- Email: [email protected]
Mutagenicity of chloropropanol in a genetic screening battery
FUNDAMENTAL AND APPLIED TOXICOLOGY 3:27-33 (1983)
Mutagenicity Of Chloropropanol In A Genetic Screening Battery ROBERT W. BILES and CHARLES E. PIPER^ Exxon Corporation, East Millstone, New Jersey; ^Hazleton Laboratories, Vienna. Virginia
ABSTRACT
Mutagenicily Of C h l o r o p r o p a n o i In A Genetic Screening Battery. Biles, R.W, and Piper, C.E. (1983). Fundam, AppL Toxicoi. 3:37-34. A 72:25 mixture of !-chioro-2-propanol and 2 - c h l o r o - l - p r o p a n o l was tested for genetic activity in a battery o f short term tests. Chloropropanol was tested over a dose range of 527-167,250/~g/plate in theSalmoneila/mammalian microsome mutagenicity assay. A dose dependent mutagenie response was observed in strains TA ! 535 and TA 100. Metabolic actisation enhanced mutagenicity in both strains. A l t h o u g h chioropropanol was mutagenic in the T K , / - mouse i y m p h o m a assay with and without metabolic activation, a smooth linear dose response relationship was not observed. Non-toxic mutagenic doses ranged from 5,000 to I0,000/,=g/ml. without activation. C b l o r o p r o p a n o l was also mutagenic in the rat bone marrow cytogenetic assay. Rats dosed orally with I 0.31 and 100 m g / k g / d a y for 5 days displayed no significant difference in mean body weight Rain or mean mitotic index when cor.tpared to controls, however, a dose-response increase (significant) in the n u m b e r o| aber;ations, mostly chromatid breaks, was observed in each case. INTRODUCTION Toxicologists may n o w augment the safety evaluation of a n e w or existing compound w i t h an appropriate genetic toxicology screening battery. While concern exists about the inap~Ptcsent Address," G.D. Sca*le & C n . Cmcal:o. ttI~n**,,
Drnprialu use of these tests or reliance on any single assay, it is n o w apparent that a number of animal carcinogens ,:an be detectedwitl~ a w e l l - c h o s e n g r o u p o f Sl~Ort-term tests Endpoints, other than cancer, currently being evaluated using appropriate groups of short-term assays a=e reproductive effects and heritable elfects in future generations. Consequently, genetic screenino tests have the potential of becoming a valuable tool to the industrial toxicologists for the purpose of safety evaluation. The application of the test battery techniauo is n o w generally recognized as an appropriate basils for using short-term tests in order to make decisions (Flamn% 1977). The battery of short-term tests most often advanced includes m e a s u r e m e n t of mutagonic potential at the subc h r o m o s o m e level (point and genP mu;.dtions) and t he chromosome level. "[his report describes an investfgation of I -chloro-2-propanol and 2 - c h l o r o - l , p l o p a n o l (~75:25 mixture) in the f o l l o w i n g battery of short-term tests: Salmonella~mammalian micro, some assay; L5178Y Mouse Lymphoma assay; and rat bone m a r r o w in rive cytogenetic assay. The t w o isomers of chloropropanol may be found in food stuffs as secondary products formed during fumigation w i t h propylene ox=de (Regelis et el., 1968; Wesley et el., 1965; Steel and Hadziyev, 1976), may r e m a i n as a residue in polypropylene following processing. and may be generated as hydrolysis products of chemical intermediates such as epich,orohydrin or chtoroanalog flame retardants (Carr and Rosenkranz. 1978). Chloropropanol has been previously tested in the Salmone//a assay and has been
TABLE 1 Response of Strains 1535 and TAIO0 Following Exposure to ChloropIopanol 48 Hour Readings S. O,phtmurium Strains TA1535 Dote (~,g/plate) 167,250 52,700 10,500 5,270 527 Organism Organism - DMSO
Ave. His .~ Fold Inc.*' without 5-9 384 172 105 b3 20 23
0 -9 3 ° "~" tO -1- 3. 2:l0 "t 6 .t: 4
0c "]o.7 t~ 7.5 4.b 2.7
TAI00 Ave. Ills.
Avg. Ills. Fold Inc. without 5-9
Fold Inc. • S-9
72¢t 280 I72 02 15 11
O -9 7b 2:30 ..t: 21 ~ 16 + 2 "J: 3
0 bS.4 25.4 15.0 5.0
0 670
2: l l l
471 314 216 171 155
± ~ .'t: ± ~
46 I8 23 5 3
0 4.4 3.0 2.0 1.4
Avg. Ills.
Fold Inc. • 5-9
74,1 471 281 170 148 I47
0 ± 55 J. 53 "£ l 0 2:13 :z 13 2:3
O 5. l 3 2 l.q l.I
^X :t: SD for three plates •Fold Increase -" Av. No. of His" Te~t Concentrations Av No. of Hi~.* Control CCompared to organism c~,ntrc,] "'Compared re solvent control Positive Cnntrols: TA 1535- MNNG 5 pglp]ate = 2193 -t. 24; 2AA 5 p g l p l a t e -- 472 ~ 30 TA100- MNNG 5 uglplate = 2748 :t: 93; 2AA 5 pg/plate = 2412 :t: 163 Copyright l¢ll$. Societyo| Toxicology
Fundamental and Applied Toxicology
(3) 1-2/83
27
81LES A N D PIPER
Response
of S t r a i n s
TABLE 2 1537, 1538 and 98 Following Exposure
to Chloropropanol
48 Hour
Readings
S. O'llhimurium S t r a i n s TA1537 D o t e f~geplate)
A v e r a g e His *'~ w i t h o u t S-9
107.250
52.700 10,500
5.270 527 Orgamsm Org,lni~,rn • D M S O
TA1538
0 o o 8 o (~
0 :r :t: :t: :L ± .f.
2 I 3 ] I 2
• S-o
Z o lO t, o ¢',
0 :t: I :t I :I: 3 -~ 3 =t: 3 ..1:2
TAgB
Average His, w i t h o u t S-9 0 It
:~ 2
14 lb 10 13 lo
:t ± ± :t: ±
3 3 3 2 4
Average His-
• S-9
2o 23 32 30 32 33
0 :t 3 .t: 3 ± 3 2: o :t: t1 .1:3
without 5-9
S-9
0 I a "J: 5
22 18 24 18 22
t- 4 :t: 5 ± 3 .1:5 1:3
32 4! 36 2Q 32 38
O =t 7 ~: q :t: 2 I. 3 ::1:3 :l: 3
"~.~ :1= S D for t h t t e pl.~h'~ Po~zr~ve C~,ntn~l,.. T A 1 5 3 7 - OAA 75 ,~glpl.~w -- ~03 = 10; 2 A A 2.5 uglpl.~te "- I57 :t 28 T A 1 5 3 8 - 2 N I " 50 p g l p l a t e -" 17o(, 3- 50, ZAA 5.0 u g l p [ a t e = 257.5 "r 82. TAoS-2NFS0uglplJle = 1557 2. 4o: 2AA S O pf, Iplate : 2008 -r l l O
reported both positive (Carr and Rosenkranz, 1978; Rosenkranz et al.. 1975. Nakamura ez' al., 1979; Pfeiffer and Dunkelburg, 1980l and negative (Poirier and Simlnon, 1978). No ether mutagenesig information was found tn the published literature. 1 -chloro-2-prol0anol produced a dose-response increase in tumors in mice by i.p. injection; however, a statistically elevated pulmonary adenoma response was not observed (Thelss et aL. 1979). Therefore. it has not been clearly estabhshed whether or not this material has carcinogenic activity.
nate increases in background revertants due to vaporization of the test agent, the highest three doses, the four lower doses, and the c.ontrol series were incubated separately. After 48 hours, the colonies (revertants to histidine prototrophy) were counted. As recommended by de Serres and Shelby, all plates were reincubated for 24 hours and recounted (deSerres and Shelby, 1979).
S a l m o n e l l a / m i c r o s o m e assay Chloropropanol was investigated in five Salmonella typhimurium strains (TA 1535, 1537, 1538, 98,100) in the plate incorporation assay which has been described in detail (Ames et al., 1975), The tester strain genotypes were confirmed prior to use. An initial toxicity assay was conducted with TA 100 to determine the maximum tolerated concentration from w h i c h the mutagenicity assay concAntratioJls would be chosen. VogeI-Bnnncr Med=um E minimal agar plates were used, to which were added 2 mL of complete top agar and 0.1 mL of appropriate overnight broth cultures of S. ~yph(mur[um. S-9 mix, prepared with Aroclor 1254-induced rat liver, was prepared fresh, The S-9 mix contained 0,05 mL S-9 (liver homogenate) per 1.0 mL of mix. The controls used were (1) negative - (a) organism alone, (b) organism plus S-9, and (c) organism plus DMSO; (2) S-9 sterility; and (3) positive - - without S-9, (a) TA 1535 and 100 MNNG (5/~g/plate), (b) TA t 5 3 8 and 98. 2-nitrofluorene {50/~g/p|ate), (c) TA 1537, 9-aminoacridine (75 ~g/plate) - - with S-9, all strains 2-aminoanthracene (2.55 ,ug/piate).
L 5 1 7 8 Y mouse l y m p h o m a assay The mouse lymphoma assay has been described in detail (Clive and Specter, 1975; Clive, et al., 1979). TK . / - mouse lymphoma cells, subline 3.7.2C, frozen and stored in liquid nitrogen were thawed and used for this assay. Cells were grown in Fisher's medium for leukemic cells of mice made up in sterile deionized water and supplemented with approximately 10% (V/V) horse serum, sodium pyruvate, pluronic F68 and oenicillin-streptomycin (complete medium FIoP}. Closing medrum consisted of the medium described above with approximately 20% (V/V) horse serum. Melted agar was added to a final concentration of approximatrely 0.32% (V/V), Selective medium consisted of cloning medium with trifluorothymidine (TFT) added at a concentration of 2 /~g/mL. S-9 was prepared as in the Salmonella assay, and the S-I:J mix contained 2.5 mL of a 1:10 dilution of the S-9 liver homogenate per 10 mL S-9 mix (1:10 determined optimal on basis of standardization experiments with 2-acetytaminofluorene). An initial toxicity assay was cond~cted w;th seven concentrations of ¢!:!crop~ opanol (range 1.0.9.960 ,~g/mL) both with and without S-9. One tenth mL of each concentration of test agent, solvent and positive control fEMS and 2AAF) were added to tubes containing 6 mL F~oP,6.0 x 10 GTK ÷/- cells with additional 4 mL of FloP or S-9 mix. The cells w e r e ~×posed for four hours, washed, resuspended in 20 mL F~0Pand incubated two days. After the first and second day of incubation, cell counts and viability in each culture were determined, and following the first day, the viable cell count adjusted to 3.0 x 10 ~ cells/mL. Based on cell counts, the percent total suspension growth was determined.
Seven concentrations (ranging from 527 to 1 6 7 , 2 5 0 / z g / plate) of the test agent wer6.evaluated in triplicate in the five bacterial strains with and without'metabolic activation. Dimethyl sulfoxide (DMSO) was used to deliver the test material in the four lowest concentrations while the material was added neat in the three highest concentrations. To elimi-
The mutagenicity assay was conducted using 15 concentrations of chloropropanol in cultures without S-9 and 18 concentrations in cultures with S-9 (Table 2), The exposure and incubation conditions were identical to the toxicity assay. After the expression period, the cultures were selected for cloning. Cell growth in these treated cultures ranged from 6 to 99% of that
MATERIALS AND METHODS Chloropropanol, designated as a 75:25 mixture of 1 -chloro2-propanol and 2-chlorn-l-propanol. was obtained from Eastman Kodak. This material was subsequently analyzed and found, by gas chromatography/mass spectrometry, to consist of 73.3% 1-chloro-2.propanol and 2 6 3 % 2-chime- 1-propanol
28
Fundam. Appl. ToxlcoL (3)
January/February, 1983
GENETIC TOXICOLOGY
OF CltI.OROPROPANOL
TABLE 3 S u m m a r y of Results: Mouse L y m p h o m : , Forward M u t a t i o n Assay of Chloropropanol
Concentration
Survival ^
Without
Metabolic
Activation
Fold
(% of c o n t r o l )
Mutation Frequency"
increa~e'"
100% 100'~ 5%
10.0 12.2 IO0.0
. . . . 15.t',
10,241
15%
47.2
3.0
244","
0,832 o,422 o,012
3o% 54% 30='0
8,603
00%
42.3 32.3 4,~.7 20 8 30 0 41 o 21 o .I 3.4 37 8 18 I
3.5 2.0 .t.O 1.7 2.5 3 4 1.8 3.5 3 I 1.5
133% 120"., 04% 1o4% I lo% 130% 114% 100% 113% 111%
2 I. I
2 0
t 00%
22.3 10.3 20.0
1.8 1.0 1.7
05°> 142at• o~%
fpgtmL) Medi., C o n t r o l S o l v e n t C o n l rol EMS-o20.O
8.0%
•q,l o 3 7,783
80% 80% u4 o., 08% ,qTet• ¢~5,"., o.1% 105¢iO3"~,
7,374
0,004
0.554 o.145 5,735 5,325 4,01() 4.500
Cloning E f f i c i e n c y (%) . .
. . --
•'~Rel,ltive s u s p e m , ion g r o w t h ,, clorHng e f f i c i e n c y =*The n u m b e r of T K -/- m u t a n t ~ p e r 1 x 10~'colony f o r m m g c e l i s p l a t e d o n t o tlae ~elc
Survival ~
Clnning t'fficiency (%)
(% of control)
Mutation F r e q u e n c y I'
increase c
! 00% 100%
3.0 2.6
. .
13%
42.2
I 0.3
--
T I'
T
T
--
500
I"
T
T
--
470
T T T 3% 5% 3% 0% 7% 28% 14%
T
T
--
T
T
--
T 24.5 13.8 ! 4.6 7.2 11,4 12.4 9.2
-54% b7% 57%
20%
55
30°/0 4bq3 38%
5.0 3.o 5..5
T q.4 5.3 5.0 2.8 4 4 4.~ 3.5 2.1 1.o 1.4 2.1
I 01% 77% J87'~ oi% IbO% 157% 127% 8Sq;,
5,0
40%
20
77%
3 6 1.7
1.4 0.7
7q% 7700,
(pglmL) Media Control Solvent Control 2AAF-50
,530
440 410 31~0 330 320 290 260 230 200 170 140 I I0 80
Fold
. .
. .
. .
"Relatives.uspens;on growth x cloning efficiency ~ThenumberofTK-I-mutantsper] x tO'colony formingcellsplaled ontothe 5elective cloning medium. CFold Increase : Mutation Frequency of Test Concentration Mutation Frequency of Solvent Control DT = Too toxic to clone of the u n t r e a t e d control. This is referred to as relative suspenstun g r o w t h . A p p r o x i m a t ' e l y 5 x 1 0 s cells w e r e plated on each of t h r e e selective m e d i u m plates (2 u g / m L TFT) and 1 0 0 cells Fundsmentsl and Applied Toxitololy
(3) 1.2/83
cloned on each of t h r e e n o n - s e l e c t i v e # a l e s for each test and control tube. The plates w e r e incubated for 1 1 days and the m u t a n t colonies c o u n t e d o n t h e selective plates, w h i l e t h e 29
BILES A N D PIPER TABLE 5
Metaphase Analysis of Bone Marrow Cells from Male and Female Rats Treated with ChIoropropanol Treatment Group
Number of Cells
Numberof Aberrant
Total Numberof
Chromatld
Chromosome Markers
Severely Damaged Cells ( > I 0 aberrations)
Analyzed
Cells
Aberrations"
Breaks
Breaks
Negative Control (H20)
350
11 (1.6 :t: .8) ^
12 (1.7 -t- .9)
7,0 1.4 ± .9)
0
2
O
10 mglkg Chloropropano]
~00
68 (8,5 +- 3,6)
92 (11.5 -t- 7.1)
51 (6.4 + 2.3)
5
26
1
31 mg/kg Chloropropano[
450
108 (12 ± 2.1)
219 (24,3 + 9.9)
59 (6.6 :k 3.3)
Z
58
10
l e o mg/kg Chloropropanol
350
93 (13.3 ± 5,2)
279 (39.8 + 44)
88 (12.6 ::t: 9.6)
4
47
8
TEM Positive Control
450
209 (23.2 ± 3.7)
949 (105 + 44)
110 (12,2 ~ 6.7)
16
163
66
^,'~ ---+ ' : ' D / o r g r o u p "Severely damaged cells were counted as exactly ten aberrations and were included in this column.
survivors were counted on the non-selective plates. Colonies were counted on a New Brunswick Biotran II, Model C-111. Rat in vivo bone m a r r o w cytogenetic assay Sexually mature male and female Sprague-Dawley, CD® rats were obtained from Charles River, Wilmington, Massachusetts. The animals were housed individually and acclimated to laboratory conditions for two weeks. Fifty rats, weighing between 200-300 grams, were randomly assigned into five groups, each consisting of 5 males and 5 females. The groups were (1) negative control - deionized water, (2) positive control - triethylene-melamine (TEM) 0.4 mg/kg, (3) chloropro~ panel - 10 mg/kg, (4) chloropropanol - 31 mg/kg and (5) chloropropanol 100 mg/kg. Chloropropanol dilutions were prepared in deionized water, and appropriate doses of vehicle or test agents were administered daily for five consecutive days via oral intubation. The animals serving as the positive control received a single intraperitoneal injection of TEM on day 5. Observations were made daily of general appearance, behavior, and toxic and pharmacologic effects. Body weights were recorded prior to initiation of treatment and on day 6, prior to administration of colchicine. All animals received a single intraperitoneal iniection of colchicine dissolved in Hanks Balanced Salt Solution (HBSS), pH 7.4. Approximately two hours after administration of colchicine, the animals were sacrificed by CO2 asphyxiation. The techniques used to process the bone marrow cells represent modifications of the techniques described by Evans (Evans, 1976) and Kilian (Kilian et al., 1977). Bone marrow was collected by needle aspiration of both femurs into 5 .mL of HBSS. Following 5 min. centrifugati0n at 1,000 rpm; the supernate was decanted and 7.0 mL 0.075 M KCI added. After 25 minutes, fiv e drops of Carnoy's fixitive were added and the cells were mixed and recentrifuged for fiv e minute s at 1,000 rpm. This process was repeated three time s prior to overnight refrigeration. The next day, the cells were pelleted, resuspended in 1-3 mL Carney's, and threedrops of this final cell suspension dropped , on a m!c~'osc0Pe slid e and al!owed to air dry. Three slides were made from eac h animal. The slides were identified and stained for 16 minutes Wit h a Giems a prel~arationl The slides we;'e then rinsed twice in tap water, air dried, 30
and mounted with cover slips. The animal identification number was covered, and the slides were coded for reading. The evaluation sequence of prenumbered slides was determined from a table of random numbers. Only those cells in metaphase stage of mitosis were analyzed for the presence of cytogenetic abnormalities. The mitotic index was also recorded for each animal. Fifty cells in metaphase were examined from each rat that provided analyzable cells. Stage vernier settings were recorded, and all cells once visualized on high power were recorded unless there was obvious mechanical damage. Chromosomal aberrations were classified into one of four basic groups: (1) chromatid breaks; (2) chromosome breaks; (3) markers, which include dicentrics, enchanges, rings, and other miscellaneous configurations; and (4) severely damaged cells, Chromatid aberrations involve a break in only one arm of the chromosome, whereas chromosomal ab6rrations involve both arms at identical sites. A break was defined as any separation that exceeds the width of the chromatid arm, or a discontinuity accompanied by disturbance of the axial integrity. Aberrant configurations, which alter the structural appearance of an entire chromosome or involve the joining together of more than one chromosome, were categorized as markers. An exchange was defined as a symmetrical or assymetrical distortion of the usual chromosomal pattern. A dicentric was defined as achromosome which possesses two centromeres. A ring chromosome was defined as one in which the ends have joined to form a circle, with or without a centremere. Those cells which had more than ten scorable aberrations were classified as severely damaged. When the total number of aberrations was determined in the cells analyzed for the individual animals, severely damaged cells were counted as exactly ten aberrations, as suggested by Kilian (Kilian et al., 1977). The mean changes in body weights and the mean mitotic indices w~re analyzed using Bartlett's test for equality 0f variance, the one-way classification of ana ys s of Va'r ance,and Scheffe's Multiple F Test procedure. The number of aberrant cells and the total number of aberrations per animal (includ!ng chr0matic breaks, chromosom e breaks, markers, and severely damaged Cells) Wei;e statistically analyzed by Wilcox0n's nonparametric comparisbn 0f group means. Fundam. A~DL Toxicol. (3)
Januarv/Februarvo 1983
GENETIC TOXICOLOGY OF CHLOROPROPANO[.
RESULTS S a l m o n e l l a / m i c r o s o m e essay The results shown in Table 1 for the two-base substitution strains TA1535 and TA100 indicate a clear dose.related mutagenic response observed w i t h and w i t h o u t activation. The quantitative difference between the response of the t w o strains may or may not have any biological significance. The presence of a background bacterial l a w n w a s seen at each test concentration and toxicity of the con=pound, as demonstrated by a "sparse l a w n " , was clearly observed at the highest concentration. TA1535 demonstrated a marked enhancement of mutagenic response w h e n S-9 was incorporated in the reaction mixture. Prolonged incubation (72 hours) and a pronounced effect in this strain, but did not appreciably affect strain T A 1 0 0 (data not shown). The results in Table 2 indicate no appreciable increased frequency of reversion to histidine prototrophy in the strains TA1537. and the frameshift strains TA1538. and TA 98. Neither metabolic activation or prolonged incubation (72 hours) had any effect in these strains. L 5 1 7 8 , ' h o u s e / y m p h o m a assay A toxic dose-related response was observed w i t h chloropropanel following the first day expression. As shown in Table 3, in the absence of metabolic activation, the total survival (as a % of control) ranged from 15% at the highest concentration to approximately 100% at the lower concentrations. Clive, et al., (1979) defines an appropriate limit as one yielding 90% kill (10% survival) w h i l e Brusick (1980) has recommended choosing 4 to 5 doses spaced from 0 to 50-90% reduction in cell growth. A greater than t w o - f o l d increase in mutation frequency over the spontaneous background levels w a s observed at eight concentrations. The response w a s not uniform as can be seen in Table 3. Table 4 indicates the mutagenic activity of chloropropanol w i t h metabolic activation. Toxicity arising at concentrations above 4 1 0 / ~ g / m L inhibited cell g r o w t h to the extent that these
test levels could not be cloned. Greater than two-fold increases in m u t a t i o n frequency over the spontaneous levels w e r e obtained at nine test concentrations, including four test concentrations at w h i c h greater than 10% total survival was achieved. Rat in vivo bona m a r r o w c y t o g e n e t i c essay No animals died during the study, and individual clinical observations indicated that chloropropanol did not produce overt toxic effects in rats at doses of 10. 31 and 100 m g / k g . Mean initial and t e r m i n a l body weights and body w e i g h t changes for the negative control and treatment groups w e r e analyzed by ANOVA (p--.05). No significant w e i g h t gains or losses wc, rA n|.~served; however, a depression in body w e i g h t gain w a s observed at the highest dose lbody w e i g h t changes in grams w e r e (1) negative control - 22*8.4; (2) 10 m g / k g 2 6 ~ 1 0 . 9 : (3) 31 m g / k g - 25+12.9;(4) 100 m g / k g - I 1.4+12.41. Five hundred representative metaphases w e r e not achieved for each group. The actual members analyzed are given in Table 5. The mean mitotic index (number of cells undergoing mitosis Der 100 cells counted) was not statistically different (p:.05) among the groups w h e n analyzed by ANOVA. The index ranged from 1-5. Values wore: (1) negative control 3.14+1.1;(2) positive control (TEM). 2.67.1.3;(3) 10 m g / k g 4.13+1.1;(4) 31 m g / k g - 3.22.0.7; (5) 100 m g / k g - 4 . 1 4 . 0 . 9 . The total number of cells analyzed, number of aberrant cells, the n u m b e r of total aberrations and certain classes of aberrations are shown in Table 5. A s u m m a r y of these data is presented in Table 6. As can be seen in these tables, the number of aberrant cells and aberrations per cell are greater in the test groups thaq in the negative control, and these responses appear to be dose related. A Mann-Whitney-Wilcoxen test w a s performed to compare the number of aberrant cells and total number of a b e r r a t i o n s / a n i m a l between the negative control and the other groups. There were significant increases (p < .05) in the number of aberrant cells and the total number of aberrations per animal i n t r e a t e d groups w h e n compared to the negative contro{s.
TABLE 6 Comparison of Frequencies of Chromosomal Aberrations in Bone M a r r o w Cells [rum Groups of Animals Treated With Chloropropanol and Controls
Cell"
Fold Increase of Aberrations/ Cell ~"
0.034
--
Average Treatment Group
Negative Control (H~O)
Total Number Cells Analyzed
% Aberrant Cells/`
Aberrations/
3SO
3.1
Chloropropal~ol
.I00
! 7.0
0.23
b,8
31 mglkg Chloropropanol
450
24.0
0.49
I4..1
Chloroprup.~/~ol
350
26.6
0.80
23.5
Positive Control (TEM)
450
46.4
2.10
61.8
10 mg/k~
I00 mglkg
"This number repre~.ent~. :he totai number of aberrant ~cells/group divided by the total number of cells analyzed per group × 100. ~This number represents the total number of aberrations/gruup divided by the lutal number of cells analyzedlglnup. CThis number represents the average aberrationslcell for the test groups divided by the value obtained for the negative control (0.034 aberrations/celt). Fundamenttl and Applied ToxicoloR)
(3) 1-2/83
31
BILES AND P}PER DISCUSSION There is no single, short-term genetic bioassay which is adequate for thorough toxicological safety evaluation. If screening for potential carcinogens is the intended use of a short-term test, then sufficient data on sensitivity and specificity already exist for many of the prominent assays to preclude reliance on any single assay in that evaluation process. In fact, recent reports recommend the use of a core battery of tests for evaluating the mutagenic and carcinogenic potential of a material. Ray (Ray, 1979) reports a core battery of tests under zvaluation which contains the three assays used in the present study, as well as an in vitro cytogenetic assay in human lymphocytes and an in vitro mammalian cell transformation assay. Further, Ray outlines the criteria for selecting a core battery and suggests that the battery should be flexible. This ongoing review demonstrates that few substances with mutagenic or direct-acting carcinogenic potential would be missed by Ray's core battery. Sobels (Sobels, 1980) believes that a minimal battery should contain at least three tests: (a) a test for gene or point mutation in bacteria, (b) two tests for point mutations in eukaryotes, or (c) one such test and a test detecting chromosome aberrations in mammalian cells in vitro. Waters, (Waters, et al., 1980) recommends (a) a point mutation assay in microorganisms, (b) a gene mutation assay in mammalian cells, (c) a primary DNA damage assay, (d) an assay for chromosomal effects in mammalian cells and (3) a transformation assay in mammalian cells. The value of the present test results, with respect to a screen for carcinogenic potential, can only be strengthened by the corroborative evidence obtained in each of the three assays. The two in vitro assays have individually been shown to have a relatively high detection capability for carcinogens based on validation experiments with chemicals of diverse structure and biological activity. The in vivo cytogenetics assay does not share this sensitive carcinogen detection capability, but does give perspective as to the potential of a chemical to have mutagenic or heritable genetic effects. Chloropropanol was positive in all three of the genetic assays. While uniformity of results in a screening battery is desirable, there are reasons for individual test result differences. The larger the battery and the more diverse the endpoints of the tests which make up the battery, the more likely that results of all the assays will not be uniform. However, consistancy should be expected within a class of similar genetic end points. For example, in the present battery, the two gene mutation assays should be required to be consistent for a meaningful evaluation. But the chromosomal assay, which measures a distinctly different endpoint, might be regarded as a complementary assay ~ one which, if positive, will cast further evidence as to a compound's toxic potential. McCann (McCann et al., 1975) reported Salmonella detected 17 of 20 previously tested animal carcinogens which were categorized as alkyl halides. In the present study, the isomers of chloropropanol were active in the base substitution strains of Salmonella'both with and without metabol!c activatio n. There was an enhanced response in TA 1535 the presence of metabolic activation. This supports most of th e previous published work with Salmonel/a, and t h e r e w a s especially good correlation with the work of Pfieffer (Pfieffer and Dunkeiburg, 1980).. However, F~i~ffer c0ncluded that l~chl0ro-2-pr0panol is totally responsible for the mutagenic activity of the isomeric mixture.Pfieffer states in hiS report that2-chlor0:-I -propanol is not mu!agenic from Work reported b y Carr/(Cal"r: and
32
Rosenkranz, 1978), but the earlier work by Carr refers to 3-chloro-1-propanol, not 2-chloro°l-propanol. In the present study, the isomeric mixture composed of 75% 2-chloro-1propanol was shown to be both a direct acting and indirect acting mutagen in bacteria. Both the toxic and mutagenic activities of the isomeric mixture of chloropropanol were enhanced in the presence of metabolic activation in the L5178Y mouse lymphoma assay. The material was also active without me!~.~:,olic activation, similar to the results obtained in the Salmonella assay. While the dose-related responses in toxicity and mutagenicity were obtained with chloropropanol, these responses were not strictly linear in nature for any of the measured parameters such as percent suspension growth, mean numoer of mutant colonies on selective plates, percent total survival or mutation frequency. This is most likely due to the relatively closely spaced dose levels which were examined. There was a dose-related effect of chloropropanol on the physical structure of the chromosomes in bone marrow cells from rodents. The oral administration of chloropropanol for 5 days showed no overt toxic effect and resulted in no significant body weight gain/loss. Furthermore, there was no significant difference in the mean mitotic index between the test groups and the negative control. There was an apparent dose-related increase in the number of total aberrations and, more specifically, in the number of chromatid breaks. While the mutagenic events described in the point mutation assays might be initiated by a single interaction with DNA, these cytogenetic responses probably involve more than one interaction, Since the cytogenetic study was done in the intact animal, it is reasonable to conclude the in vivo defense/repair mechanisms are not sufficient to protect the somatic cells from genetic damage. An interesting extension of this is the possible interaction of the materials with germinal cells. Materials which are structurally similar to chloropropanol, i.e. alphachlorohydrin and 3-chloro-l-propanediol have been shown to have specific effects on germinal cells and also cause sterility in rats while not affecting libido or spermatogenesis (Cooper et aL, 1974; Erickson, 1970). In addition to this, a mutation within a germinal cell which still allows viability might effect a heritible event; a genetic alteration which is expressed in a future generation. The genetic activity of an isomeric mixture of 1-chloro-2propanol and 2-chloro-1 -propanol, demonstrated in the present study, predict the possibility of two, possibly three, chronic health problems which may arise from exposure to the compound. Since short-term tests are currently best considered screening devices, the potential carcinogenic, reproductive and heritable mutagenic effects need to be evaluated by classical long termanimal test methods for a definitive assessment of the toxicity of c'~toroDropanol. REFERENCES
Ames, B.N,, McCann, J, and Yamasaki, E. (1975). Methods for Detecting Carcinogens and Mutagens with the SalmoneliaMicrosome Mutagenicity Test, Mur. Res. 31:347-364, Brusick, .D. (1980), Principles of Genetic Toxicology~ Plenum Press, New York and London. pp~ 204-211. Carr, H.S. and R0senkranz; H.S. (1978). Mutagenicity of Derivative of {:he.Flame Retardant Tris (2;3-~Iibromopropyl) PhosL phat6: Halogenated Propan0ls. MuL Res~ 57:381-384. Clive, D. and Spector, jr; (1975). Laboratory Procedure for Assessing Specific' Locus Mutati0ns at f~he TK L0cu~s in Cultured L5i784 Morose Lymphom;i Cells.. ~{fut, Res. 31:17-29. Fundam. At~DL Toxicol. (3)
Januar~/Februar~v.'1983
GENETIC TOXICOLOGY OF CHLOROPROPANOL Clive, D., johnson, K.O., Spector, J.F.S., Batson, A.G. and Brown, M.M.: (1979). Validation and Characterization of the L5178Y/ TK +l- Mouse Lymphoma Mutagen Assay System. Mul. Res. 59:61-108. Cooper, E., Jones, A. and Jackson, H. (1974). Effect of AlphaChlorohydrin and Related Compounds on the Reproductive Organs and Fertility of the Male Rat. J. Reprod. Fertil. 38:379-386.
deSerres, F.J. and Shelby, M.D. (1979). The Salmonella Mutagenicity Assay: Recommendations. Science 203:563-565. Ericson, R.J. (1970). Male Antifertility Compounds: U-5897 as a rat chemosterilant../. Reprod. ]'~,rliL 22:213-222. Evans, H.J. (1976). Cytological Methods for Detecting Chemical Mutagens; Chemical Mutagens; Principles and Methods for Their Detection, Volume 4, ed. by A. Hollaender, Plenum Press, New York. pp. 1-30. Flamm, W.G., Chairman (1977). Subcommittee on Environmental Mutagenesis Report: Approaches to Deternfinlng the Mutagenic Properties of Chemicals: Risk to Future Generations. Prepared for the DHEW Committee to Coordinate Toxicology and Related Programs..L Environ. Path. ToxicoL 1:301-352. Kilian, D.J., Moreland, F.M., Benge, M.C., Legator, M.S. and Whorton, E.B. (1977). A Collaborative study to Measure Interlaboratory Variation with the In Vivo Bone Marrow Metaphase Procedure, Handbook of Mutagen Testiog, Elsevier/ North-Holland, Amsterdam, pp. 243-260. McCann, l., Choi, E., Yamasaki, E. and Ames, B. (1975). Detection of Carcinogens as Mutagens in the Sa|monellalMicrosome Test: Assay of 300 Chemicals. Proc. Nat. Acad. Sci. USA 72:5135-5139. Nakamura, A., Tateno, N., Kojima, S., Kaniwa, M. and Kawamura, T. (1979). The Mutagenicity of Halogenated Alkanols and their Phosphoric Acid Esters for Salmonella Typhimurium. Mut. Res. 66: 373-380.
Pfeiffer, E. and Dunkelburg, H. (lO80). Mutagenicity of Ethylene Oxide and Propylene Oxide and their Glycols and Halohydrins Formed from them during the Fu,. igation of Foodstuffs. Fd. CosmeL Tu.vicoL 18:115-1 ]8. Poirier, L. and Simmon, V. (1978). ]'he Mutage,~icity of Substituted Organo-Halides to S. Typhimurium TA 100. Presented to the International Congress in Buenos Aires, October 1978. Ray, V. (1979). Application of Microbial and Mammalian Cells to the Assessment of Mutagenicity. Pharn~acoloR.c Reviews 30:537-546. Regells, E.P., Fisher, B.S. and Kllmeck, A.B. (1908). Isolation and Determinalion o/t Chlorohydrlns in Foods Fumigated with Ethylene Oxide or with Propylene Oxide. J. Ass. Offic. Anal. Chem. 51:707-715. Rosenkranz, ILS., Wlodkowski, T.]. and Bodine, S.R. (1975). Chloropropanol, ,¢Mutagienic Residue Resuhing from Propylene Oxide Sterilization. Mut. P,es. 30:303-304. Sobels, F,H. (1980). Evaluating the Mutagenie Potential of Chemicals: The Minimal Battery and Extrapolation Problems. Given at International Conference "Mutagetficity Testing of Pharmaceuticals: Present Status" Paris 12-14, March 1980. Steele, L. and Hadziyev, D. (1976). Sterilization of Dehydrated Potato Granules with Propylene Oxide. Z. l.ebens.Miltehmters. U-Forsell. 162:387. Theiss, J., Shimkin, M. and Polrler, L. (1979). Induction of Pulmonary Adenomas in Strain A Mice by Substituted Organo HMides. Cancer Research 39:391-395. Wailers, M., Simmon, V., Mitchell, A. and lorgenson, T. (1980). An overview of Short-term Tests for the Mutagenic and Carcinogenic Potential of Pesticides../. Environ. ScL lleallh B] 5(6):867-906. Wesley, F., Roarke, B. and Darbishire, O. (1965), Formation of Persislent Toxic Chlorohydrins in Foods by Fumigation with Ethylene Oxide and Propylene Oxide. J. Food Sci. 30:1037-1042.
33
Mutagenicity Of Chloropropanol In A Genetic Screening Battery ROBERT W. BILES and CHARLES E. PIPER^ Exxon Corporation, East Millstone, New Jersey; ^Hazleton Laboratories, Vienna. Virginia
ABSTRACT
Mutagenicily Of C h l o r o p r o p a n o i In A Genetic Screening Battery. Biles, R.W, and Piper, C.E. (1983). Fundam, AppL Toxicoi. 3:37-34. A 72:25 mixture of !-chioro-2-propanol and 2 - c h l o r o - l - p r o p a n o l was tested for genetic activity in a battery o f short term tests. Chloropropanol was tested over a dose range of 527-167,250/~g/plate in theSalmoneila/mammalian microsome mutagenicity assay. A dose dependent mutagenie response was observed in strains TA ! 535 and TA 100. Metabolic actisation enhanced mutagenicity in both strains. A l t h o u g h chioropropanol was mutagenic in the T K , / - mouse i y m p h o m a assay with and without metabolic activation, a smooth linear dose response relationship was not observed. Non-toxic mutagenic doses ranged from 5,000 to I0,000/,=g/ml. without activation. C b l o r o p r o p a n o l was also mutagenic in the rat bone marrow cytogenetic assay. Rats dosed orally with I 0.31 and 100 m g / k g / d a y for 5 days displayed no significant difference in mean body weight Rain or mean mitotic index when cor.tpared to controls, however, a dose-response increase (significant) in the n u m b e r o| aber;ations, mostly chromatid breaks, was observed in each case. INTRODUCTION Toxicologists may n o w augment the safety evaluation of a n e w or existing compound w i t h an appropriate genetic toxicology screening battery. While concern exists about the inap~Ptcsent Address," G.D. Sca*le & C n . Cmcal:o. ttI~n**,,
Drnprialu use of these tests or reliance on any single assay, it is n o w apparent that a number of animal carcinogens ,:an be detectedwitl~ a w e l l - c h o s e n g r o u p o f Sl~Ort-term tests Endpoints, other than cancer, currently being evaluated using appropriate groups of short-term assays a=e reproductive effects and heritable elfects in future generations. Consequently, genetic screenino tests have the potential of becoming a valuable tool to the industrial toxicologists for the purpose of safety evaluation. The application of the test battery techniauo is n o w generally recognized as an appropriate basils for using short-term tests in order to make decisions (Flamn% 1977). The battery of short-term tests most often advanced includes m e a s u r e m e n t of mutagonic potential at the subc h r o m o s o m e level (point and genP mu;.dtions) and t he chromosome level. "[his report describes an investfgation of I -chloro-2-propanol and 2 - c h l o r o - l , p l o p a n o l (~75:25 mixture) in the f o l l o w i n g battery of short-term tests: Salmonella~mammalian micro, some assay; L5178Y Mouse Lymphoma assay; and rat bone m a r r o w in rive cytogenetic assay. The t w o isomers of chloropropanol may be found in food stuffs as secondary products formed during fumigation w i t h propylene ox=de (Regelis et el., 1968; Wesley et el., 1965; Steel and Hadziyev, 1976), may r e m a i n as a residue in polypropylene following processing. and may be generated as hydrolysis products of chemical intermediates such as epich,orohydrin or chtoroanalog flame retardants (Carr and Rosenkranz. 1978). Chloropropanol has been previously tested in the Salmone//a assay and has been
TABLE 1 Response of Strains 1535 and TAIO0 Following Exposure to ChloropIopanol 48 Hour Readings S. O,phtmurium Strains TA1535 Dote (~,g/plate) 167,250 52,700 10,500 5,270 527 Organism Organism - DMSO
Ave. His .~ Fold Inc.*' without 5-9 384 172 105 b3 20 23
0 -9 3 ° "~" tO -1- 3. 2:l0 "t 6 .t: 4
0c "]o.7 t~ 7.5 4.b 2.7
TAI00 Ave. Ills.
Avg. Ills. Fold Inc. without 5-9
Fold Inc. • S-9
72¢t 280 I72 02 15 11
O -9 7b 2:30 ..t: 21 ~ 16 + 2 "J: 3
0 bS.4 25.4 15.0 5.0
0 670
2: l l l
471 314 216 171 155
± ~ .'t: ± ~
46 I8 23 5 3
0 4.4 3.0 2.0 1.4
Avg. Ills.
Fold Inc. • 5-9
74,1 471 281 170 148 I47
0 ± 55 J. 53 "£ l 0 2:13 :z 13 2:3
O 5. l 3 2 l.q l.I
^X :t: SD for three plates •Fold Increase -" Av. No. of His" Te~t Concentrations Av No. of Hi~.* Control CCompared to organism c~,ntrc,] "'Compared re solvent control Positive Cnntrols: TA 1535- MNNG 5 pglp]ate = 2193 -t. 24; 2AA 5 p g l p l a t e -- 472 ~ 30 TA100- MNNG 5 uglplate = 2748 :t: 93; 2AA 5 pg/plate = 2412 :t: 163 Copyright l¢ll$. Societyo| Toxicology
Fundamental and Applied Toxicology
(3) 1-2/83
27
81LES A N D PIPER
Response
of S t r a i n s
TABLE 2 1537, 1538 and 98 Following Exposure
to Chloropropanol
48 Hour
Readings
S. O'llhimurium S t r a i n s TA1537 D o t e f~geplate)
A v e r a g e His *'~ w i t h o u t S-9
107.250
52.700 10,500
5.270 527 Orgamsm Org,lni~,rn • D M S O
TA1538
0 o o 8 o (~
0 :r :t: :t: :L ± .f.
2 I 3 ] I 2
• S-o
Z o lO t, o ¢',
0 :t: I :t I :I: 3 -~ 3 =t: 3 ..1:2
TAgB
Average His, w i t h o u t S-9 0 It
:~ 2
14 lb 10 13 lo
:t ± ± :t: ±
3 3 3 2 4
Average His-
• S-9
2o 23 32 30 32 33
0 :t 3 .t: 3 ± 3 2: o :t: t1 .1:3
without 5-9
S-9
0 I a "J: 5
22 18 24 18 22
t- 4 :t: 5 ± 3 .1:5 1:3
32 4! 36 2Q 32 38
O =t 7 ~: q :t: 2 I. 3 ::1:3 :l: 3
"~.~ :1= S D for t h t t e pl.~h'~ Po~zr~ve C~,ntn~l,.. T A 1 5 3 7 - OAA 75 ,~glpl.~w -- ~03 = 10; 2 A A 2.5 uglpl.~te "- I57 :t 28 T A 1 5 3 8 - 2 N I " 50 p g l p l a t e -" 17o(, 3- 50, ZAA 5.0 u g l p [ a t e = 257.5 "r 82. TAoS-2NFS0uglplJle = 1557 2. 4o: 2AA S O pf, Iplate : 2008 -r l l O
reported both positive (Carr and Rosenkranz, 1978; Rosenkranz et al.. 1975. Nakamura ez' al., 1979; Pfeiffer and Dunkelburg, 1980l and negative (Poirier and Simlnon, 1978). No ether mutagenesig information was found tn the published literature. 1 -chloro-2-prol0anol produced a dose-response increase in tumors in mice by i.p. injection; however, a statistically elevated pulmonary adenoma response was not observed (Thelss et aL. 1979). Therefore. it has not been clearly estabhshed whether or not this material has carcinogenic activity.
nate increases in background revertants due to vaporization of the test agent, the highest three doses, the four lower doses, and the c.ontrol series were incubated separately. After 48 hours, the colonies (revertants to histidine prototrophy) were counted. As recommended by de Serres and Shelby, all plates were reincubated for 24 hours and recounted (deSerres and Shelby, 1979).
S a l m o n e l l a / m i c r o s o m e assay Chloropropanol was investigated in five Salmonella typhimurium strains (TA 1535, 1537, 1538, 98,100) in the plate incorporation assay which has been described in detail (Ames et al., 1975), The tester strain genotypes were confirmed prior to use. An initial toxicity assay was conducted with TA 100 to determine the maximum tolerated concentration from w h i c h the mutagenicity assay concAntratioJls would be chosen. VogeI-Bnnncr Med=um E minimal agar plates were used, to which were added 2 mL of complete top agar and 0.1 mL of appropriate overnight broth cultures of S. ~yph(mur[um. S-9 mix, prepared with Aroclor 1254-induced rat liver, was prepared fresh, The S-9 mix contained 0,05 mL S-9 (liver homogenate) per 1.0 mL of mix. The controls used were (1) negative - (a) organism alone, (b) organism plus S-9, and (c) organism plus DMSO; (2) S-9 sterility; and (3) positive - - without S-9, (a) TA 1535 and 100 MNNG (5/~g/plate), (b) TA t 5 3 8 and 98. 2-nitrofluorene {50/~g/p|ate), (c) TA 1537, 9-aminoacridine (75 ~g/plate) - - with S-9, all strains 2-aminoanthracene (2.55 ,ug/piate).
L 5 1 7 8 Y mouse l y m p h o m a assay The mouse lymphoma assay has been described in detail (Clive and Specter, 1975; Clive, et al., 1979). TK . / - mouse lymphoma cells, subline 3.7.2C, frozen and stored in liquid nitrogen were thawed and used for this assay. Cells were grown in Fisher's medium for leukemic cells of mice made up in sterile deionized water and supplemented with approximately 10% (V/V) horse serum, sodium pyruvate, pluronic F68 and oenicillin-streptomycin (complete medium FIoP}. Closing medrum consisted of the medium described above with approximately 20% (V/V) horse serum. Melted agar was added to a final concentration of approximatrely 0.32% (V/V), Selective medium consisted of cloning medium with trifluorothymidine (TFT) added at a concentration of 2 /~g/mL. S-9 was prepared as in the Salmonella assay, and the S-I:J mix contained 2.5 mL of a 1:10 dilution of the S-9 liver homogenate per 10 mL S-9 mix (1:10 determined optimal on basis of standardization experiments with 2-acetytaminofluorene). An initial toxicity assay was cond~cted w;th seven concentrations of ¢!:!crop~ opanol (range 1.0.9.960 ,~g/mL) both with and without S-9. One tenth mL of each concentration of test agent, solvent and positive control fEMS and 2AAF) were added to tubes containing 6 mL F~oP,6.0 x 10 GTK ÷/- cells with additional 4 mL of FloP or S-9 mix. The cells w e r e ~×posed for four hours, washed, resuspended in 20 mL F~0Pand incubated two days. After the first and second day of incubation, cell counts and viability in each culture were determined, and following the first day, the viable cell count adjusted to 3.0 x 10 ~ cells/mL. Based on cell counts, the percent total suspension growth was determined.
Seven concentrations (ranging from 527 to 1 6 7 , 2 5 0 / z g / plate) of the test agent wer6.evaluated in triplicate in the five bacterial strains with and without'metabolic activation. Dimethyl sulfoxide (DMSO) was used to deliver the test material in the four lowest concentrations while the material was added neat in the three highest concentrations. To elimi-
The mutagenicity assay was conducted using 15 concentrations of chloropropanol in cultures without S-9 and 18 concentrations in cultures with S-9 (Table 2), The exposure and incubation conditions were identical to the toxicity assay. After the expression period, the cultures were selected for cloning. Cell growth in these treated cultures ranged from 6 to 99% of that
MATERIALS AND METHODS Chloropropanol, designated as a 75:25 mixture of 1 -chloro2-propanol and 2-chlorn-l-propanol. was obtained from Eastman Kodak. This material was subsequently analyzed and found, by gas chromatography/mass spectrometry, to consist of 73.3% 1-chloro-2.propanol and 2 6 3 % 2-chime- 1-propanol
28
Fundam. Appl. ToxlcoL (3)
January/February, 1983
GENETIC TOXICOLOGY
OF CltI.OROPROPANOL
TABLE 3 S u m m a r y of Results: Mouse L y m p h o m : , Forward M u t a t i o n Assay of Chloropropanol
Concentration
Survival ^
Without
Metabolic
Activation
Fold
(% of c o n t r o l )
Mutation Frequency"
increa~e'"
100% 100'~ 5%
10.0 12.2 IO0.0
. . . . 15.t',
10,241
15%
47.2
3.0
244","
0,832 o,422 o,012
3o% 54% 30='0
8,603
00%
42.3 32.3 4,~.7 20 8 30 0 41 o 21 o .I 3.4 37 8 18 I
3.5 2.0 .t.O 1.7 2.5 3 4 1.8 3.5 3 I 1.5
133% 120"., 04% 1o4% I lo% 130% 114% 100% 113% 111%
2 I. I
2 0
t 00%
22.3 10.3 20.0
1.8 1.0 1.7
05°> 142at• o~%
fpgtmL) Medi., C o n t r o l S o l v e n t C o n l rol EMS-o20.O
8.0%
•q,l o 3 7,783
80% 80% u4 o., 08% ,qTet• ¢~5,"., o.1% 105¢iO3"~,
7,374
0,004
0.554 o.145 5,735 5,325 4,01() 4.500
Cloning E f f i c i e n c y (%) . .
. . --
•'~Rel,ltive s u s p e m , ion g r o w t h ,, clorHng e f f i c i e n c y =*The n u m b e r of T K -/- m u t a n t ~ p e r 1 x 10~'colony f o r m m g c e l i s p l a t e d o n t o tlae ~elc
Survival ~
Clnning t'fficiency (%)
(% of control)
Mutation F r e q u e n c y I'
increase c
! 00% 100%
3.0 2.6
. .
13%
42.2
I 0.3
--
T I'
T
T
--
500
I"
T
T
--
470
T T T 3% 5% 3% 0% 7% 28% 14%
T
T
--
T
T
--
T 24.5 13.8 ! 4.6 7.2 11,4 12.4 9.2
-54% b7% 57%
20%
55
30°/0 4bq3 38%
5.0 3.o 5..5
T q.4 5.3 5.0 2.8 4 4 4.~ 3.5 2.1 1.o 1.4 2.1
I 01% 77% J87'~ oi% IbO% 157% 127% 8Sq;,
5,0
40%
20
77%
3 6 1.7
1.4 0.7
7q% 7700,
(pglmL) Media Control Solvent Control 2AAF-50
,530
440 410 31~0 330 320 290 260 230 200 170 140 I I0 80
Fold
. .
. .
. .
"Relatives.uspens;on growth x cloning efficiency ~ThenumberofTK-I-mutantsper] x tO'colony formingcellsplaled ontothe 5elective cloning medium. CFold Increase : Mutation Frequency of Test Concentration Mutation Frequency of Solvent Control DT = Too toxic to clone of the u n t r e a t e d control. This is referred to as relative suspenstun g r o w t h . A p p r o x i m a t ' e l y 5 x 1 0 s cells w e r e plated on each of t h r e e selective m e d i u m plates (2 u g / m L TFT) and 1 0 0 cells Fundsmentsl and Applied Toxitololy
(3) 1.2/83
cloned on each of t h r e e n o n - s e l e c t i v e # a l e s for each test and control tube. The plates w e r e incubated for 1 1 days and the m u t a n t colonies c o u n t e d o n t h e selective plates, w h i l e t h e 29
BILES A N D PIPER TABLE 5
Metaphase Analysis of Bone Marrow Cells from Male and Female Rats Treated with ChIoropropanol Treatment Group
Number of Cells
Numberof Aberrant
Total Numberof
Chromatld
Chromosome Markers
Severely Damaged Cells ( > I 0 aberrations)
Analyzed
Cells
Aberrations"
Breaks
Breaks
Negative Control (H20)
350
11 (1.6 :t: .8) ^
12 (1.7 -t- .9)
7,0 1.4 ± .9)
0
2
O
10 mglkg Chloropropano]
~00
68 (8,5 +- 3,6)
92 (11.5 -t- 7.1)
51 (6.4 + 2.3)
5
26
1
31 mg/kg Chloropropano[
450
108 (12 ± 2.1)
219 (24,3 + 9.9)
59 (6.6 :k 3.3)
Z
58
10
l e o mg/kg Chloropropanol
350
93 (13.3 ± 5,2)
279 (39.8 + 44)
88 (12.6 ::t: 9.6)
4
47
8
TEM Positive Control
450
209 (23.2 ± 3.7)
949 (105 + 44)
110 (12,2 ~ 6.7)
16
163
66
^,'~ ---+ ' : ' D / o r g r o u p "Severely damaged cells were counted as exactly ten aberrations and were included in this column.
survivors were counted on the non-selective plates. Colonies were counted on a New Brunswick Biotran II, Model C-111. Rat in vivo bone m a r r o w cytogenetic assay Sexually mature male and female Sprague-Dawley, CD® rats were obtained from Charles River, Wilmington, Massachusetts. The animals were housed individually and acclimated to laboratory conditions for two weeks. Fifty rats, weighing between 200-300 grams, were randomly assigned into five groups, each consisting of 5 males and 5 females. The groups were (1) negative control - deionized water, (2) positive control - triethylene-melamine (TEM) 0.4 mg/kg, (3) chloropro~ panel - 10 mg/kg, (4) chloropropanol - 31 mg/kg and (5) chloropropanol 100 mg/kg. Chloropropanol dilutions were prepared in deionized water, and appropriate doses of vehicle or test agents were administered daily for five consecutive days via oral intubation. The animals serving as the positive control received a single intraperitoneal injection of TEM on day 5. Observations were made daily of general appearance, behavior, and toxic and pharmacologic effects. Body weights were recorded prior to initiation of treatment and on day 6, prior to administration of colchicine. All animals received a single intraperitoneal iniection of colchicine dissolved in Hanks Balanced Salt Solution (HBSS), pH 7.4. Approximately two hours after administration of colchicine, the animals were sacrificed by CO2 asphyxiation. The techniques used to process the bone marrow cells represent modifications of the techniques described by Evans (Evans, 1976) and Kilian (Kilian et al., 1977). Bone marrow was collected by needle aspiration of both femurs into 5 .mL of HBSS. Following 5 min. centrifugati0n at 1,000 rpm; the supernate was decanted and 7.0 mL 0.075 M KCI added. After 25 minutes, fiv e drops of Carnoy's fixitive were added and the cells were mixed and recentrifuged for fiv e minute s at 1,000 rpm. This process was repeated three time s prior to overnight refrigeration. The next day, the cells were pelleted, resuspended in 1-3 mL Carney's, and threedrops of this final cell suspension dropped , on a m!c~'osc0Pe slid e and al!owed to air dry. Three slides were made from eac h animal. The slides were identified and stained for 16 minutes Wit h a Giems a prel~arationl The slides we;'e then rinsed twice in tap water, air dried, 30
and mounted with cover slips. The animal identification number was covered, and the slides were coded for reading. The evaluation sequence of prenumbered slides was determined from a table of random numbers. Only those cells in metaphase stage of mitosis were analyzed for the presence of cytogenetic abnormalities. The mitotic index was also recorded for each animal. Fifty cells in metaphase were examined from each rat that provided analyzable cells. Stage vernier settings were recorded, and all cells once visualized on high power were recorded unless there was obvious mechanical damage. Chromosomal aberrations were classified into one of four basic groups: (1) chromatid breaks; (2) chromosome breaks; (3) markers, which include dicentrics, enchanges, rings, and other miscellaneous configurations; and (4) severely damaged cells, Chromatid aberrations involve a break in only one arm of the chromosome, whereas chromosomal ab6rrations involve both arms at identical sites. A break was defined as any separation that exceeds the width of the chromatid arm, or a discontinuity accompanied by disturbance of the axial integrity. Aberrant configurations, which alter the structural appearance of an entire chromosome or involve the joining together of more than one chromosome, were categorized as markers. An exchange was defined as a symmetrical or assymetrical distortion of the usual chromosomal pattern. A dicentric was defined as achromosome which possesses two centromeres. A ring chromosome was defined as one in which the ends have joined to form a circle, with or without a centremere. Those cells which had more than ten scorable aberrations were classified as severely damaged. When the total number of aberrations was determined in the cells analyzed for the individual animals, severely damaged cells were counted as exactly ten aberrations, as suggested by Kilian (Kilian et al., 1977). The mean changes in body weights and the mean mitotic indices w~re analyzed using Bartlett's test for equality 0f variance, the one-way classification of ana ys s of Va'r ance,and Scheffe's Multiple F Test procedure. The number of aberrant cells and the total number of aberrations per animal (includ!ng chr0matic breaks, chromosom e breaks, markers, and severely damaged Cells) Wei;e statistically analyzed by Wilcox0n's nonparametric comparisbn 0f group means. Fundam. A~DL Toxicol. (3)
Januarv/Februarvo 1983
GENETIC TOXICOLOGY OF CHLOROPROPANO[.
RESULTS S a l m o n e l l a / m i c r o s o m e essay The results shown in Table 1 for the two-base substitution strains TA1535 and TA100 indicate a clear dose.related mutagenic response observed w i t h and w i t h o u t activation. The quantitative difference between the response of the t w o strains may or may not have any biological significance. The presence of a background bacterial l a w n w a s seen at each test concentration and toxicity of the con=pound, as demonstrated by a "sparse l a w n " , was clearly observed at the highest concentration. TA1535 demonstrated a marked enhancement of mutagenic response w h e n S-9 was incorporated in the reaction mixture. Prolonged incubation (72 hours) and a pronounced effect in this strain, but did not appreciably affect strain T A 1 0 0 (data not shown). The results in Table 2 indicate no appreciable increased frequency of reversion to histidine prototrophy in the strains TA1537. and the frameshift strains TA1538. and TA 98. Neither metabolic activation or prolonged incubation (72 hours) had any effect in these strains. L 5 1 7 8 , ' h o u s e / y m p h o m a assay A toxic dose-related response was observed w i t h chloropropanel following the first day expression. As shown in Table 3, in the absence of metabolic activation, the total survival (as a % of control) ranged from 15% at the highest concentration to approximately 100% at the lower concentrations. Clive, et al., (1979) defines an appropriate limit as one yielding 90% kill (10% survival) w h i l e Brusick (1980) has recommended choosing 4 to 5 doses spaced from 0 to 50-90% reduction in cell growth. A greater than t w o - f o l d increase in mutation frequency over the spontaneous background levels w a s observed at eight concentrations. The response w a s not uniform as can be seen in Table 3. Table 4 indicates the mutagenic activity of chloropropanol w i t h metabolic activation. Toxicity arising at concentrations above 4 1 0 / ~ g / m L inhibited cell g r o w t h to the extent that these
test levels could not be cloned. Greater than two-fold increases in m u t a t i o n frequency over the spontaneous levels w e r e obtained at nine test concentrations, including four test concentrations at w h i c h greater than 10% total survival was achieved. Rat in vivo bona m a r r o w c y t o g e n e t i c essay No animals died during the study, and individual clinical observations indicated that chloropropanol did not produce overt toxic effects in rats at doses of 10. 31 and 100 m g / k g . Mean initial and t e r m i n a l body weights and body w e i g h t changes for the negative control and treatment groups w e r e analyzed by ANOVA (p--.05). No significant w e i g h t gains or losses wc, rA n|.~served; however, a depression in body w e i g h t gain w a s observed at the highest dose lbody w e i g h t changes in grams w e r e (1) negative control - 22*8.4; (2) 10 m g / k g 2 6 ~ 1 0 . 9 : (3) 31 m g / k g - 25+12.9;(4) 100 m g / k g - I 1.4+12.41. Five hundred representative metaphases w e r e not achieved for each group. The actual members analyzed are given in Table 5. The mean mitotic index (number of cells undergoing mitosis Der 100 cells counted) was not statistically different (p:.05) among the groups w h e n analyzed by ANOVA. The index ranged from 1-5. Values wore: (1) negative control 3.14+1.1;(2) positive control (TEM). 2.67.1.3;(3) 10 m g / k g 4.13+1.1;(4) 31 m g / k g - 3.22.0.7; (5) 100 m g / k g - 4 . 1 4 . 0 . 9 . The total number of cells analyzed, number of aberrant cells, the n u m b e r of total aberrations and certain classes of aberrations are shown in Table 5. A s u m m a r y of these data is presented in Table 6. As can be seen in these tables, the number of aberrant cells and aberrations per cell are greater in the test groups thaq in the negative control, and these responses appear to be dose related. A Mann-Whitney-Wilcoxen test w a s performed to compare the number of aberrant cells and total number of a b e r r a t i o n s / a n i m a l between the negative control and the other groups. There were significant increases (p < .05) in the number of aberrant cells and the total number of aberrations per animal i n t r e a t e d groups w h e n compared to the negative contro{s.
TABLE 6 Comparison of Frequencies of Chromosomal Aberrations in Bone M a r r o w Cells [rum Groups of Animals Treated With Chloropropanol and Controls
Cell"
Fold Increase of Aberrations/ Cell ~"
0.034
--
Average Treatment Group
Negative Control (H~O)
Total Number Cells Analyzed
% Aberrant Cells/`
Aberrations/
3SO
3.1
Chloropropal~ol
.I00
! 7.0
0.23
b,8
31 mglkg Chloropropanol
450
24.0
0.49
I4..1
Chloroprup.~/~ol
350
26.6
0.80
23.5
Positive Control (TEM)
450
46.4
2.10
61.8
10 mg/k~
I00 mglkg
"This number repre~.ent~. :he totai number of aberrant ~cells/group divided by the total number of cells analyzed per group × 100. ~This number represents the total number of aberrations/gruup divided by the lutal number of cells analyzedlglnup. CThis number represents the average aberrationslcell for the test groups divided by the value obtained for the negative control (0.034 aberrations/celt). Fundamenttl and Applied ToxicoloR)
(3) 1-2/83
31
BILES AND P}PER DISCUSSION There is no single, short-term genetic bioassay which is adequate for thorough toxicological safety evaluation. If screening for potential carcinogens is the intended use of a short-term test, then sufficient data on sensitivity and specificity already exist for many of the prominent assays to preclude reliance on any single assay in that evaluation process. In fact, recent reports recommend the use of a core battery of tests for evaluating the mutagenic and carcinogenic potential of a material. Ray (Ray, 1979) reports a core battery of tests under zvaluation which contains the three assays used in the present study, as well as an in vitro cytogenetic assay in human lymphocytes and an in vitro mammalian cell transformation assay. Further, Ray outlines the criteria for selecting a core battery and suggests that the battery should be flexible. This ongoing review demonstrates that few substances with mutagenic or direct-acting carcinogenic potential would be missed by Ray's core battery. Sobels (Sobels, 1980) believes that a minimal battery should contain at least three tests: (a) a test for gene or point mutation in bacteria, (b) two tests for point mutations in eukaryotes, or (c) one such test and a test detecting chromosome aberrations in mammalian cells in vitro. Waters, (Waters, et al., 1980) recommends (a) a point mutation assay in microorganisms, (b) a gene mutation assay in mammalian cells, (c) a primary DNA damage assay, (d) an assay for chromosomal effects in mammalian cells and (3) a transformation assay in mammalian cells. The value of the present test results, with respect to a screen for carcinogenic potential, can only be strengthened by the corroborative evidence obtained in each of the three assays. The two in vitro assays have individually been shown to have a relatively high detection capability for carcinogens based on validation experiments with chemicals of diverse structure and biological activity. The in vivo cytogenetics assay does not share this sensitive carcinogen detection capability, but does give perspective as to the potential of a chemical to have mutagenic or heritable genetic effects. Chloropropanol was positive in all three of the genetic assays. While uniformity of results in a screening battery is desirable, there are reasons for individual test result differences. The larger the battery and the more diverse the endpoints of the tests which make up the battery, the more likely that results of all the assays will not be uniform. However, consistancy should be expected within a class of similar genetic end points. For example, in the present battery, the two gene mutation assays should be required to be consistent for a meaningful evaluation. But the chromosomal assay, which measures a distinctly different endpoint, might be regarded as a complementary assay ~ one which, if positive, will cast further evidence as to a compound's toxic potential. McCann (McCann et al., 1975) reported Salmonella detected 17 of 20 previously tested animal carcinogens which were categorized as alkyl halides. In the present study, the isomers of chloropropanol were active in the base substitution strains of Salmonella'both with and without metabol!c activatio n. There was an enhanced response in TA 1535 the presence of metabolic activation. This supports most of th e previous published work with Salmonel/a, and t h e r e w a s especially good correlation with the work of Pfieffer (Pfieffer and Dunkeiburg, 1980).. However, F~i~ffer c0ncluded that l~chl0ro-2-pr0panol is totally responsible for the mutagenic activity of the isomeric mixture.Pfieffer states in hiS report that2-chlor0:-I -propanol is not mu!agenic from Work reported b y Carr/(Cal"r: and
32
Rosenkranz, 1978), but the earlier work by Carr refers to 3-chloro-1-propanol, not 2-chloro°l-propanol. In the present study, the isomeric mixture composed of 75% 2-chloro-1propanol was shown to be both a direct acting and indirect acting mutagen in bacteria. Both the toxic and mutagenic activities of the isomeric mixture of chloropropanol were enhanced in the presence of metabolic activation in the L5178Y mouse lymphoma assay. The material was also active without me!~.~:,olic activation, similar to the results obtained in the Salmonella assay. While the dose-related responses in toxicity and mutagenicity were obtained with chloropropanol, these responses were not strictly linear in nature for any of the measured parameters such as percent suspension growth, mean numoer of mutant colonies on selective plates, percent total survival or mutation frequency. This is most likely due to the relatively closely spaced dose levels which were examined. There was a dose-related effect of chloropropanol on the physical structure of the chromosomes in bone marrow cells from rodents. The oral administration of chloropropanol for 5 days showed no overt toxic effect and resulted in no significant body weight gain/loss. Furthermore, there was no significant difference in the mean mitotic index between the test groups and the negative control. There was an apparent dose-related increase in the number of total aberrations and, more specifically, in the number of chromatid breaks. While the mutagenic events described in the point mutation assays might be initiated by a single interaction with DNA, these cytogenetic responses probably involve more than one interaction, Since the cytogenetic study was done in the intact animal, it is reasonable to conclude the in vivo defense/repair mechanisms are not sufficient to protect the somatic cells from genetic damage. An interesting extension of this is the possible interaction of the materials with germinal cells. Materials which are structurally similar to chloropropanol, i.e. alphachlorohydrin and 3-chloro-l-propanediol have been shown to have specific effects on germinal cells and also cause sterility in rats while not affecting libido or spermatogenesis (Cooper et aL, 1974; Erickson, 1970). In addition to this, a mutation within a germinal cell which still allows viability might effect a heritible event; a genetic alteration which is expressed in a future generation. The genetic activity of an isomeric mixture of 1-chloro-2propanol and 2-chloro-1 -propanol, demonstrated in the present study, predict the possibility of two, possibly three, chronic health problems which may arise from exposure to the compound. Since short-term tests are currently best considered screening devices, the potential carcinogenic, reproductive and heritable mutagenic effects need to be evaluated by classical long termanimal test methods for a definitive assessment of the toxicity of c'~toroDropanol. REFERENCES
Ames, B.N,, McCann, J, and Yamasaki, E. (1975). Methods for Detecting Carcinogens and Mutagens with the SalmoneliaMicrosome Mutagenicity Test, Mur. Res. 31:347-364, Brusick, .D. (1980), Principles of Genetic Toxicology~ Plenum Press, New York and London. pp~ 204-211. Carr, H.S. and R0senkranz; H.S. (1978). Mutagenicity of Derivative of {:he.Flame Retardant Tris (2;3-~Iibromopropyl) PhosL phat6: Halogenated Propan0ls. MuL Res~ 57:381-384. Clive, D. and Spector, jr; (1975). Laboratory Procedure for Assessing Specific' Locus Mutati0ns at f~he TK L0cu~s in Cultured L5i784 Morose Lymphom;i Cells.. ~{fut, Res. 31:17-29. Fundam. At~DL Toxicol. (3)
Januar~/Februar~v.'1983
GENETIC TOXICOLOGY OF CHLOROPROPANOL Clive, D., johnson, K.O., Spector, J.F.S., Batson, A.G. and Brown, M.M.: (1979). Validation and Characterization of the L5178Y/ TK +l- Mouse Lymphoma Mutagen Assay System. Mul. Res. 59:61-108. Cooper, E., Jones, A. and Jackson, H. (1974). Effect of AlphaChlorohydrin and Related Compounds on the Reproductive Organs and Fertility of the Male Rat. J. Reprod. Fertil. 38:379-386.
deSerres, F.J. and Shelby, M.D. (1979). The Salmonella Mutagenicity Assay: Recommendations. Science 203:563-565. Ericson, R.J. (1970). Male Antifertility Compounds: U-5897 as a rat chemosterilant../. Reprod. ]'~,rliL 22:213-222. Evans, H.J. (1976). Cytological Methods for Detecting Chemical Mutagens; Chemical Mutagens; Principles and Methods for Their Detection, Volume 4, ed. by A. Hollaender, Plenum Press, New York. pp. 1-30. Flamm, W.G., Chairman (1977). Subcommittee on Environmental Mutagenesis Report: Approaches to Deternfinlng the Mutagenic Properties of Chemicals: Risk to Future Generations. Prepared for the DHEW Committee to Coordinate Toxicology and Related Programs..L Environ. Path. ToxicoL 1:301-352. Kilian, D.J., Moreland, F.M., Benge, M.C., Legator, M.S. and Whorton, E.B. (1977). A Collaborative study to Measure Interlaboratory Variation with the In Vivo Bone Marrow Metaphase Procedure, Handbook of Mutagen Testiog, Elsevier/ North-Holland, Amsterdam, pp. 243-260. McCann, l., Choi, E., Yamasaki, E. and Ames, B. (1975). Detection of Carcinogens as Mutagens in the Sa|monellalMicrosome Test: Assay of 300 Chemicals. Proc. Nat. Acad. Sci. USA 72:5135-5139. Nakamura, A., Tateno, N., Kojima, S., Kaniwa, M. and Kawamura, T. (1979). The Mutagenicity of Halogenated Alkanols and their Phosphoric Acid Esters for Salmonella Typhimurium. Mut. Res. 66: 373-380.
Pfeiffer, E. and Dunkelburg, H. (lO80). Mutagenicity of Ethylene Oxide and Propylene Oxide and their Glycols and Halohydrins Formed from them during the Fu,. igation of Foodstuffs. Fd. CosmeL Tu.vicoL 18:115-1 ]8. Poirier, L. and Simmon, V. (1978). ]'he Mutage,~icity of Substituted Organo-Halides to S. Typhimurium TA 100. Presented to the International Congress in Buenos Aires, October 1978. Ray, V. (1979). Application of Microbial and Mammalian Cells to the Assessment of Mutagenicity. Pharn~acoloR.c Reviews 30:537-546. Regells, E.P., Fisher, B.S. and Kllmeck, A.B. (1908). Isolation and Determinalion o/t Chlorohydrlns in Foods Fumigated with Ethylene Oxide or with Propylene Oxide. J. Ass. Offic. Anal. Chem. 51:707-715. Rosenkranz, ILS., Wlodkowski, T.]. and Bodine, S.R. (1975). Chloropropanol, ,¢Mutagienic Residue Resuhing from Propylene Oxide Sterilization. Mut. P,es. 30:303-304. Sobels, F,H. (1980). Evaluating the Mutagenie Potential of Chemicals: The Minimal Battery and Extrapolation Problems. Given at International Conference "Mutagetficity Testing of Pharmaceuticals: Present Status" Paris 12-14, March 1980. Steele, L. and Hadziyev, D. (1976). Sterilization of Dehydrated Potato Granules with Propylene Oxide. Z. l.ebens.Miltehmters. U-Forsell. 162:387. Theiss, J., Shimkin, M. and Polrler, L. (1979). Induction of Pulmonary Adenomas in Strain A Mice by Substituted Organo HMides. Cancer Research 39:391-395. Wailers, M., Simmon, V., Mitchell, A. and lorgenson, T. (1980). An overview of Short-term Tests for the Mutagenic and Carcinogenic Potential of Pesticides../. Environ. ScL lleallh B] 5(6):867-906. Wesley, F., Roarke, B. and Darbishire, O. (1965), Formation of Persislent Toxic Chlorohydrins in Foods by Fumigation with Ethylene Oxide and Propylene Oxide. J. Food Sci. 30:1037-1042.
33