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The in vivo rat micronucleus test: integration with a 14-day study
Genetic Toxicology
ELSEVIER
Mutation Research 342 (1995) 71-76
The in vivo rat micronucleus test: integration with a 14-day study Michael L. Garriott *, Jamie D. Brunny, Delinda E.F. Kindig, Joseph W. Parton, Linda S. Schwier Lilly Research Laboratories, A Division of Eli Lilly and Company, Greenfield, IN 46140 USA Received 13 June 1994; revised 2 November 1994; accepted 28 November 1994
Abstract
A 14-day subchronic toxicity study is routinely conducted in Fischer 344 rats at the Lilly Research Laboratories. This study is done to gather preliminary toxicological information about chemical entities showing efficacy in various pharmacological screens. This manuscript describes the validation of a method for evaluating micronuclei in the bone marrow polychromatic erythrocytes of animals from this test in order to obtain additional information about the genotoxic potential of these compounds without incurring the cost of additional animals or the use of additional test article, which is often in limited supply. Compounds selected for evaluation were acetylsalicylic acid, mitomycin C, cyciophosphamide, colchicine, 6-mercaptopurine, and etoposide. With the exception of colchicine, the results obtained were as expected with acetylsalicylic acid yielding negative results and the other compounds yielding positive results. These findings are consistent with those published for mice (MacGregor et al., Fund. Appl. Toxicol., 14, 513-522, 1990) and show that a bone marrow micronucleus test can be successfully integrated into a routine subchronic rat toxicology study.
Keywords: Micronucleus test; Rat; Integration; Subchronic
I. Introduction
The mouse micronucleus test has been recommended for the safety evaluation of new pharmaceuticals, agrichemicals, and industrial chemicals in various regulatory guidelines worldwide. The test was developed in the early seventies (Matter and Schmid, 1971; Schmid, 1973; Heddle, 1973) and though mostly unchanged during that decade, the protocol has received considerable attention
* Corresponding author. Tel.: (317) 277-4800; Fax: (317) 277-4436. Elsevier Science B.V.
SSDI 0 1 6 5 - 1 2 1 8 ( 9 4 ) 0 0 0 8 1 - 6
during the last ten years (Salamone and Heddle, 1983; M a c G r e g o r et al., 1987; Garriott et al., 1988; Tinwell, 1990). Most recently M a c G r e g o r et al. (1990) have reported the existence of a steady state frequency for micronuclei following repeated daily dosing regimens and they have suggested the integration of micronucleus studies with routine toxicological studies. Their work was conducted in mice though they made recommendations for the use of rats as well. At Lilly Research Laboratories, pilot studies are conducted with potential pharmaceuticals very early after the discovery process to obtain preliminary toxicity information about the compound.
72
M.L. Garriott et al. / Mutation Research 342 (1995) 71-76
These studies are routinely conducted in rats and consist of daily treatment of the animals for two weeks followed by necropsy 24 hr after the last treatment and examination of tissues. Although various factors are considered when choosing doses for this testing, a preferred factor is to use a multiple of the anticipated human clinical dose when possible. This study was conducted to verify the conclusions of MacGregor et al. (1990), to gather data in rats, and to expand the data base by investigating additional compounds prior to beginning to routinely integrate or "piggy-back" a micronucleus test onto this preliminary toxicity screen. The purpose of integrating this study would be to gather preliminary information about the potential genotoxicity of a test article while obtaining general toxicity data since this could be done without incurring the cost of additional animals or the use of additional test material. The compounds tested are all human therapeutics and included a non-genotoxin (acetylsalicyclic acid), direct- (mitomycin C and 6-mercaptopurine) and indirect- (cyclophosphamide) acting clastogens, and two spindle poisons (colchicine and etoposide).
2. Materials and methods Acetylsalicyclic acid (ASA: CAS No. 50-78-2), mitomycin C (MMC: CAS No. 50-07-7), cyclophosphamide (CP: CAS No. 6055-19-2), 6mercaptopurine (6MP: CAS No. 6112-76-1), and etoposide (ET: CAS No. 33419-42-0) were obtained from Sigma Chemical Company, St. Louis, MO, and were solubilized in 10% acacia. Colchicine (COL: CAS No. 64-86-8) was obtained from Eli Lilly and Company, Indianapolis, IN, and was dissolved in 0.9% saline. With the exception of MMC, all compounds were administered daily by oral gavage for 14 consecutive days. Animals receiving the low-and mid-dose of M M C were treated for 14 days while those receiving the high dose were treated for five days. Male and female Fischer 344 rats were obtained from Harlan Sprague Dawley, Indianapolis, IN, at 4-5 weeks of age and were acclimated
for approximately 7 days prior to test initiation. Groups of five animals were housed in ventilated, stainless steel cages with wire mesh bottoms and Lexan ® fronts that were maintained in a room with a temperature of 24 +_ 2°C and at least 40% humidity with a 12/12 hr l i g h t / d a r k cycle. Animals were individually identified by ear punch and given free access to Purina Certified Rodent Chow No. 5002 and tap water. Doses utilized in this study were based on the human clinical dose, or the normal human dose (ASA), of the compound as described in the Physician's Desk Reference (1990) and on median lethal dose (MLD) data from the Merck Index (1983). Doses were not extrapolated from humans to rats, but were determined on an equivalent m g / k g basis. Doses for M M C were 0.1, 1, and 10 × the human clinical dose, while doses for 6MP and ASA were 1, 10, and 50 x the clinical and normal human dose, respectively. The top dose of 750 m g / k g for ASA is also equivalent to one-half the oral M L D in rats. Doses for C O L were 1, 10, and 100 × the human dose while CP was dosed at levels equal to 0.0002, 0.02, and 1 × the human dose. The doses for E T were 0.1, 1, and 5 × the clinical dose. As noted in the Introduction, doses for the preliminary subchronic study at Lilly are based, when possible, on the anticipated clinical dose and tested at the types of dose intervals shown above. These intervals are much wider than those for a definitive micronucleus test conducted according to existing regulatory guidelines, but they are determined by the Chronic Study Area and not by the Genetic
Table 1 Summary of 14-day subchronic results Compound Toxicity a Genotoxicityb Expected c Acetylsalicylicacid Mitomycin C Cyclophosphamide Colchicine 6-Mercaptopurine Etoposide
+ + + +
+ + + +
+ + + + +
Animals exhibited clinical signs of toxicity and/or death at one or more dose levels. b Measured by induction of micronuclei (+) or not (-). c Genotoxicity result anticipated from the literature. a
M.L. Garriott et al./ Mutation Research 342 (1995) 71-76 Table 2 Results of the micronucleus tests
a
Compound
Sex
Acetylsalicylic acid
M M M M F F F F
Dose (mg/kg) 0 15 150 750 0 15 150 750
M P C E b per 1000 P C E c 1.4 1.8 1.0 2.0 0.8 0.6 1.8 3.0
Trend test two-tailed p value
+ 1.7 o + 1.3 + 0.7 +- 0 e + 0.8 _+ 0.5 + 0.4 + 2.7
0.59
pooled g Mitomycin C
Experiment 1
M M M F F F
0 0.05 0.5 0 0.05 0.5
2.2 1.6 2.8 2.2 2.4 1.6
+ 1.6 + 2.1 + 1.9 _+ 2.5 + 1.7 + 0.9
Experiment 2
M M F F
0 5.0 0 5.0
2.8+_2.6 6.4 + 2.6 2.6 _+ 1.3 5.8 +- 2.2
h h h h
0 0.05 0.5 5.0 0 0.05 0.5 5.0
2.5 1.6 2.8 6.4 2.4 2.4 1.6 5.9
+ 2.0 + 2.1 + 1.9 _+ 2.6 + 1.9 + 1.7 + 0.9 + 2.2
M M M M F F F F
0 0.001 0.1 5.0 0 0.001 0.1 5.0
2.4 2.0 1.8 19.4 2.0 2.4 1.8 8.4
+ 2.6 _+ 1.9 + 1.6 + 7.7 + 0.7 + 2.5 + 1.3 + 2.1
M M M M F F F F
0 2.5 25 125 0 2.5 25 125
0.0060 < 0.0001
< 0.0001
pooled g 6-Mercaptopurine
0.014 0.0003
0.0003
pooled g Cyclophosphamide
0.51 1.0 0.008
pooled g M M M M F F F F
0.002 f 0.016 f
0.52
pooled g
Combined
73
1.6 + 0.9 3.0 + 2.0 155.4 + 73.9 - J 1.2 + 0.8 2.0 + 1.6 76.4 + 16.6 - J
< 0.0001 < 0.0001
< 0.0001
pooled g
< 0.0001 < 0.0001
74
M.L. Garriott et aL / Mutation Research 342 (1995) 71-76
Table 2 (continued) Compound
Sex
Colchicine
M M M M F F F F
Dose (mg/kg) 0 k 0.06 0.6 6.0 0k 0.06 0.6 6.0
MPCE b per 1000 PCE c
Trend test two-tailed p value
1.2 + 1.3 2.4 _+ 1.5 1.2 _+ 1.1 - J 1.6 ± 1.1 1.4 0.5 1.2 ± 0.3 - J
1.0
pooled g Etoposide
M M M M F F F F
0 1.14 11.36 57 0 1.14 11.36 57
0.8 + 0.8 1.0 _+0.7 3.4 + 2.1 87.2 _+ 17.8 1.0 _+ 1.0 1.4 _+ 1.1 2.4 _+2.5 18.6 + 4.2
0.59 0.72
< 0.0001
pooled g
< 0.0001 < 0.0001
a Fourteen equal treatments ~ 24 h apart with harvest ~ 24 h after the last treatment. Ten percent aqueous acacia used for the vehicle and delivered at 10 ml/kg. b MPCE, micronucleated polychromatic erythrocyte. c PCE, polychromatic erythrocyte. d Values are mean _+SD for 5 animals/treatment group. e Only 1 animal survived. Statistically significant due to low background in female vehicle control group. Not considered positive for micronucleus induction. g Pooled, Mantel-Haenszel pooled across sex. h Five equal treatments ~ 24 h apart with harvest ~ 24 h after the last treatment. Values are mean + SD for 10 animals. i No survivors. k 0.9% saline used for the vehicle at a dose volume of 12 ml/kg.
Toxicology A r e a . T h e r e f o r e , it was desirable to know w h e t h e r e x p e c t e d results c o u l d be o b t a i n e d at th es e dose levels and dose intervals f r o m comp o u n d s k n o w n for t h e ir genotoxicity, or lack thereof. T h r e e t r e a t m e n t groups and o n e v e h i c l e control g r o u p e a c h consisting of 5 males and 5 fema l es w e r e utilized for each test c o m p o u n d . A p prox i m at el y 24 h after th e last t r e a t m e n t , animals w e r e e u t h a n i z e d by c a r b o n dioxide asphyxiation. B o t h f e m u r s w e r e r e m o v e d by t r i m m i n g away th e skin and muscle and cutting at the pelvic socket and k n e e joint. U s i n g a pair of shears, t h e proximal e n d of the f e m u r was cut at a p p r o x i m a t e l y a 30 ° angle to expose th e b o n e marrow. A b r u s h (00 sable hair artist brush) was d i p p e d into b o v i n e calf serum, t h e n inserted into the b o n e m a r r o w
cavity, and gently r o t a t e d . T h e brush tip was t h e n d i p p e d and r o t a t e d in a d r o p l e t o f s e r u m previously ap p l i ed n e a r o n e en d o f a m i c r o s c o p e slide. T h e slide was t h e n p l a c e d in a M i n i P r e p ® bloods m e a r i n g i n s t r u m e n t and a s m e a r was m a d e , o n e p er e a c h femur. F o l l o w i n g o v e r n i g h t air-drying an d fixation for 5 m i n in 100% m e t h a n o l , t h e slides w e r e stained with acridine o r a n g e (Hayashi et al., 1983). Slides w e r e c o d e d for p r e v e n t i o n of bias af t er b e i n g s e p a r a t e d into two groups for e x a m i n a t i o n by two observers. Slides w e r e examined with a Zeiss Lab 16 m i c r o s c o p e e q u i p p e d with e p i f l u o r e s c e n c e . A t least o n e t h o u s a n d anuc l e a t e p o l y c h r o m a t i c erythrocytes w e r e c o u n t e d for e a c h animal to d e t e r m i n e the f r e q u e n c y o f m i c r o n u c l e u s induction. A t r e n d test for Poisson distribution ( M a c G r e -
M.L. Garriott et al. /Mutation Research 342 (1995) 71-76
gor et al., 1987) was performed on micronucleated polychromatic erythroc3,te values from individual animals. The M a n t e l - H a e n s z e l test (Mantel, 1963) was used to pool the inference across sexes (Tamura et al., 1990).
3. Results and discussion Results from the testing are summarized in Table 1 and compound-specific data are shown in Table 2. With the exception of COL, the results were consistent with expectations. Although this spindle poison was expected to elicit a positive response for micronucleus induction, the results were negative. A possible explanation for the negative response may be the wide dose interval utilized since C O L is active only over a narrow dose range. Another possible explanation may be that the compound was dosed orally instead of intraperitoneally as in the study by MacGregor et al. (1990). The top dose in that study and the mid-dose in this study were nearly equivalent and might reasonably be expected to produce a similar result. No animals survived at the high dose. To ensure that the methodology could detect a spindle poison, a second aneugen, ET, was evaluated. This compound did elicit a positive response. When the data collected for A S A were evaluated statistically, a significant, dose-related increase in micronucleus frequency was obtained for the data collected from females and for the combined data. Inspection of the data showed that the incidence of M P C E in the female vehicle control group was quite low (0.8 M P C E per 1000 PCE). Furthermore, only one female of the five in the top dose group had an incidence (7 M P C E per 1000 PCE) of micronuclei greater than that found in the control data for all compounds. Therefore, it was concluded that although statistically significant, the increase in the incidence of M P C E in female rats was not biologically meaningful and that A S A was negative for the induction of MPCE. MMC was actually tested twice. The two lower doses were originally tested following the 14-day treatment regimen. When negative results were
75
obtained, contrary to expectation, the experiment was repeated, employing a vehicle control group and a single treatment group receiving 5.0 m g / k g . Animals were only treated for 5 days to reduce the cost of the study. This procedural change was justified by both the published work of MacGregor et al. (1990) and the results previously obtained in this study with other compounds which confirmed the existence of steady state kinetics for micronucleated polychromatic erythrocyte production. Since the integrated micronucleus test was also being evaluated as a replacement assay for the in vitro unscheduled D N A synthesis (UDS) assay in a two-test battery used for early development screening at Lilly, it was desirable to test a compound previously shown to be positive in the modified Ames test used for screening and negative in the UDS assay. 6MP was selected and the results suggest that the replacement of the UDS with the micronucleus test may yield a more sensitive test battery. However, more compounds would need to be tested to verify this conclusion, especially since 6MP is a clastogen and that class of compounds is not active in the UDS assay. In conclusion, the results from this study confirm that, as suggested by MacGregor et al. (1990), the micronucleus test can be integrated with routine toxicity studies for the identification of genotoxic compounds. This approach results in a reduced use of both animals and test article.
References Garriott, M.L., C.E. Piper and A.J. Kokkino (1988) A simplified protocol for the mouse bone marrow micronucleus test, J. Appl. Toxicol., 8, 141-144. Hayashi, M., T. Sofuni and M. Ishidate, Jr. (1983) An application of acridine orange fluorescent staining to the micronucleus test, Mutation Res., 120, 241-247. Heddle, J.A. (1973) A rapid in vivo test for chromosomal damage, Mutation Res., 18, 187-190. MacGregor, J.T., J.A. Heddle, M. Hite, B.H. Margolin, C. Ramel, M.F. Salamone, R.R. Tice and D. Wild (1987) Guidelines for the conduct of micronucleus assays in mammalian bone marrow erythrocytes, Mutation Res., 189, 103-112. MacGregor, J.T., C. Wehr, P.R. Henika and M.D. Shelby (1990) The in vivo erythrocytemicronucleus test: measure-
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M.L. Garriott et al. / Mutation Research 342 (1995) 71-76
ment at steady state increases assay efficiency and permits integration with toxicity studies, Fund. Appl. Toxicol., 14, 513-522. Mantel, N. (1963) Chi-square tests with one degree of freedom; extensions of the ManteI-Haenszel procedure, J. Am. Stat. Assoc., 58, 690-700. Matter, B.E. and W. Schmid (1971) Trenimon-induced chromosomal damage in bone-marow cells of six mammalian species evaluated by the micronucleus test, Mutation Res., 12, 417-425. Merck Index, 10th Ed. (1983) Merck and Co., Inc., Rahway, NJ. Physician's Desk Reference, 44th Ed. (1990) Medical Economics Data, Montvale, NJ.
Salamone, M.F. and J.A. Heddle (1983) The bone marrow micronucleus assay: Rationale for a revised protocol, in: F.J. de Serres (Ed.), Chemical mutagens. Principles and Methods For Their Detection, Vol. 8, Plenum Press, New York, pp. 111-149. Schmid, W. (1973) Chemical mutagen testing on in vivo somatic mammalian cells, Agents Actions, 3, 77-85. Tamura, R.N., M.L Garriott and J.W. Parton (1990) Pooled inference across sexes for the in vivo micronucleus assay, Mutation Res., 240, 127-133. Tinwell, H. (Ed.) (1990) Serial versus single dosing protocols for the rodent bone-marrow micronuclear assay, Mutation Res., 234, 111-261.
ELSEVIER
Mutation Research 342 (1995) 71-76
The in vivo rat micronucleus test: integration with a 14-day study Michael L. Garriott *, Jamie D. Brunny, Delinda E.F. Kindig, Joseph W. Parton, Linda S. Schwier Lilly Research Laboratories, A Division of Eli Lilly and Company, Greenfield, IN 46140 USA Received 13 June 1994; revised 2 November 1994; accepted 28 November 1994
Abstract
A 14-day subchronic toxicity study is routinely conducted in Fischer 344 rats at the Lilly Research Laboratories. This study is done to gather preliminary toxicological information about chemical entities showing efficacy in various pharmacological screens. This manuscript describes the validation of a method for evaluating micronuclei in the bone marrow polychromatic erythrocytes of animals from this test in order to obtain additional information about the genotoxic potential of these compounds without incurring the cost of additional animals or the use of additional test article, which is often in limited supply. Compounds selected for evaluation were acetylsalicylic acid, mitomycin C, cyciophosphamide, colchicine, 6-mercaptopurine, and etoposide. With the exception of colchicine, the results obtained were as expected with acetylsalicylic acid yielding negative results and the other compounds yielding positive results. These findings are consistent with those published for mice (MacGregor et al., Fund. Appl. Toxicol., 14, 513-522, 1990) and show that a bone marrow micronucleus test can be successfully integrated into a routine subchronic rat toxicology study.
Keywords: Micronucleus test; Rat; Integration; Subchronic
I. Introduction
The mouse micronucleus test has been recommended for the safety evaluation of new pharmaceuticals, agrichemicals, and industrial chemicals in various regulatory guidelines worldwide. The test was developed in the early seventies (Matter and Schmid, 1971; Schmid, 1973; Heddle, 1973) and though mostly unchanged during that decade, the protocol has received considerable attention
* Corresponding author. Tel.: (317) 277-4800; Fax: (317) 277-4436. Elsevier Science B.V.
SSDI 0 1 6 5 - 1 2 1 8 ( 9 4 ) 0 0 0 8 1 - 6
during the last ten years (Salamone and Heddle, 1983; M a c G r e g o r et al., 1987; Garriott et al., 1988; Tinwell, 1990). Most recently M a c G r e g o r et al. (1990) have reported the existence of a steady state frequency for micronuclei following repeated daily dosing regimens and they have suggested the integration of micronucleus studies with routine toxicological studies. Their work was conducted in mice though they made recommendations for the use of rats as well. At Lilly Research Laboratories, pilot studies are conducted with potential pharmaceuticals very early after the discovery process to obtain preliminary toxicity information about the compound.
72
M.L. Garriott et al. / Mutation Research 342 (1995) 71-76
These studies are routinely conducted in rats and consist of daily treatment of the animals for two weeks followed by necropsy 24 hr after the last treatment and examination of tissues. Although various factors are considered when choosing doses for this testing, a preferred factor is to use a multiple of the anticipated human clinical dose when possible. This study was conducted to verify the conclusions of MacGregor et al. (1990), to gather data in rats, and to expand the data base by investigating additional compounds prior to beginning to routinely integrate or "piggy-back" a micronucleus test onto this preliminary toxicity screen. The purpose of integrating this study would be to gather preliminary information about the potential genotoxicity of a test article while obtaining general toxicity data since this could be done without incurring the cost of additional animals or the use of additional test material. The compounds tested are all human therapeutics and included a non-genotoxin (acetylsalicyclic acid), direct- (mitomycin C and 6-mercaptopurine) and indirect- (cyclophosphamide) acting clastogens, and two spindle poisons (colchicine and etoposide).
2. Materials and methods Acetylsalicyclic acid (ASA: CAS No. 50-78-2), mitomycin C (MMC: CAS No. 50-07-7), cyclophosphamide (CP: CAS No. 6055-19-2), 6mercaptopurine (6MP: CAS No. 6112-76-1), and etoposide (ET: CAS No. 33419-42-0) were obtained from Sigma Chemical Company, St. Louis, MO, and were solubilized in 10% acacia. Colchicine (COL: CAS No. 64-86-8) was obtained from Eli Lilly and Company, Indianapolis, IN, and was dissolved in 0.9% saline. With the exception of MMC, all compounds were administered daily by oral gavage for 14 consecutive days. Animals receiving the low-and mid-dose of M M C were treated for 14 days while those receiving the high dose were treated for five days. Male and female Fischer 344 rats were obtained from Harlan Sprague Dawley, Indianapolis, IN, at 4-5 weeks of age and were acclimated
for approximately 7 days prior to test initiation. Groups of five animals were housed in ventilated, stainless steel cages with wire mesh bottoms and Lexan ® fronts that were maintained in a room with a temperature of 24 +_ 2°C and at least 40% humidity with a 12/12 hr l i g h t / d a r k cycle. Animals were individually identified by ear punch and given free access to Purina Certified Rodent Chow No. 5002 and tap water. Doses utilized in this study were based on the human clinical dose, or the normal human dose (ASA), of the compound as described in the Physician's Desk Reference (1990) and on median lethal dose (MLD) data from the Merck Index (1983). Doses were not extrapolated from humans to rats, but were determined on an equivalent m g / k g basis. Doses for M M C were 0.1, 1, and 10 × the human clinical dose, while doses for 6MP and ASA were 1, 10, and 50 x the clinical and normal human dose, respectively. The top dose of 750 m g / k g for ASA is also equivalent to one-half the oral M L D in rats. Doses for C O L were 1, 10, and 100 × the human dose while CP was dosed at levels equal to 0.0002, 0.02, and 1 × the human dose. The doses for E T were 0.1, 1, and 5 × the clinical dose. As noted in the Introduction, doses for the preliminary subchronic study at Lilly are based, when possible, on the anticipated clinical dose and tested at the types of dose intervals shown above. These intervals are much wider than those for a definitive micronucleus test conducted according to existing regulatory guidelines, but they are determined by the Chronic Study Area and not by the Genetic
Table 1 Summary of 14-day subchronic results Compound Toxicity a Genotoxicityb Expected c Acetylsalicylicacid Mitomycin C Cyclophosphamide Colchicine 6-Mercaptopurine Etoposide
+ + + +
+ + + +
+ + + + +
Animals exhibited clinical signs of toxicity and/or death at one or more dose levels. b Measured by induction of micronuclei (+) or not (-). c Genotoxicity result anticipated from the literature. a
M.L. Garriott et al./ Mutation Research 342 (1995) 71-76 Table 2 Results of the micronucleus tests
a
Compound
Sex
Acetylsalicylic acid
M M M M F F F F
Dose (mg/kg) 0 15 150 750 0 15 150 750
M P C E b per 1000 P C E c 1.4 1.8 1.0 2.0 0.8 0.6 1.8 3.0
Trend test two-tailed p value
+ 1.7 o + 1.3 + 0.7 +- 0 e + 0.8 _+ 0.5 + 0.4 + 2.7
0.59
pooled g Mitomycin C
Experiment 1
M M M F F F
0 0.05 0.5 0 0.05 0.5
2.2 1.6 2.8 2.2 2.4 1.6
+ 1.6 + 2.1 + 1.9 _+ 2.5 + 1.7 + 0.9
Experiment 2
M M F F
0 5.0 0 5.0
2.8+_2.6 6.4 + 2.6 2.6 _+ 1.3 5.8 +- 2.2
h h h h
0 0.05 0.5 5.0 0 0.05 0.5 5.0
2.5 1.6 2.8 6.4 2.4 2.4 1.6 5.9
+ 2.0 + 2.1 + 1.9 _+ 2.6 + 1.9 + 1.7 + 0.9 + 2.2
M M M M F F F F
0 0.001 0.1 5.0 0 0.001 0.1 5.0
2.4 2.0 1.8 19.4 2.0 2.4 1.8 8.4
+ 2.6 _+ 1.9 + 1.6 + 7.7 + 0.7 + 2.5 + 1.3 + 2.1
M M M M F F F F
0 2.5 25 125 0 2.5 25 125
0.0060 < 0.0001
< 0.0001
pooled g 6-Mercaptopurine
0.014 0.0003
0.0003
pooled g Cyclophosphamide
0.51 1.0 0.008
pooled g M M M M F F F F
0.002 f 0.016 f
0.52
pooled g
Combined
73
1.6 + 0.9 3.0 + 2.0 155.4 + 73.9 - J 1.2 + 0.8 2.0 + 1.6 76.4 + 16.6 - J
< 0.0001 < 0.0001
< 0.0001
pooled g
< 0.0001 < 0.0001
74
M.L. Garriott et aL / Mutation Research 342 (1995) 71-76
Table 2 (continued) Compound
Sex
Colchicine
M M M M F F F F
Dose (mg/kg) 0 k 0.06 0.6 6.0 0k 0.06 0.6 6.0
MPCE b per 1000 PCE c
Trend test two-tailed p value
1.2 + 1.3 2.4 _+ 1.5 1.2 _+ 1.1 - J 1.6 ± 1.1 1.4 0.5 1.2 ± 0.3 - J
1.0
pooled g Etoposide
M M M M F F F F
0 1.14 11.36 57 0 1.14 11.36 57
0.8 + 0.8 1.0 _+0.7 3.4 + 2.1 87.2 _+ 17.8 1.0 _+ 1.0 1.4 _+ 1.1 2.4 _+2.5 18.6 + 4.2
0.59 0.72
< 0.0001
pooled g
< 0.0001 < 0.0001
a Fourteen equal treatments ~ 24 h apart with harvest ~ 24 h after the last treatment. Ten percent aqueous acacia used for the vehicle and delivered at 10 ml/kg. b MPCE, micronucleated polychromatic erythrocyte. c PCE, polychromatic erythrocyte. d Values are mean _+SD for 5 animals/treatment group. e Only 1 animal survived. Statistically significant due to low background in female vehicle control group. Not considered positive for micronucleus induction. g Pooled, Mantel-Haenszel pooled across sex. h Five equal treatments ~ 24 h apart with harvest ~ 24 h after the last treatment. Values are mean + SD for 10 animals. i No survivors. k 0.9% saline used for the vehicle at a dose volume of 12 ml/kg.
Toxicology A r e a . T h e r e f o r e , it was desirable to know w h e t h e r e x p e c t e d results c o u l d be o b t a i n e d at th es e dose levels and dose intervals f r o m comp o u n d s k n o w n for t h e ir genotoxicity, or lack thereof. T h r e e t r e a t m e n t groups and o n e v e h i c l e control g r o u p e a c h consisting of 5 males and 5 fema l es w e r e utilized for each test c o m p o u n d . A p prox i m at el y 24 h after th e last t r e a t m e n t , animals w e r e e u t h a n i z e d by c a r b o n dioxide asphyxiation. B o t h f e m u r s w e r e r e m o v e d by t r i m m i n g away th e skin and muscle and cutting at the pelvic socket and k n e e joint. U s i n g a pair of shears, t h e proximal e n d of the f e m u r was cut at a p p r o x i m a t e l y a 30 ° angle to expose th e b o n e marrow. A b r u s h (00 sable hair artist brush) was d i p p e d into b o v i n e calf serum, t h e n inserted into the b o n e m a r r o w
cavity, and gently r o t a t e d . T h e brush tip was t h e n d i p p e d and r o t a t e d in a d r o p l e t o f s e r u m previously ap p l i ed n e a r o n e en d o f a m i c r o s c o p e slide. T h e slide was t h e n p l a c e d in a M i n i P r e p ® bloods m e a r i n g i n s t r u m e n t and a s m e a r was m a d e , o n e p er e a c h femur. F o l l o w i n g o v e r n i g h t air-drying an d fixation for 5 m i n in 100% m e t h a n o l , t h e slides w e r e stained with acridine o r a n g e (Hayashi et al., 1983). Slides w e r e c o d e d for p r e v e n t i o n of bias af t er b e i n g s e p a r a t e d into two groups for e x a m i n a t i o n by two observers. Slides w e r e examined with a Zeiss Lab 16 m i c r o s c o p e e q u i p p e d with e p i f l u o r e s c e n c e . A t least o n e t h o u s a n d anuc l e a t e p o l y c h r o m a t i c erythrocytes w e r e c o u n t e d for e a c h animal to d e t e r m i n e the f r e q u e n c y o f m i c r o n u c l e u s induction. A t r e n d test for Poisson distribution ( M a c G r e -
M.L. Garriott et al. /Mutation Research 342 (1995) 71-76
gor et al., 1987) was performed on micronucleated polychromatic erythroc3,te values from individual animals. The M a n t e l - H a e n s z e l test (Mantel, 1963) was used to pool the inference across sexes (Tamura et al., 1990).
3. Results and discussion Results from the testing are summarized in Table 1 and compound-specific data are shown in Table 2. With the exception of COL, the results were consistent with expectations. Although this spindle poison was expected to elicit a positive response for micronucleus induction, the results were negative. A possible explanation for the negative response may be the wide dose interval utilized since C O L is active only over a narrow dose range. Another possible explanation may be that the compound was dosed orally instead of intraperitoneally as in the study by MacGregor et al. (1990). The top dose in that study and the mid-dose in this study were nearly equivalent and might reasonably be expected to produce a similar result. No animals survived at the high dose. To ensure that the methodology could detect a spindle poison, a second aneugen, ET, was evaluated. This compound did elicit a positive response. When the data collected for A S A were evaluated statistically, a significant, dose-related increase in micronucleus frequency was obtained for the data collected from females and for the combined data. Inspection of the data showed that the incidence of M P C E in the female vehicle control group was quite low (0.8 M P C E per 1000 PCE). Furthermore, only one female of the five in the top dose group had an incidence (7 M P C E per 1000 PCE) of micronuclei greater than that found in the control data for all compounds. Therefore, it was concluded that although statistically significant, the increase in the incidence of M P C E in female rats was not biologically meaningful and that A S A was negative for the induction of MPCE. MMC was actually tested twice. The two lower doses were originally tested following the 14-day treatment regimen. When negative results were
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obtained, contrary to expectation, the experiment was repeated, employing a vehicle control group and a single treatment group receiving 5.0 m g / k g . Animals were only treated for 5 days to reduce the cost of the study. This procedural change was justified by both the published work of MacGregor et al. (1990) and the results previously obtained in this study with other compounds which confirmed the existence of steady state kinetics for micronucleated polychromatic erythrocyte production. Since the integrated micronucleus test was also being evaluated as a replacement assay for the in vitro unscheduled D N A synthesis (UDS) assay in a two-test battery used for early development screening at Lilly, it was desirable to test a compound previously shown to be positive in the modified Ames test used for screening and negative in the UDS assay. 6MP was selected and the results suggest that the replacement of the UDS with the micronucleus test may yield a more sensitive test battery. However, more compounds would need to be tested to verify this conclusion, especially since 6MP is a clastogen and that class of compounds is not active in the UDS assay. In conclusion, the results from this study confirm that, as suggested by MacGregor et al. (1990), the micronucleus test can be integrated with routine toxicity studies for the identification of genotoxic compounds. This approach results in a reduced use of both animals and test article.
References Garriott, M.L., C.E. Piper and A.J. Kokkino (1988) A simplified protocol for the mouse bone marrow micronucleus test, J. Appl. Toxicol., 8, 141-144. Hayashi, M., T. Sofuni and M. Ishidate, Jr. (1983) An application of acridine orange fluorescent staining to the micronucleus test, Mutation Res., 120, 241-247. Heddle, J.A. (1973) A rapid in vivo test for chromosomal damage, Mutation Res., 18, 187-190. MacGregor, J.T., J.A. Heddle, M. Hite, B.H. Margolin, C. Ramel, M.F. Salamone, R.R. Tice and D. Wild (1987) Guidelines for the conduct of micronucleus assays in mammalian bone marrow erythrocytes, Mutation Res., 189, 103-112. MacGregor, J.T., C. Wehr, P.R. Henika and M.D. Shelby (1990) The in vivo erythrocytemicronucleus test: measure-
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ment at steady state increases assay efficiency and permits integration with toxicity studies, Fund. Appl. Toxicol., 14, 513-522. Mantel, N. (1963) Chi-square tests with one degree of freedom; extensions of the ManteI-Haenszel procedure, J. Am. Stat. Assoc., 58, 690-700. Matter, B.E. and W. Schmid (1971) Trenimon-induced chromosomal damage in bone-marow cells of six mammalian species evaluated by the micronucleus test, Mutation Res., 12, 417-425. Merck Index, 10th Ed. (1983) Merck and Co., Inc., Rahway, NJ. Physician's Desk Reference, 44th Ed. (1990) Medical Economics Data, Montvale, NJ.
Salamone, M.F. and J.A. Heddle (1983) The bone marrow micronucleus assay: Rationale for a revised protocol, in: F.J. de Serres (Ed.), Chemical mutagens. Principles and Methods For Their Detection, Vol. 8, Plenum Press, New York, pp. 111-149. Schmid, W. (1973) Chemical mutagen testing on in vivo somatic mammalian cells, Agents Actions, 3, 77-85. Tamura, R.N., M.L Garriott and J.W. Parton (1990) Pooled inference across sexes for the in vivo micronucleus assay, Mutation Res., 240, 127-133. Tinwell, H. (Ed.) (1990) Serial versus single dosing protocols for the rodent bone-marrow micronuclear assay, Mutation Res., 234, 111-261.