000
7/205
3.3
7/221
***
Precipitate in cultures treated with 312.5, 625 and 1250 pg/ml on dosing
Precipitate in cultures treated with 625 and 1250 ug/ml at end of treatment (3 hours)
***
i:; Otherwise
P<O.OOI PX).01
18 : Company Sanitized. Does not contain TSCA CBI
DPT 435/984104
TABLE 2
Mitotic index data - first test (continued)
With S9 mix, 3 hours treatment and 18 hours recovery
Concentration q^
(tig/mi) 0
(Water)
Mitotic index
Incidence
148/1000 156/1000
%Mean
15.2
Relative "initotie-iadex--
(%)
100
Polyploidy
Incidence
0/500 2/500
% Mean 0.2
9.8
16771000
16.3
107
158/1000
19.5
111/1000
13.7
90
162/1000
39.1
150/1000
15.5
102
160/1000
78.1
155/1000
16.2
107
168/1000
156.3
127/1000
13.4
88
140/1000
312.5
120/1000
10.9
72
98/1000
625 1250
57/1000
6.4
42
11/438
3.4
70/1000
21/500
***
0/0
-
0/0
Precipitate in cultures treated with 625 and 1250 ug/ml on dosing
* '" po.OOl Otherwise P>0.01
19 ' Company Sanitized. Does not contain TSCA CBr
DPT 435/984104
TABLE 3
Metaphase analysis data - first test
Without S9 mix, 3 hours treatment and 18 hours recovery
Concentration of Otg/ml)
0
(Water)
No. cells examined Chromatid
type ctb cte
Aberrations Chromosome Others
type csb cse
Gaps ctg csg
No. of aberrant cells Exc. Mean Inc. Mean
gaps % gaps %
Relative Mitotfc index
% .;
100
0
0.0 0
0.0
100
100
0
0
312.5
100
100
625
100
7
ioo
7
1
3
1
1250
100
10
2
100
12
3
0 0.0 0 0.0
95
0
0
4 5.5 4 5.5
64
7 *** 7 ***
8 7.5 8 7.5
43
7 *** 7 ***
0.8 Mitomycin C
50
14
2
2
50
21
2
1
10 20.0 10 20.0
10 *** 10 ***
ctb
Chromatid break
csb
Chromosome break
ctg
Chromatid gap
***
Otherwise,,
P<0.001 P>0.01
cte cse csg others
Chromatid exchange Chromosome exchange
Chromosome gap Cells with greater than 10 aberrations, pulverised cells and pulverised chromosomes
^, 20: Company Sanitized. Doss not coRtem T.^A
Concentration of
Qtg/nd)
0
(Water)
DPT 435/984104
TABLE 3
Metaphase analysis data - first test (continued) With S9 mix, 3 hours treatment and 18 hours recovery
No. cells examined
Chromatic! type
ctb cte
Aberrations Chromosome Others
type csb cse
Gaps ctg csg
No. of aberrant cells Exc. Mean Inc. Mean
gaps % gaps %
Relative -RGioac
index %
100
0
0.0
0
0.0
100
100
0
0
156.3 312.5 625
100
100
1
2
100
100
1
100
2
1
4
100
17
1
9
30 Cyclophosphamide
50
9
3
2
50
4
4
7
1
0
1.5
1 2.0
88
3
3
1
0
0.5
1
1.5
72
1
1
2
3
6
9.0
8 10.0
42
1
12 *** 12 ***
1
11 20.0 12 22.0
1
9 *** 10 ***
ctb
Chromatid break
csb
Chromosome break
ctg
Chromatid gap
**
Otherwise
P<0.001 P>0.01
cte cse csg others
Chromatid exchange
Chromosome exchange
Chromosome gap Cells with greater than 10 aberrations, pulverised cells and pulverised chromosomes
21 : Company Sanitized. Doss not contain TSCA CBii
TABLE 4
DPT 435/984104
Mitotic index data - second test
Without S9 mix, 3 hours treatment and 18 hours recovery
Concentration Q^,
(Ug/ml)
0
(Water)
Mitoticmdex
Incidence
183/1000 190/1000
% Mean 18.7
- - --ftelative mitotic index (%)
100
Petypleidy- - - -
Incidence
0/500 0/500
% Mean 0.0
156.3 312.5 450 550 625 750 WQQ 1250
173/1000
15.7
84
141/1000
170/1000
15.8
84
146/1000
100/1000
10.2
55
103/1000
70/1000
8.0
43
90/1000
81/1000
7.0
37
58/1000
61/1000
5.9
32
57/1000
37/1000
3.8
20
39/1000
20/1000
1.8
10
16/1000
15/500
3.2
17/500
***
Precipitate in cultures treated from 312.5 to 1250 pg/ml on dosing
** .
Otherwise
p<o.ooi P>0.01
11 ^omPany SanEtissd. Does not contafn TSCA CB8
DPT 435/984104
TABLE 4
Mitotic index data - second test (continued)
With S9 mix, 3 hours treatment and 18 hours recovery
Concentration o^
(Ug/ml)
0
(Water)
Mitotic index
Incidence
127/1000 108/1000
% Mean 11.8
Relative mitotic index
(%)
100
Polyploidy
Incidence
0/500 2/500
% Mean 0.2
78.1 156.3 312.5 450 550 625 750 1250
101/1000
10.5
89
109/1000
117/1000
11.4
97
111/1000
98/1000
10.0
85
101/1000
84/1000
7.9
67
73/1000
61/1000
6.3
53
65/1000
57/1000
5.8
59/1000
47/1000
3.8
28/1000
49
15/500
3.6
21/500
***
32
35/1000
3.4
29
33/1000
Precipitate in cultures treated with 450 and 1250 p,g/nu on dosing
**;
Otherwise
PO.OOl P>0.01
23 : Company Sanitized. Doss not contain TSCA CB
DPT 435/984104
TABLES
Metaphase analysis data - second test
Without S9 mix, 3 hours treatment and 18 hours recovery
Concentration of (fig/sal)
0
(Water)
No. cells examined
Chromatic! type
cib cte
Aberrations Chromosome Others
type csb ese
100
1
100
2
Gaps ctg csg
No. of aberrant cells Exc. Mean Inc. Mean
gaps % gaps %
Relative Mitotic index
.%--
1
1.0 1
1.0
100
1
1
312.5 450 550
100
100
1
100
14
100
7
1
100
7
2
1
100
22
1 1
0 0.5 0 0.5
84
1
1
5 4.0 5 4.5
55
3
4
8 6.5 8 6.5
43
5 ** 5 **
0.8 Mitomycin C
50
19
3
2
50
10
8
1
1
12 24.0 12 24.0
12 *** 12 ***
ctb
Chromatid break
csb
Chromosome break
ctg
Chromatid gap
*** **
Otherwise ,*
P0.001
P<0.01 PX).01
cte cse csg others
Chromatid exchange Chromosome exchange
Chromosome gap Cells with greater than 10 aberrations, pulverised cells and pulverised chromosomes
24:
Company Sanitized. Dcss not contain TSCA CBi
Concentration of
Otg/ml)
0
(Water)
DPT 435/984104
TABLES
Metaphase analysis data - second test (continued) With S9 mix, 3 hours treatment and 18 hours recovery
No. cells
examined
Chromatid type
ctb cte
- AbeHatieas--- - - Chromosome Others
type csb cse
Gaps ctg csg
- -Nor Q^abersaBt cells- -
Exc. Mean Inc. Mean
gaps % gaps %
-RelativeMitotic
index %
100
1
100
1
1
1.0
1
1.0
100
1
1
312.5 450 625
100
1
100
4
100
8
100
10
2
100
4
2
100
6
30 Cyclophosphamide
100
12 3
3
100
11
2
6
1
1 1
1 2.0 1 2.0
85
3
3
6 7.0 6 7.0
67
g *** g ***
6 5.5 6 5.5
49
5 ** 5 **
14 15.5 15 16.0
17 *** YI ***
ctb
Chromatid break
csb
Chromosome break
ctg
Chromatid gap
*** **
wise
P<0.001 P<0.01 P>0.01
cte cse csg others
Chromatid exchange Chromosome exchange
Chromosome gap Cells with greater than 10 aberrations, pulverised cells and pulverised chromosomes
: 25 : Company Sanitized. Doss not contain TSCA CB!
DPT 435/984104 APPENDIX
Historical control data Aberrant cells seen in human lymphocytes in whole blood culture, treated with solvent controls from February 1995 until May 1998.
Total no. of cells
Without gaps
No. of aberrant
cells
%mean
Range of means (%)
min
max
Upper 95% confidence
limit
(%)
-S9
80780
477
0.59
0
3.68
1.5
+S9
75600
429
0.57
0
3
1.5
-S9 +S9
Total no. of cells
80780
With gaps
No. of
aberrant cells
%mean
609
0.75
Range of means (%)
min
max
0
4.47
Upper 95% confidence
limit (%)
1.76
75600
542
0.72
0
3
2
Polyploid cells seen in human lymphocytes in whole blood culture, treated with solvent controls from February 1995 until May 1998.
Total no.
No. of %mean Range of means
Upper 95%
of cells polyploid
(%)
confidence
cells
limit
min
max
(%)
-S9
33391
37
0.11
0
- 0.5
0.5
*
+S9
30433
66
0.22
0
0.7
0.63
: 26 : 'Company SanEfizad. Doss not csniap:n TSCA CBl
APPENDIX
DPT 435/984104
Historical control data (continued)
Aberrant cells seen in human lymphocytes in whole blood culture, treated with positive controls from February 1995 until May 1998.
Total no. of cells
No. of
aberrant cells -
%mean
Without gaps Range of means (%)
min
max
-S9
29876
4663
15.61
8
48
Lower 95%
confidence limit
8.75
Upper 95% confidence
limit (%)
31
+S9
27064
4184
15.46
9
42
10
27.95
Total no. of
cells
No. of
aberrant cells
%mean
With gaps Range of means (%)
min
max
Lower 95%
confidence limit
-S9
29876
4851
16.24
8.5
51.5
9.25
Upper 95% confidence
limit (%)
32
+S9
27064
4342
16.04
9
42
10
29
CompanySa^ed.OoesnotcontalnTSCACBS
27 =