APPENDIX 2

ACTIVITY PROFILESFOR GENETIC AND RELATED EFFECTS

APPENDIX 2

ACTIVTY PROFILESFOR GENETIC AND RELATED EFFECTS

Methods

The x-axis of the activity profile (Waters et. al., 1987, 1988) represents thebioassays in phylogenetic sequence by endpoint, and the values on the y-axis

represent the logarithmically transformed lowest effective doses (LED) and highestineffective doses (HID) tested. The term 'dose', as used in this report, does not takeinto consideration length of treatment or exposure and may therefore be consideredsynonymous with concentration. ln practice, the concentrations used in all thein-vitro tests were converted to l1g/ml, and those for in-vivo tests were expressedas mg/kg bw. Because dose units are plotted on a log scale, differences in molecularweights of compounds do not, in most cases, greatly influence comparisons of theiractivity profiles. Conventions for dose conversions are given below.

Profile-line height (the magnitude of each bar) is a function of the LED orHID, which is associated with the characteristics of each individual test system -such as population size, cell-cycle kinetics and metabolic competence. Thus, thedetection limit of each test system is different, and, across a given activity profile,responses wil vary substantially. No attempt is made to adjust or relate responsesin one test system to those of another.

Line heights are derived as follows: for negative test results, the highest dosetested without appreciable toxicity is defined as the HID. If there was evidence ofextreme toxicity, the next highest dose is used. A single dose tested with a negativeresult is considered to be equivalent to the HID. Similarly, for positive results, theLED is recorded. If the original data were analysed statistically by the authorJ thedose recorded is that at which the response was significant (p .( 0.05). If theavailable data were not analysed statistically, the dose required to produce an effectis estimated as follows: when a dose-related positive response is observed with twoor more doses, the lower of the doses is taken as the LED; a single dose resultingin a positive response is considered to be equivalent to the LED.

ln or der to accommodate both the wide range of doses encountered andpositive and negative responses on a continuous scale, doses are transformed

-491-

492 lARe MONOGRAHS VOLUME 52

logarithmically, so that effective (LED) and ineffective (HID) doses are representedby positive and negative numbers, respectively. The response, or logarithmic doseunit (L~Uij), for a given test system i and chemical j is represented by theexpressions

LDUij = -log1o (dose), for HID values; LDU ~Oand

5LDUij = -log1o (dose x 10-), for LED values; LDU ):0.These simple relationships define a dose range of 0 to -5 logarithmic units for

ineffective doses (1-100 00 iiglml or mg/kg bw) and 0 to + 810garithmic units foreffective doses (100 00.001 iig/ml or mg/kg bw). A sc ale ilustrating the LDUvalues is shown in Figure 1. Negative responses at doses less than 1 iiglml (mg/kgbw) are set equal to 1. Effectively, an LED value ~ 100 00 or an HID value ~1produces an LDU = 0; no quantitative information is gained from such extremevalues. The dotted lines at the levels of log dose units 1 and -1 define a 'zone ofuncertainty' in which positive results are reported at such high doses (between1000 and 100 00 iiglml or mg/kg bw) or negative results are reported at such lowdose levels (1 to 10 iig/ml or mg/kg bw) as to caII into question the adequacy of thetest.

Fig. 1. Scale of log dose units used on the y-axis or activity profiles

Positive Log dose(J.glml or mg/kg bw) units

0.001 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80.01 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70.1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61.0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

10 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4100 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

100 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210 00 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

100 00 . . . . . . . . . . . . . . . .. 1 .... . . . . . . . . . . . 0. . . . . . . . . . . . . . .. 10 .. . . . . . . . . . . . . . - 1. . . . . . . . . . . . . .. 100 . . . . . . . . . . . . . . . -2. . . . . . . . . . . . .. 100 . . . . . . . . . . . . . . . -3. . . . . . . . . . . . . 10 00 . . . . . . . . . . . . . . . -4. . . . . . . . . . . . 100 00 . . . . . . . . . . . . . . . -5

(1)

Negative

(J.glml or mg/kg hw)LED and HID are expressed as llg/ml or mg/kg bw.

APPENDIX 2493

ln practice, an activity profie iscomputer generated. A data entry programmeis used to store abstracted data from published reports. A sequential file (in ASCII)is created for each compound, and a record within that file consists of the nameand Chemical Abstracts Service number of the compound, a three-Ietter code forthe test system (see below), the qualitative test result (with and without anexogenous metabolic system), dose (LED or HID), citation number and addition alsource information. An abbreviated citation for each publication is stored in asegment of a record accessing both the test data file and the citation file. Duringprocessing of the data file, an average of the logarithmic values of the data subsetis calculated, and the length of the profile line represents this average value. AlI dosevalues are plotted for each profie line, regardless of whether results are positiveor negative. Results obtained in the absence of an exogenous metabolic system areindicated by a bar (-), and results obtained in the presence of an exogenous

metabolic system are indicated by an upward-directed arrow (t). When aIl resultsfor a given assay are either positive or negative, the mean of the LDU values isplotted as a solid line; when conflcting data are reported for the same assay (i.e.,both positive and negative results), the majority data are shown by a solid line andthe minority data by a dashed line (drawn to the extreme conflcting response). lnthe few cases in which the numbers of positive and negative results are equal, thesolid line is drawn in the positive direction and the maximal negative response isindicated with a dashed line.

Profile lines are identified by three-Ietter code words representing thecommonly used tests. Code words for most of the test systems in current use ingenetic toxicology were defined for the US Environmental Protection Agency'sGENE-TOX Program (Waters, 1979; Waters & Auletta, 1981). For !ACMonographs Supplement 6, Volume 44 and subsequent volumes, including thispublication, codes were redefined in a manner that should facilitate inclusion ofadditional tests. Naming conventions are described below.

Data listings are presented in the text and include end point and test codes,a short test code definition, results reither with (M) or without (NM) an exogenousactivation system), the associated LED or HID value and a short citation. Testcodes are organized phylogeneticaIly and by endpoint from left to right across eachactivity profile and from top to bottom of the corresponding data listing. Endpointsare defined as follows: A, aneuploidy; C, chromosomal aberrations; D, DNAdamage; F: assays of body fluids; G, gene mutation; H, host-mediated assays; l,inhibition of intercellular communication; M, micronuclei; :P sperm morphology;R, mitotic recombination or gene conversion; S, sister chromatid exchange; and 'fcell transformation.

494 lARe MONOGRAHS VOLUME 52

Dose conversions for activity profiles

Doses. are converted to J.g/ml for in-vitro tests and to mg/kg bw per day forin-vivo experiments.

1. ln-vitro test systems

(a) Weight/volume converts directly to J.g/ml.

(b) Molar (M) concentration X molecular weight = mg/ml = HP J.g/ml; mMconcentration X molecular weight = J.g/ml.

(c) Soluble solids expressed as % concentration are assumed to be in units

of mass per volume (i.e., 1% = 0.01 g/ml = 10 00 llg/ml; also, 1 ppm= 1 llg/ml).

(d) Liquids and gases expressed as % concentration are assumed to be givenin units of volume per volume. Liquids are converted to weight per volumeusing the density (D) of the solution (D = g/ml). Gases are convertedfrom volume to mass using the ideal gas law, PV = nR'I For exposureat 20-37°C at standard atmospheric pressure, 1% (v/v) = 0.4 llg/ml Xmolecular weight of the gas. Also, 1 ppm (v/v) = 4 x 10-5 l1g/ml xmolecular weight.

(e) ln microbial plate tests, it is usual for the doses to be reported asweight/plate, whereas concentrations are required to enter data on theactivity profile chart. While remaining cognisant of the errors involvedin the process, it is assumed that a 2-ml volume of top agar is deliveredto each plate and that the test substance remains in solution within it;concentrations are derived from the reported weight/plate values by

dividing by this arbitrary volume. For spot tests, a 1-ml volume is usedin the calculation.

(f Conversion of particulate concentrations given in J.g/cm2 are based onthe area (A) of the dish and the volume of medium per dish; i.e., for a100mm dish: A = 11R2 = 11 x (5 cm)2 = 78.5 cm2. If the volume ofmedium is 10 ml, then 78.5 cm2 = 10 ml and 1 cm2 = 0.13 mL.

2. ln-vitro systems using in-vivo activation

For the body fIuid-urine (BF-) test, the concentration used is the dose (inmg/kg bw) of the compound administered to test animaIs or patients.

3. ln-vivo test systems

(a) Doses are converted to mg/kg bw per day of exposure, assuming 100%absorption. Standard values are used for each sex and species of rodent,including body weight and average intake per day, as reported by Gold

APPENDIX 2495

et al. (1984). For example, in a test using male mice fed 50 ppm of the agentin the diet, the standard food intake per day is 12% of body weight, andthe conversion is dose = 50 ppm X 12% = 6 mg/kg bw per day.Standard values used for hum ans are: weight - males, 70 kg; females,

55 kg; surface area, 1.7 m2; inhalation rate, 20 l/min for light work, 30 l/minfor mild exercise.

(h) When reported, the dose at the target site is used. For example, dosesgiven in studies of lymphocytes of humans exposed in vivo are themeasured blood concentrations in l1g/mI.

Codes for test systemsFor specific nonmammalian test systems, the first two letters of the

three-symbol code word define the test organism (e.g., SA- for Salmonellatyhimunum, EC- for Eschenchia coli). If the species is not known, the conventionused is -S-. The third symbol may be used to define the tester strain (e.g., SA8 forS. tyhimunum TA1538, ECW for E. coli WP2uvrA). When strain designation is notindicated, the third letter is used to define the specific genetic end point underinvestigation (e.g., -D for differential toxicity, -F for forward mutation, -G forgene conversion or genetic crossing-over, -N for aneuploidy, -R for reverse

mutation, -U for unscheduled DNA synthesis). The third letter may also be usedto define the general endpoint under investigation when a more complete definitionis not possible or relevant (e.g., --M for mutation, -C for chromosomalaberration).

For mammalian test systems, the first letter of the three-Ietter code worddefines the genetic endpoint under investigation: A- for aneuploidy, B- forbinding, C- for chromosomal aberration, D- for DNA strand breaks, G- forgene mutation, 1- for inhibition of intercellular communication, M- formicronucleus formation, R- for DNA repair, S- for sister chromatid exchange,T - for cell transformation and U- for unscheduled DNA synthesis.

For animal (i.e., non-hum an) test systems in vitro, when the cell type is notspecified, the code letters -lA are used. For such assays in vivo, when the animalspecies is not specified, the code letters -VA are used. Commonly used animalspecies are identified by the third letter (e.g., -C for Chinese hamster, -M formou se, -R for rat, -S for Syrian hamster).

For test systems using human cells in vitro, when the cell type is not specified,the code letters -IH are used. For assays on humans in vivo, when the cell type isnot specified, the code letters - VH are used. Otherwise, the second letter specifiesthe cell type under investigation (e.g., -BR for bone marrow, -LH for lymphocytes).

Sorne other specific coding conventions used for mammalian systems are asfollows: BF- for body fluids,RM- for host-mediated, -L for leucocytes or

496 lARe MONOGRAHS VOLUME 52

lymphocytes in vitro (-AL, animaIs; -HL, humans), -L-for leucocytes in vivo (-LA,animaIs; -LH, humans), -T for transformed cells.

Note that these are examples of major conventions used to define the assaycode words. The alphabetized listing of codes must be examined to confirm aspecific code word. As might be expected from the limitation to three symbols,sorne codes do not fit the naming conventions precisely. ln a few cases, test systemsare defined by first-Ietter code words, for example: MS1; mouse spot test; SL~mouse specific locus test, postspermatogonia; SLO, mouse specific locus test, otherstages; DLM, dominant lethal test in mice; DLR, dominant lethal test in rats; MHrrmouse heritable translocation test.

The genetic activity profiles and listings that follow were prepared incollaboration with Environmental Health Research and Testing Inc. (EHRT) undercontract to the US Environmental Protection Agency; EHRT also determined thedoses used. The references cited in each genetic activity profile listing can be foundin the list of references in the appropriate monograph.

Rererences

Garrett, N.E., Stack, H.P., Gross, M.R. & Waters, M.D. (1984) An analysis of the spectraof genetic activity produced by known or suspected human carcinogens. Mutat. Res.,134, 89-111

Gold, L.S., Sawyer, C.B., Magaw, R., Backman, G.M., de Veciana, M., Levison, R.,Hooper, N.K., Havender, W.R., Bernstein, L., Peto, R., Pie, M.C. & Ames, B.N.(1984) A carcinogenic potency database of the standardized results of animal bioassays.Environ. Health Perspect., 58, 9-319

Waters, M.D. (1979) The GENE- TOX program. ln: Hsie, A.W., O'Neil, J.P. & McElheny,\iK., eds, Mammalian Cel! Mutagenesis: The Maturation of Test Systems (Banbury Report2), Cold Spring Harbr, NY, CHS Press, pp. 449-47

Waters, M.D. & Auletta, A. (1981) The GENE-TOX program: genetic activity evaluation.1 chem. ln! comput. Sei., 21,35-38

Waters, M.D., Stack, H.P., Brady, A.L., Lohman, P.H.M., Haroun, L. & Vainio, H. (1987)Appendix 1: Activity profies for genetic and related tests. ln: lAC Monographs on theEvaluation of the Careinogenic Risk of Chemicals to Humans, Suppl. 6, Genetic andRelated Effects: An Update of Selected lARe Monographs from Volumes 1 to 42, Lyon,lARe, pp. 687-696

Waters, M.D., Stack, H.P., Brady, A.L., Lohman, P.H.M., Haroun, L. & Vainio, H. (1988)Use of computeried data listings and activity profies of genetic and related effectsin the reviewof 195 compounds. Mutat. Res., 205,295-312

U1i--z::wU1ooLlo..

8

6

4

2

o

-2

-4

SODIUM CHLORITE 7758-19-2

5

A

o

C

1

C

H

V

H

1111- -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ~ - ~- - - - - ~ - - - - - - - - --T~

C AB VA A

PROKARYOTES 1 Lm/ER EUKAR. MAMMALS lN VITRO HUMANS 1 F /HI MAMMALS lN ViVOlN VITRO

~""trZU-:xN

+:1...

4(J1--Z:J 2w(J00t. 00..

-2

8

6

-4

SODIUM HYPOCHLORITE

E 555C RRRD 035

7681-52-9

C

1

C

5

H

F

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - -. - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - --

- - - - - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - -. - - - - - - - - - - - - - - - - --

5

R

8

PROKARYOTES 1 LOWER EUKAR.

C

H

F

H CV BH R

MAMMAlS lN VITRO HUMANS 1 F /HI MAMMAlS lN ViVOlN VITRO

R

V

R

.i1.00

~

~s:ozoo

~~CI

â5s:tIVlN

t. :: a:

t. = -'

"3l

l"rul

l.l"'- - '-

u: Ln l-

wzcr:i1-t. u: 0:

W:Lt. .. u:

0et0-l:iut. a: t.

..t. a: ..

0t. a: ..

0:L0et(D

APPE

ND

IX 2

~-------------1----J1111.

11111---~-----------~

1111~

coru

"31

lO"3

51INn 3500 :JOL

oru1

499

l.OZ :;

a: -:: :;:: Z:i _a:;:;2:l.

:: :: ::-ia:::::a::::i..u.l. az

eta:

l-::

:;:::i

2:

Det1-:;2:l.-ia:t. _ '-

::::a:::1-UWl.2:l.1-Za:-ic.cia::1::WetW=-D-il.w

t. a: ""

l-Dt. = Ln

)-ett.=

oa::1aetc.

8

6

4U1f--Z=i

wU1ooLlo-i

2

o

-2

-4

BROMOFORM

5

A

o

A

C

C

75-25-2

o

"x

G 55 1T C

C

1

C

5

H

L

5"VV

A"

- -- - - - -- - - - -- -- -- - -- - - -- - -- - - --- - - - - --- - -. -- - - --- - ---- - - -- - - --- -- - - - - - - --. -- - - - -- - - - -- - - - ------- -- - -- - - -------~

111,-------,--,11,11

.L,1

A If

PROKARYDTES

,11

- -- -- - - -- - - - -- -- - _ _ _ _ _ _ _ -- -- _!-1,,,1111,

!l!lIlll!Y!!

Lm/ER EUKAR.

1,,

- -- -- - - - --- - - - - -- - - - -- -- - - - - __ __ ___ _ _ _ _ _ _ _ _ __ _ _ J__11,,1,,11~

o

M

H

MAMMALS lN VITRO

C

B

A

HUMANS

lN VITROF /HI MAMMALS lN ViVO

8

~na:ozoo~:ien~oBa:trVIN

U'i-l-Z::wU'ooLJo-l

8

6

4

2

o

-2

-4

CH LOR 0 DIB R 0 MO ME T HAN E 124-48-1

5 $5A fiAo &'9

5

C

G

C

lC

5

H

L

5

V

A

- -- - - - - - -~ N - - - - - - ~ - - - - - - - - - - _ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ N _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ ~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _" _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _.

11 III1 III.___ 1 III___,._U.J__: ::: --------1-----1 Il 1 --------1 Il 1 ------1 Il 1 ---------1 Il 1 ------, '" --------. '" ----------n '" -------!li ------Xv, -----"------------------

5

C

R

PROKARYOTES LOWER EUKAR. MAMMLS lN VITRO HUMANS

lN VITRO

H

V

H

F /HI MAMMALS lN VIVO

?;"'trZU~~N

~~

502lA

Re M

ON

OG

RA

HS

VO

LUM

E 52

cutO

a-ru

oru1

a-l

51INn 3500 ~OL

APPE

ND

IX 2

503

ui DZ

:;cr -

i: :;

:: zI_D:;;;~ui

x:~x:-.cri:i:cri:i"-L

L

ui DZ

etcr !:

i: :;

::I~ru

=-=

1

a-Det

..~

1;;

£'~

a..ui-.cr

ln _ '-i:i:cri:~uwui~ui~zcr-.Q

.

ricrW

l£::-l

w..

et0:

w1-

~..

DZ

-.01-W

uiU

Wln cr 0)

~a:

D0

U1 cr U"

)-et0:

U1 cr C)

cr0

l£D-l

et:i

Q.

u

cotO

a-ru

aru1

a-1

51INn 3500 ~OL

50lA

Re M

ON

OG

RA

HS

VO

LUM

E 52

ln Dz:;a: -I: :;::zI_D:;;;2;ln

:E =

- :E-ia:I:I:a:I:I"-i.ln DzO

:a: !:I: :;::I2;

L.1

t0"3D

10:1-

ru;;

L.

2;rut0

ln-iui-L

.a:I:I:a:I:1-UW

=x:::

ln2;ln1-Za:-ia.Iiw

a:::-.

::..

wfY

0:f-

w..

~Z

DD

-if-WU

lna:

wu: = en

l-D

DL

u: = Ln

~0:D

u:==

a:fY

::Drn

0:..

a.D

co1O

"3ru

oru1

"31

51INn 3500 ~OL

APPE

ND

IX 2

505

ln c:

z ~

II -1: ~:: :z

:i_a~:;~lnX

:~%:

..~1:II1::i"-laln c:

zci~ S;

:::i~a

1=

-=ru

a..

Il1

1-

C(

:;..

~at0

ln..IIUl _ '-

E~1-UWln=

:z_~ln1-ZII..a.

WiiII

-.li

..::

0:w

~Il

..W

Z~

0a.J

~WUln

crW

0U

l = en

1-a0:

Ula:~

r0

ciUl a: c:

II-.

li:i

aIlU

a...0

C(

1O.;

rua

ru1

.;i

51INn 3500 ~OL

4uii---Z:: 2wui00Ll 00..

-2

8

6

-4

TRICHLOROACETONITRILE 545-06-2

5

1

C

- - - - - - - -- - -- - - -- - - -- - - - - - - - - -- - - - -- - - --- - - - -- - - - - - - -. - - - - - - - -- - - - -- - - - -- -- -- -- -- - - - -- - -- --- - - --- - --- - -- ---- -- ~

- - - - - -. -. -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - - - - - - - - -. - - - - - - - - - - - - -- - - --

5 5 5

A A A

o 5 9

PROKARYOTES 1 Lm/ER EUKAR. MAMMALS lN VITRO

H

V

H

HUMANS 1 F /HI MAMMALS lN vivolN VITRO

~

~~ozoo~ttC/

~o8~t'UlN

4

Ul~-Z:: 2w(J00Ll 00-l

-2

8

6

BROMOETHRNE

5 5R Ro 5

74-96-4

5

1

C

------~---------------------------------------------~--------------------------------------------------------_.

1 11 11 1

------j-r'------------------------------'---------------,-----------------------------------------------------1 11 11 1r 11 1-4 -- A

55RR

79

PROKARYOTES

o

H

X

LOWER EUKAR.

c

ic

MAMMALS lN VITRO HUMANS

lN VITRO

F /HI MAMMALS lN ViVO

~"'tTZU~~N

Vlo..

508lA

Re M

ON

OG

RA

HS

VO

LUM

E 52

ui 0z~cr

-1: ~:: zI_0~:;2!ui..cr1:1:cr1:I"-i.ui 0Z

Ilcr !:1: ~::I2!

i-10

aIl

i- = z:

l-a

:;1

2!1.f'

ui..cr1:1:cr1:

l-UWui2!ui1-Zcr..a.Iicr:l::wIlw:xW

0..Zcr:r

ui1-

wW

1-0a

)-0:

Ilcr0

:l-l

0Il:r

a.U

entD

'aru

aru1

'a1

51INn 3500 :JOL

U1f-..Z.::WU1ooLJo..

8

6

4

2

o

-2

-4

TRICHLOROETHRNE, 1,1,2-

A

N

N

79-005

T

8

H

----------------------------.---------------------------------------------------------------------------------

- - - - - -. -. -- - - - - - - - ~- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ~- - - - - - - - - - - - - - - - - - - - - - --

s SSA A Ro 5 9

A

N

G

PROKARYOTES 1 Lm/ER EUKAR. MAMHLS lN VITRO

u

V

H

HUMANS 1 F /HI MAMMALS lN vivolN VITRO

2;~trZU-;XN

(jf5

510lA

Re M

ON

OG

RA

HS

VO

LUM

E 52

colD

~ru

oru1

~1

51INn 3500 ~OL

APPE

ND

IX 2

511

uiOz ;:cr -

i: ;:

::z:i_C

J;::;3ln-icri:i:cri::i"-i.uiOz 0:

~ S;

:::i3'\1

inCO

CJ

0:1

1-l"

:;0

31"..

ui-icri:i:cri:1-uWui3ln1-Zcr-i0-cicr:.::U

Jw

1-0:

.-w~

a:C

JU

J-i

,...ln

....w

..1-a

l-r0:

.-cr

a::.a

m=

u:=0:

00-

U

coin

'\ru

oru1

'\1

51INn 3500 :JOL

512lA

Re M

ON

OG

RA

HS

VO

LUM

E 52

011ui 0

1Z

:;1

CI -

1E

:;1

::z1

:I_111

01

:;1

s:111

2.11

ui11

-.1

CI

1E

1E

1C

I1

EJ11

:I1

'-1

i.1111

ui 01

zn:1

CI !:

11E

:;1

::1

:I2.

1L

O1

11i

ru1

"l11

01

l- '- U)

1n:

11-

~1

s:1

..1

M1

2.1

..1

ui11

-.1

CI

1E

1E

1C

I1

E11

=-=

11l-

IU

1W

1ui

12.

1111ln

1l-

IZ

1C

I1

-.1

0.1111

ci11

CI

1:.

1::

1w

11n:

W1

W

01

:31

0.-

1-.

Li

1

-l11

::1

l.1

ln1

W1

l-i

0l-

i)-

-l1

n:a:

iC

I1

:.(I

10

01

n:i

0.U

1.

C1

ru"l1

LO

"l

51INn 3500 :JOL

oru1