Lack of in vivo and in vitro genotoxicity with the nonsteroid, antiinflammatory agent sodium meclofenamate

Teratogenesis, Carcinogenesis, and Mutagenesis 4:329-340 (1984)

Lack of In Vivo and In Vitro Genotoxicity With the Nonsteroid, Antiinflammatory Agent Sodium Meclofenamate

Melvin L. Kropko, J.E. Fitzgerald, and Felix A. de la lglesia Department of Pathology and Experimental Toxicology, Warner-LamberVParke- Davis Pharmaceutical Research, Ann Arbor, Michigan

Sodium meclofenamate (Meclomen@), CI-583, is an anthranilic acid salt devel- oped as a nonsteroidal, antiinflammatory agent. A multitest battery of short-term tests was employed to characterize the genotoxic and mutagenic potential of the compound by measuring point mutations in bacteria, induction of sister-chromatid exchange, chromosome aberrations, and gene mutations in mammalian cells in vitro. In vitro assays included metabolic activation. The in vivo assay was for chromosome aberrations in bone marrow cells from rats. At toxicity-limited doses for each assay, no activity was detected in the bacterial or mammalian cell mutation assays, and sister-chromatid exchange frequencies were not increased over control rates. When sodium meclofenamate was evaluated in vitro, chromosome aberrations were induced under metabolic activation in CHO cells. Chro- mosome aberrations and clastogenic activity were not demonstrated after oral administration of Meclomen@ to male rats. It was concluded that sodium meclofenamate does not possess overt mutagenic potential under these conditions. The activity seen in vitro with CHO cells after metabolic activation did not correlate with the results from the other tests and was attributed to the formation of reactive metabolites not present or formed in in vivo systems.

Key words: bacterial mutagenesis, sister-chromatid exchange, chromosome aberration, mammalian cell gene mutation, nonsteroid antiinflammatory drug

INTRODUCTION

Meclomen@, the sodium salt of meclofenamic acid, is a member of anthranilic acid derivatives with potent antiinflammatory, analgesic, and antipyretic actions [ 11. Chemically, the drug is not related to other antiarthritic agents such as steroids, phenylbutazone, or salicylate compounds.

Address reprint requests to Dr. F.A. de la Iglesia, Department of Pathology and Experimental Toxicol- ogy, 2800 Plymouth Road, Ann Arbor, MI 48105.

0 1984 Alan R. Liss, Inc.

330 Kropko, Fitzgerald, and de la Iglesia

Against yeast-induced pyrexia in rats, meclofenamic acid demonstrated 80 times more antipyretic activity than aspirin [2]. Extensive in vivo toxicological data col- lected from rats, rabbits, dogs, and monkeys provided data for safety extrapolations to man [3,4]. Advantages of short-term mutagenesis tests reside in an efficient, economical manner in which results are available within short time periods [5,6].

The present genotoxicity evaluation included a battery of short-term tests performed to evaluate the mutagenic potential of sodium meclofenamate [7-101, measuring point mutations in bacterial and mammalian cells, induction of sister-chromatid exchange, and chromosome aberrations in Chinese hamster cells. In vivo studies included chromosome aberrations in bone marrow cells from rats. The results of these studies are reported herein.

MATERIALS AND METHODS Chemicals and Reagents

Sodium meclofenamate (MeclomerP), CI-583, is designated N-(2,6-dichloro- m-toly1)anthranilic acid, monosodium salt. The structure is shown in Figure 1. The compound, a white crystalline solid, soluble in water, was 99.4% pure by ultraviolet assay.

Positive control mutagens used in these studies included N-methyl-N’-nitro-N- nitrosoguanidine (MNNG, Aldrich Chemical Co.); dimethylnitrosamine (DMN, Sigma Chemical Co.); methyl methanesulfonate (MMS, Aldrich Chemical Co.); cyclophosphamide (CP, Mead Johnson Co.); ethyl methanesulfonate (EMS, Eastman Organic Chemicals); benzo(a)pyrene (BP, Aldrich Chemical Co.); triethylenemelamine (TEM, Polyscience Inc.); sodium azide (SA, Difco); 9-aminoacridine (9-AA, ICN, Nutritional Biochemicals); 2-nitrofluorene (2-NF, ICN); 2-aminoanthracene (2-AA, Aldrich). Mammalian Cell Cultures

Chinese hamster ovary cells (CHO) were obtained from American Type Culture Collection (#CCL61) at passage 10. Cells were grown exponentially in Ham’s nutrient mixture F-12 (Gibco) with L-glutamine, without thymidine, and supplemented with 10% fetal bovine serum and 50 pg/ml Gentamicin@ (Schering). Chinese hamster lung cells (V-79) were obtained from Dr. Ernest H.Y. Chu, University of Michigan, at an undetermined passage level. Cells were grown exponentially in Eagle’s minimum essential medium (EMEM) with 1.0 mM sodium pyruvate, nonessential amino acids, 2 mM L-glutamine, 10% fetal bovine serum (Gibco), and 50 pgIml Gentamicin@.

, COOH

I N A SALT LL . H20

Fig. 1. Structural formula for sodium meclofenamate.

Lack of Genotoxicity With Sodium Meclofenamate 331

Metabolic Activation Fraction A commercially available S9 supernatant from liver tissue of adult male rats

induced with Aroclor 1254 was used. This fraction contained 32.7 mg protein/ml and had a relative benzo(a)pyrene hydroxylase activity of 10.8 nmol hydroxybenzpyrene/ 20 min/mg protein. In preassay bacterial mutagenesis titrations, this lot activated 2-anthramine and benzo(a)pyrene to their mutagenic forms.

Sister-Chromatid Exchange

For the nonactivation assay flasks were seeded at 2 x lo6 cells in 10 ml of complete medium prior to treatment. The maximum tolerated dose and 5 half-log dilutions below this level were included.

For treatment, the medium was brought to 5 pM BrdUrd with appropriate drug amounts added to yield the desired final concentrations. Positive control in the nonactivation phase was MMS at 6.5 pg/ml. Metabolic activation assay was accom- plished by concurrent treatment with CP at 10 pg/ml for 1 h in the presence of S-9 mixture in serum-free medium. Cells were harvested, slides prepared, and 20 cells were scored according to standard techniques [ 11,121.

In Vitro Chromosome Aberration Exponentially growing CHO cells were treated with the drug at 200, 100, and

31 pg/ml or solvent control (HBSS) for 2 h in the presence of S9 mixture in serum- free culture medium. The positive control substance was DMN at 4.0 mg/ml in the presence of metabolic activation.

In the nonactivation phase, cells were exposed to the drug at 100, 31, and 10 pglml for 18 h with MNNG as positive control at 4 pg/ml.

Cells from both stages of the assay were harvested after 18 h in culture. Slides were stained with Giemsa, and 50 metaphase cells per group were scored for chromosome aberrations [ 131.

Mammalian Cell Mutation V-79 cell cultures were grown in Ham’s F-12 with 10-5M hypoxanthine,

10-5M aminopterin, and 3 X 1Op6M deoxythymidine (HAT) to select for the wild type, then returned to normal growth medium prior to use [ 141.

The assay consisted of HBSS solvent control, positive controls, and six test concentrations of the compound, ranging from 400 to 12.5 pg per 1 ml. These concentrations, controls, and S9 mix when required, were incubated with lo6 cells for 3 h (day 0). Medium was replaced and incubation continued for 1 day, after which a posttreatment plating efficiency (PE,) was determined. The cells from the various treatment and control groups were subcultured for mutant expression on days 1 and 5 . Eight days after initiation of the cells in culture, cells carried through expression were replaced at 1 X lo5 cells per dish in medium containing 10 p g / d of 6-thioguanine (6-TG) and a second plating efficiency determined (PE2). Cultures were incubated for 11 days in medium containing 6-TG and refed after 4 and 7 days. Mutation frequencies were computed from colony counts and expressed as mutants per lo6 clonable cells [15-171.


In Vivo Rat Bone Marrow Cytogenetic Analysis

Random bred SPF albino Wistar male rats, approximately 42 days of age and weighing 90- 120 g, were obtained from Hilltop Laboratory Animals Inc., Scottdale, Pennsylvania. This strain was selected since it is used in the on-going carcinogenicity studies (unpublished results). The animals were observed for at least 5 days prior to initiation of the study, and those acceptable were assigned to dose groups on the basis of approximate body weight equivalency. Housing was in stainless steel single cages, and food and water were offered ad libitum.

TEM, the positive control, was given at 0.5 mg/kg. Sodium meclofenamate was administered orally as a single dose at 15, 11, 7, and 0 mg/kg. Dose level selection was based on the maximum 2-day tolerated dose of 15.0 mg/kg in rodents, whereas 7.0 mg/kg was the recommended level in clinical trials. Four to 5 h prior to sacrifice, the animals were injected intraperitoneally with 4.0 mg/kg colchicine prepared in HBSS and adjusted to pH 7.4. The rats were euthanized with carbon dioxide, the bone marrow from the femur was aspirated, and standard chromosome preparations obtained. Chromosome aberrations were scored from 50 adequate metaphase spreads from each animal, and 500 cells were counted for calculation of the mitotic index. Numerical data were analyzed for dose-related effects using analysis of variance at the 5 % significance level.

Bacterial Mutagenicity

This assay included metabolic activation using five Salmonella typhimurium strains obtained from the laboratory of Dr. Bruce Ames, (University of California, Berkeley, CA). Master cultures were monitored for retention of strain characteristics and renewed monthly from frozen stocks according to recommended procedures [ 18- 221. Cytotoxicity was determined for each tester strain over a series of half-log dilutions under activation and nonactivation conditions. Five concentrations were then selected for the plate incorporation assay based on the lowest dose that inhibited background revertant count for each strain in the cytotoxicity test. Concurrent positive and solvent controls were included. These compounds were used as positive mutagens at levels expected to produce at least a threefold increase in the number of revertants per plate (Table I).

For statistical analysis, the best fit relating the square root of the revertant count to the logarithm of the dose was determined for each strain with and without S9 and

TABLE I. Salmonella Strains and Chemicals Used as Positive Controls in the Plate Incorporation Assav

Indicator Gene Mutation Metabolic phase strain affected detected Nonactivation Activation

TA-1535 his G46 Base-pair Sodium azide (1)" 2-Aminoantracene (5)

TA-1537 his C3076 Franieshift 9-Arninoacridine (75) 2-Aminoantrdcene (5) TA-1538 his D3052 Frameshift 2-Nitrofluorene (1) 2-Aminoantracene (5) TA-98 his D3052 Frameshift 2-Nitrofluorene (1) 2-Aminoantracene (5) TA-I00 his G46 Base-pair Sodium azide ( 5 )

aNumbers in parentheses indicate micrograms per plate.

substitution

substitution


yielding acceptable results. Only the positive dose responses were used to determine the slope of the best fit line. The two statistical hypotheses tested were mean response of all drug concentrations within a strain versus controls and the slope of the dose response.

RESULTS

The assay in CHO cells indicated that 10 pg/ml was tolerated before overt cytotoxicity .

In the sister-chromatid exchange (SCE) assay, 31 pglml in the nonactivation phase retarded cell cycle progression to the extent that all metaphases were M1 and hence could not be scored for SCE. At concentrations of 10, 3.1, 1.0, and 0.31 pg/ ml, sodium rneclofenamate did not increase the SCE frequency above solvent control values, whereas the positive control induced a fourfold increase in SCE frequency under these conditions (Table 11).

In the activation phase cells exhibited only slight cell cycle delay at 31 and 100 pg/ml. At concentrations of 100, 31, 10, 3.1, and 1.0 pg/ml, the SCE frequency was not significantly elevated by sodium meclofenamate above solvent control values. CP at 10 pg/ml, the positive control for this phase, induced a 2.4-fold increase above background SCE frequency in the solvent control group under concurrent conditions.

TABLE 11. Sister-Chromatid Exchange in Chinese Hamster Ovary Cells Exposed to Sodium Meclofenamate

Treatment and Cells % SCEI SCEI dose (pg/ml) scored MI chromosomea Cell"

With metabolic activation Vehicle controlb 20 13 Cy clophosphamide

(10) 20 30 Sodium meclofenamate

(100) 19 15 (31) 19 12 (10) 20 8 (3.1) 20 8 (1.0) 19 15

Without metabolic activation Vehicle control 20 7 Methyl methanesulfonate

(6.5) 20 6

(31) 0 100 (10) 20 5 (3.1) 20 5 (1 .O) 20 5 (0.31) 18 2

Sodium meclofenamate

aValues are mean f SD. bSterile distilled water. 'Significant (P < 0.05) increase above control values. dInsufficient number of M2 cells for SCE scoring.

0.79 f 0.2 14.7 + 5.0

1.9 k 0.3' 33.0 k 9.1'

0.81 f 0.3 14.4 f 5.7 0.83 0.3 15.2 f 5 .5 0.91 f 0.2 17.2 f 4.0 0.90 f 0.4 15.7 f 6.0 0.80 f 0.2 14.7 f 4.6

0.69 f 0.2 14.3 f 5.3

2.8 f 0.6' 53.4 f 10.5'

d d - -

0.60 5 0.2 11.9 5 3.6 0.64 f 0.2 11.6 4.3 0.56 f 0.1 10.1 f 2.8 0.67 k 0.2 12.6 f 4.1


In the in vitro chromosome aberration assay, 18 h of exposure of cells to sodium meclofenamate at 10, 31, and 100 p g / d induced no significant increases in chromosome counts, aberrations per cell, or number of micronuclei (Table 111). Under concurrent conditions, MNNG at 4 pg/ml induced an 8.3-fold increase in the mean number of aberrations per cell above solvent control values.

In the metabolic activation phase, dose-related increases in the number of aberrations per cell and number of micronuclei were apparent at 3 1, 100, and 200 pg/ ml, while no consistent changes in chromosome number were detected in any dose group. Under concurrent conditions, DMN at 4.0 m g / d induced a 40-fold increase in aberrations per cell above solvent controls.

As distinct from the metaphases of the positive control group, sodium meclofenamate-treated cells exhibited proportionately more gaps and breaks than chromosome exchanges or reunion figures.

The cytotoxicity portion of the point mutation assay included a maximally tolerable level for V-79 cells below 400 pg/ml, producing a null relative plating efficiency at 24 h posttreatment. Dose-related cytotoxicity was observed over the three concentrations used in the assay.

Spontaneous mutation frequency in the nonactivation phase was 22 mutants/ lo6 clonable cells and 31 mutants/106 clonable cells in the activation phase (Table IV). Postexpression plating efficiency in the sodium meclofenamate-treated V-79 cells was reduced but not in a dose-related fashion. At 400 pglml, the plating efficiency was 46-49% compared to a 58-68% plating efficiency in solvent controls. Despite this treatment effect, the mutant incidence in the postexpression population was not appreciably different than solvent control values at any dose level with or without

TABLE 111. In Vitro Chromosome Aberration Analysis of Chinese Hamster Ovary Cells Exposed to Sodium Meclofenamate

Treatment and Cells Chromosome Abberrationd Micronuclei dose (pg/ml) scored counta cella %

With metabolic activation Vehicleb - 50 22.3 f 6.6 0.02 * 0.1 3.5 DMN‘ (4,000) 50 21.0 k 5.1 0.80 f I.7d 10.0

(200) 50 20.8 4.3 0.70 f l.Od 7.5 (100) 50 22.5 It 8.4 0.62 l .Od 3.5


(31) 50 21.5 f 5.9 0.12 f 0.4d 1.5

Vehicleh - 48 21.4 k 5.1 0.21 f 0.5 4.0


Without metabolic activation

MNNGC (4) 48 22.7 f 7.1 2.25 f 2.1d 7.5

(100) 50 24.1 f 9.8 0.30 f 0.7 7.5 (31) 50 19.7 It 2.3 0.16 k 0.4 7.5 (10) 50 19.6 f 1.3 0.06 f 0.2 4.0

aValues expressed as means f SD. bHank’s balanced salt solution. ‘DMN, Dimethylnitrosarnine; MNNG, N-methyl-” -nitro-N-nitrosoguanidine. ‘Significant (P < 0.05) increase over vehicle control.


TABLE IV. In Vitro Point Mutation Assay With V-79 Cells Exposed to Sodium Meclofenamate

6-TG Resistant Plating efficiency colonies

Treatment" Per ten lo6 Clonable and dose W m I ) Relativeb Absolute' dishesd cellse

Without metabolic activation 15 2 2 k 8 Vehicle - 100 68

EMS (800) > loo 52 34 1 650 k 54 Sodium meclofenamate

(400) 0 50 2 4 + 3 (200) 13 75 30 3 9 + 8 (100) > 100 67 8 12 i - 6

Vehicle - 100 58 18 31 f 10 BP (10) 33 55 390 709 99

(400) 0 46 3 6 - 1 3 (200) 30 88 8 9 + 4 (100) > loo 58 21 4 6 + 6

With metabolic activation

Sodium medofenamate

"Cells were exposed for 3 h; vehicle is Hank's balanced salt solution; EMS, ethyl methanesulfonate; BP, Benzo(a)pyrene.

'As % at the end of the expression period. dColonies > 0.5 mm only. 'Mutant frequency calculated from the quotient of 6-TG resistant colonies per ten dishes/absolute plating efficiency; values expressed as mean f SE.

% of control values, 24 h, after exposure.

metabolic activation. The largest increase was 1.8-fold at 200 kg/ml in the metabolic activation phase. The positive controls EMS and BP had normal plating efficiencies accompanied by 30- and 23-fold increases above the respective control values.

In the in vivo rat bone marrow cytogenetic assay, the negative control and the 7.0-15.0 mg/kg groups exhibited no abnormal metaphases at 24 or 48 h posttreatment. The positive control exhibited low incidence of aberrations at 48 h, and 8.8% metaphase cells with breaks, 0.4% metaphase cells with exchanges, and 0.16 lesions per metaphase were seen at 24 h posttreatment. No significant aneuploidy and no statistically significant differences in mitotic indices were found at 24 or 48 h (Table V).

In the bacterial assay, the concentration of 2,000 pg/plate induced a precipitate in the top agar, and pinpoint colonies were apparent in the background lawn accompanied by a slight decrease in the number of TA-1538 and TA-1537 revertants both with and without S9 mixture. The cytotoxicity test showed that 632 figlplate partially inhibited the background lawn of all strains.

The definitive phase of plate incorporation assay thus included 1,0oO, 200, 40, 8, and 1.6 pg/plate. Solvent control counts for each strain were within the historical range established in this and other laboratories [8-10,18,23]. Triplicate plate counts for each strain showed no significant statistical effects. Under parallel conditions, positive control mutagens for each respective strain and test phase increased the revertant count at least 14-fold above solvent control values (Table VI).

336 Kropko, Fitzgerald, arid de la Iglesia

TABLE V. Cytogenetic Study of Bone Marrow Cells From Rats Receiving Sodium Meclofenamate

Chromosome Treatment Time" Mitotic Count frequency Aberrationsc and dose (hr) indexh 0 - 39 40 - 43 44 - 80 %B %T L/M

Sodium meclofenamate (15.0) 24 4.08 k 2.39 4 246

48 3.92 & 2.88 9 240 1 (11.0) 24 6.88 & 2.53 5 244 1

48 4.68 2.68 1 248 1 (7.0) 24 2.88 f 1.60 6 243 1

48 1.76 f 1.32 6 244 -

- - - - - - - - - -

- - -

- - -

- - -

- - - - Control 24 3.84 & 2.64 4 246 (-1 48 3.92 k 1.89 3 246 1 - - -

TEM 24 5.44 & 0.85 9 240 1 8.80 0.40 0.16 (0.5) 48 3.40 + 2.42 7 240 3 3.20 - 0.06

mgikg given orally; controls received distilled water; TEM, triethylenemelamine, intraperitoneally . Cytogenetic assay times in hours postdose. bValues indicate percentage of cells in metaphase k SD, based on 500 cells per animal, five animals at each group and posttreatment interval. 'Based on 250 metaphase cells examined from five animals/group; %B, percentage of metaphases with breaks; %T, percentage of metaphases with exchanges; L/M, average number of lesions per metaphase (break score).

TABLE VI. SalmonellalMammalian Microsome Mutagenicitv Plate Assav With Sodium Meclofenamate

Vehicle Sodium meclofenamateb

1.6 8.0

40.0 200.0

1Ooo.o

Positive control'

Salmonella revertants" TA-1535 - TA-1537 TA-1538 TA- 98 TA-100 - f - + - -k - -k - -k

33 24 32 23 32 39 38 42 88 91

29 17 16 22 25 34 34 49 97 82 32 16 25 19 18 42 31 42 100 65 29 20 22 20 23 26 34 54 78 78 25 19 24 18 17 24 35 46 85 63 19 14 10 8 14 13 27 34 52 53

1,003 1,440 577 383 12,960 3,140 8,040 2,400 3,040 3,320

aValues represent the average of triplicate plates. Treatment phase: +, with metabolic activation; -, without metabolic activation. bExpressed in pg/plate. 'Positive controls listed in Table I.


DISCUSSION

The present battery of in vitro and in vivo tests for establishing mutagenic risk was constructed to evaluate sodium meclofenamate and included chromosome aberrations, point mutations, and DNA damage and/or repair [7,24,27-32,371. Ancillary metabolic activation was employed to facilitate the formation of reactive metabolites. [ 18,25,26].

The individual assays were performed at doses approaching toxicity for maximum exposure and at the same time providing metabolic activation. Concurrent positive control mutagens induced significant increases in all endpoints implying that appropriate conditions existed to detect a response by the drug. Conclusively negative results were found in in vitro assays for mutation and sister-chromatid exchange in mammalian cells and for mutation in bacteria. In vitro, the chromosome aberration results appeared equivocal. However, sodium meclofenamate and the positive control DMN induced increases in chromosome gaps and breaks in cells in the presence of metabolic activation in vitro. This response was not observed in vitro without metabolic activation or in vivo when bone marrow cells from intact animals were examined for cytogenetic abnormalities.

The literature contains several examples of noncarcinogenic clastogens such as potassium sorbate, sodium benzoate, saccharin, and caffeine among others [33,34]. Since nonsteroidal antiinflammatory agents in particular represent a heterogeneous group of chemicals, the correlation between short-term tests and carcinogenesis in rodents with other nonsteroidal antiinflammatory drugs was investigated from literature reports (Table VII) . Among nonsteroidal, antiinflammatory agents examined in short-term mutagenicity tests, a wide variety of results have been obtained. Examples of compounds inducing chromosome aberrations but without bacterial mutation potential include acetylsalicylic acid and phenylbutazone [35]. Conversely, indomethacin induces chromosome aberrations in rat bone marrow [35] but not in cultured human cells [36]. The closest structural analogue of Meclomen@ is mefenamic acid, which is negative for chromosome aberrations both in vivo and in vitro (unpublished data). Phenacetin, a recognized rodent carcinogen, causes point mutation in bacteria, SCE induction, and results in chromosomal aberrations in hamster cells in vitro but not in rat bone marrow cells [29,35].

Sodium meclofenamate-generated reactants in the microsome system induced only interlocus events under in vitro conditions. The specificity and contribution of this intrinsic property to actual somatic or germinal mutation in animals was not corroborated by the in vivo cytogenetic study in rodents. In vitro tests for chromosome aberrations may be subject to nonspecific conditions such as redox states, pH changes, or osmotic imbalance brought about by ionic concentrations which may not occur under in vivo conditions [28]. As such, we did not ascribe significant biological relevance to the chromosome aberrations found. This was supported by the lack of clastogenic activity under direct assay conditions. Thus, the in vitro chromosome abnormalities represent an unexpected phenomenon which is shared with other compounds of this class (ie, acetaminophen, acetylsalicylic acid, and phenacetin) and makes the correlation of this test with other in vivo cytogenetic assays equivocal (Table VII). In addition, in recently completed studies sodium meclofenamate did not demonstrate tumorigenic potential in rodents (unpublished data), suggesting that the results from the in vitro chromosome study were not indicative of in vivo effects.

w

w

00

TABL

E V

II. S

umm

arv

of R

oden

t Car

cino

eene

sis.

Mut

aeen

esis

. and

Cvt

oeen

etic

s Stu

dies

With

Non

ster

oid.

Ant

iinfla

mm

ator

v A

gent

s

Ham

ster

in v

itro

Hum

an in

vitr

o R

at in

viv

o B

acte

rial

Com

poun

d C

arci

noge

nesi

sa

mut

agen

esis

A

berr

atio

ns

SCE

Abe

rrat

ions

SC

E B

one

Mar

row

Sodi

um

mec

lofe

nam

ate

-b

NT

N

T

+ A

ceta

min

ophe

n N

T +

NT

NT

NT

A

cety

lsal

icyl

ic

-

-

-

-

-

-

+ N

T N

T

NT

+ +

-

acid

-

-

NT

N

T

NT

Arn

inop

yrin

e -

- -

-

-

-

-

NT

Indo

rnet

haci

n N

T M

efen

amic

aci

d N

T Ph

enac

etin

+

+ +

+ N

T N

T Ph

enyl

buta

zone

N

T

+ N

T

aRod

ent c

arci

noge

nesi

s bio

assa

y: -

, ne

gativ

e; +

, pos

itive

; NT,

not

test

ed.

Dat

a fr

om G

ranb

erg-

Ohm

an e

t a1

[29]

, Kaw

achi

et a

1 [35], a

nd S

asak

i et a

1 [3

6].

bAut

hors

' unp

ublis

hed

resu

lts.

-

-

-

-

-

-

-

-

-

-


ACKNOWLEDGMENTS

The participation of Dr. R.S. Lake in this study is acknowledged. Ms. S. Wold and Dr. C. Heifetz contributed the bacterial assays.

REFERENCES

1. Winder CV: Experimental observations on flufenamic, mefenamic, and meclofenamic acids. Phar-

2. Wax J: Laboratory pharmacology of Meclofenamic acid. Curr Ther Res 23:3-13, 1978. 3. Fitzgerald JE, Kurtz SM, Schardein JL, Fisken RA, Reutner TF: Animal toxicology studies with

sodium meclofenamate (MecIomen@). Curr Ther Res 23: 14-21, 1978. 4. Schardein JL, Blatz AT, Woosley ET, Kaump DH: Reproduction studies on sodium meclofenamate

in comparison with phenylbutazone. Toxic01 Appl Pharmacol 15:46-55, 1969. 5 . Brusick D: Description of genetic toxicology assays. In Brusick D: “Principles of Genetic Toxicol-

ogy.” New York: Plenum Press, 1980, pp 161-190. 6. Sram, RJ: Evaluation of the Drug Mutagenic Activity. In Benesovi 0, Rychter Z, Jelinek R:

“Evaluation of Embryotoxicity, Mutagenicity, and Carcinogenicity Risks in New Drugs. ” Praha: Univerzita Karlova, 1979, pp 241-250.

7. de la Iglesia FA, Lake RS, Fitzgerald JE: Short-term tests for mutagenesis and carcinogenesis in drug toxicology. What to test and when to test is the question. Drug Metab Rev 11:103-146, 1980.

8. de Serres FJ, Shelby MD: Recommendations on data production and analysis using the Salmonella/ microsome mutagenicity assay. Mutat Res 64: 159-165, 1979.

9. de Serres FJ, Shelby MD: The Salmonella mutagenicity assay: Recommendation. Science 203563- 565, 1979.

10. de Serres FJ: Problems associated with the application of short-term tests for mutagenicity in mass- screening programs. Environ Mutagen 1 :203-208, 1979.

11. Goto K, Maeda S, Kano Y, Sugiyama T: Factors involved in differential Giemsa-staining of sister- chromatids. Chromosoma 66:351-359, 1978.

12. Perry P, Evans HJ: Cytological detection of mutagenkarcinogen exposure by sister-chromatid exchange. Nature 258: 121-125, 1975.

13. Au W, Pathak S, Collie CF, Hsu TC: Cytogentic toxicity of gentian violet md crystal violet on mammalian cells in vitro. Mutat Res 58:269-276, 1978.

14. Shaw EI, Hsie A: Conditions necessary for quantifying ethyl-methanesulfonate-induced mutations to purine-analogue resistance in Chinese Hamster V-79 cells. Mutat Res 51 :237-254, 1978.

15. Bradley MO, Sharkey NA, Kohn KW, Layard MW: Mutgenicity and cytotoxicity of various nitrosoureas in V-79 Chinese hamster cells. Cancer Res 40:27 19-2725, 1980.

16. Irr JD, Snee RD: Statistical evaluation of mutagenicity in the CHO/HGPRT system. In Hsie AW, O’Neill JP, McElheny VK: “Mammalian Cell Mutagenesis: The Maturation of Test Systems. ” Banbury Report 2. Cold Spring Harbor Laboratory, 1979, pp 263-275.

17. Krahn DR, Heidelberger C: Liver homogenate-mediated mutagenesis in Chinese hamster V-79 cells by polycyclic aromatic hydrocarbons and aflatoxins. Muta Res 46:27-44. 1977.

18. Ames BN, McCann J, Yamasaki E: Methods for detecting carcinogens and mutagens with the Sulmonella/mammalian-microsome mutagenicity test. Mutat Res 3 1 :347-364, 1975.

19. Belser WL, Shaffer SD, Bliss RD, Hynds PM, Yammamoto L, Pitts JN, Winer, JA: A standardized procedure for quantification of the Ames Salmonella/mammalian microsome mutagenicity test. Environ Mutagen 3: 123-139, 1981.

20. McMahon RE, Cline JC, Thompson CZ: Assay of 855 test chemicals in ten tester strains using a new modification of the Ames test for bacterial mutagens. Cancer Res 39:682-693, 1979.

21. Rinkus SJ, Legator MS: Chemical characterization of 465 known or suspected carcinogens and their correlation with mutagenic activity in the Salmonella typhimurium system. Cancer Res 39:3289- 3318, 1979.

22. Seiler JP, Matern IE, Green MHL, Anderson D: Meeting report: Second European workshop on bacterial in vitro mutagenicity test systems. Environ Mutagen 2:97-100, 1980.

23. Dunkel VC: Collaborative studies on the Salmonelldmicrosome mutagenicity assay. J Assoc Off Anal Chem 62:874-882, 1979.

macology of fenamates in medicine. Ann Phys Medicine IX (Suppl):7-16, 1967.

340

24. Hollstein M, McCann J, Angelosanto FA, Nichols WW: Short-term tests for carcinogens and mutagens. Mutat Res 65:133-226, 1979.

25. Bartsch H, Malaveille C, Camus AM, Martel-Planche G , Brun G , Hautefeville A, Sabadie N, Barbin A, Kuroki T, Drevon C, Piccoli C, Montesano R: Validation and comparative studies on 180 chemicals with S. typhimurium strains and V-79 Chinese hamster cells in the presence of various metabolizing systems. Mutat Res 76: 1-50, 1980.

26. McCann J, Ames BN: The SalmokeEEulmicrosome mutagenicity test: Predictive value for animal Carcinogenicity. In Hiatt HH, Watson JD, Winsten JA: “Origins of Human Cancer, Book C, Human Risk Assessment.” Cold Spring Harbor Laboratory, 1977, pp 1431-1450.

27. Clayson DB: Comparison between in vitro and in vivo tests for carcinogenicity, an overview. ICPEMC Working Paper 2/1. Mutat Res 75:205-213, 1980.

28. Preston RJ, Au W, Bender MA, Brewen JG, Carrano AV, Heddie JA, McFee AF, Wolff S, Wassom JS: Mammalian in vivo and in vitro cytogenetic assays. Mutat Res 87:143-188, 1981.

29. Granberg-Ohman I, Johansson S, Hjerpe A: Sister-chromatid exchanges and chromosomal aberrations in rats treated with phenacetin, phenagone, and caffeine. Mutat Res 79: 13-18, 1980.

30. Legator MS, Palmer KA, Green S, Peterson KW: Cytogenetic studies in rats of cyclohexyIamine, a metabolite of cyclamate. Science 165: 1139-1 140, 1969.

31. Legator MS, Palmer KA, Ilse-Dore A: A collaborative study of in vivo cytogenetic analysis. I. Interpretation of slide preparations. Toxic01 Appl Pharmacol 24:337-350, 1973.

32. Schmid W, Arakaki DT, Breslau NA, Culbertson JC: Chemical mutagenesis: The Chinese hamster bone marrow as an in vivo test system. Humangenetik 11:103-118, 1971.

33. Abe S, Sasaki M: Chromosome aberrations and sister-chromatid exchanges in Chinese hamster cells exposed to various chemicals. J Natl Cancer Inst 58: 1635-1641, 1977.

34. Abe S, Sasaki M: SCE as an index of mutagenesis and/or carcinogenesis. In Sandburg A: “Sister Chromatid Exchange, Progress and Topics in Cytogenetics, Vol. 2.” New York: Alan R. Liss,

35. Kawachi T, Yahagi T, Kada T, Tazima Y, Ishidate M, Sasaki M, Sugiyama T: Cooperative programme on short-term assays for carcinogenicity in Japan. In Montesano R , Bartch H, Tomatis L: “Molecular and Cellular Aspects of Carcinogen Screening Tests. ” International Agency for Research on Cancer, IARC Pub No. 27, Lyon, 1980, p 323-330.

36. Sasaki M, Sugimura K, Yoshida MA, Abe S: Cytogenetic effects of 60 chemicals on cultured human and Chinese hamster celk. La Kromosome 574: 11-20, 1980.

37. Hsie AW, O’Neill JP, San Sebastian JR, Brimer P: The CHOIHGPRT mutation assay: Progress with quantitative mutagenesis and mutagen screening. In Hsie AW, O’Neill JP, McElheny VK: “Mammalian Cell Mutagenesis: The Maturation of Test Systems.” Banbury Report No. 2, 1979, pp 407-420. Cold Spring Harbor Symposium.

Kropko, Fitzgerald, and de la Iglesia

1982, pp 461-514.

Documents

Lack of in vivo and in vitro genotoxicity with the nonsteroid, antiinflammatory agent sodium meclofenamate