Teratology and multigeneration reproduction studies with maleic anhydride in rats

FUNDAMENTALANDAPPLIEDTOXICOLOGY7,359-366(1986)

Teratology

and Multigeneration Reproduction with Maleic Anhydride in Rats’

Studies

ROBERTD.SHORT,*FREDERICKR.JOHANNSEN,* GEORGE J. LEVINSRAS,* DEANE. RODWELL,~,'ANDJAMES L. SCHARDEIN~ *Monsanto Company, 800 N. Lindbergh Boulevard, St. Louis, Missouri 63167, and jlnternational Research and Development Corporation, Mattawan, Michigan 49071

Teratology and Multigeneration SHORT,R.D.,JOHANNSEN,F.

Reproduction

Studies with Maleic Anhydride in Rats.

R.,LEvINsKAs,G.J.,RoDwELL,D.E.,

ANDSCHARDEIN,J.L.

(1986). Fundam. Appl. Tonicol. 7,359-366. These studies were initiated to evaluate the effects of maleic anhydride on development and reproduction in CD rats. In the teratology study, pregnant rats (19-23/group) received 0, 30,90, or 140 mg/kg/day maleic anhydride in corn oil orally from Days 6- 15 of gestation and fetuses were examined for gross soft tissue and skeletal defects. A reduced weight gain or weight loss was observed in all maleic anhydride-treated groups between Days 6 and 9; however, mean weights ofall groups were within 5% ofcontrol on Days 15 and 20. No treatment-related effects on fetal development were observed. In the multigeneration study, rats (10 males and 20 females/group) received 0,20, 55, or 150 mg/kg/day maleic anhydride in corn oil orally and were mated to produce two generations, each with two litters. Groups of the same size from the second litter were used for subsequent generations and were given the same dose of maleic anhydride as were their parents. The high-dose group was terminated during the second generation due to treatment-related mortality in adults. Renal cortical necrosis occurred in high-dose Fo males and females. Increased kidney weights were observed in low- and mid-dose adult Fl females. No treatment-related effects on reproduction were observed with maleic anhydride at doses up to 55 mg/kg/day over two generations. o 1986 Society ofToxicology.

Currently about 300 million pounds of maleic anhydride is produced in the United States each year (U.S. International Trade Commission, 198 1). This material is used in the production of polyester resins, agricultural chemicals, oil additives, and food acidulants. The American Conference of Govemmental Industrial Hygienists (ACGIH) has adopted a threshold limit value (TLV) for maleic anhydride. This value is currently 1 mg/m3 and was established in order to provide workers with protection against respiratory tract irritation (ACGIH 1982a,b). Unfortunately when the TLV was adopted ’ A portion of this work was presented at the 22nd annual meeting of the Society of Toxicology (Las Vegas, Nev.. 1983)., 2 Present address: WIL Research Laboratories, Inc., 1407 Montgomery, Ashland, Ohio 44805.

in 1968, the published literature contained limited information on maleic anhydride which could be used to assessits health effects and to evaluate the suitability of the TLV using a variety of endpoints. In an effort to remedy this situation, various groups independently have been examining the toxicity of maleic anhydride. Recently, a Chemical Industry Institute of Toxicology (CIIT)-sponsored study suggested that maleic anhydride, which was administered in the diet, was not carcinogenic to rats (CIIT, 1983). The present studies were initiated to contribute to this data base and, specifically, to provide information to assessthe potential teratogenic and reproductive effects of maleic anhydride. METHODS Animals. Adult CD rats (Charles River Breeding Laboratories, Wilmington, Mass.), approximately 12 weeks of

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0272X)590/86 $3.00 Copyright 0 1986 by the Society of Toxicology. All rights of reproduction in any form reserved.

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SHORT ET AL.

age, were used in the teratology study. The multigeneration study was conducted with weanling rats of the same strain. All rats were acclimated to the laboratory for at least 10 days prior to initiating a study. Rats were individually housed, except during mating and lactation, in wire mesh cages or plastic cages with corn-cob bedding. All animals were maintained in environmentally controlled rooms with 12-hr photoperiods and given free accessto feed (Purina Rodent Chow, Ralston-Purina, St. Louis, MO.) and water. Maleic anhydride was supplied by Monsanto Company as white briquettes with a purity of greater than 99%. Teratology study. Briquettes of maleic anhydride were finely ground with a mortar and pestle and suspended in corn oil with the aid of a tissue homogenizer. All doses were prepared daily in corn oil in order to minimize problems with stability. A 1% (w:v) concentration was used to administer all doses. One male and one female were housed together for mating. The day of mating was determined by daily inspection for a copulatory plug or a sperm-positive vaginal smear. This day was designated Day 0 of gestation. Rats (25 mated females/group) were treated orally with 30,90, or 140 mg/kg/day of maleic anhydride from Day 6 through Day 15 of gestation. Mated females in the control group were treated in a similar manner with 14 ml/kg of corn oil. Body weights were recorded at intervals during gestation. All females were sacrificed with carbon dioxide on Day 20 of gestation, and the fetuses were delivered by cesarean section. All fetuses were weighed and examined for external abnormalities. Approximately one-third of the fetuses were placed in Bouin’s fixative and examined for soft tissue abnormalities (Wilson, 1965). The remaining fetuseswere fixed in alcohol, cleared with potassium hydroxide, stained with Alizarin Red S (Dawson, 1926), and examined for skeletal abnormalities. Multigeneration study. Maleic anhydride was suspended in corn oil as described previously; however, the concentration was varied so that the desired dose could be administered orally in a volume of 10 ml/kg. Rats, in groups of 10 males and 20 females, received 0,20,55, or 150 mg/kg/day of maleic anhydride. These rats represented the Fo generation, and females were bred twice with males in the same dose group to produce Fla and Fl b litters. Then 10 males and 20 females were randomly selected from the F 1b litter to become parents of the F2a and F2b litters. Each generation was dosed a minimum of 80 days with maleic anhydride before its members were mated. Treatment began when Fo rats were 5 to 6 weeks and Fl animals were 22 days ofage, and continued until the generation was terminated. During the mating period each male was housed with two females for up to 15 days. The females were examined daily for evidence of mating as revealed by vaginal plugs or sperm-positive vaginal smears. The day evidence of copulation was observed was identified as Day 0 of

gestation and the female was transferred to an individual plastic cage containing nesting material. Males were returned to their wire-mesh cages at the end of the mating period. Females for which no evidence of copulation was detected after the 15day mating period were individually housed in plastic cages with bedding. Rats were observed for signs of toxicity and body weights were recorded at intervals during the study. Litters with more than 10 pups were reduced to five males and five females, when possible, on Day 4 of lactation. A histopathological evaluation was performed on approximately 30 tissues from each parent that died during the study, selected parents from all groups in the Fo generation, selected parents from the control and mid-dose groups in the Fl generation, and 10 pups/sex from the control and mid-dose groups from the F2b litters. Statistical analysis. Several different statistical methods were used to compare measurements made on test animals to the corresponding values determined for controls. The methods and the measurements to which they were applied are analyis of variance and Dunnett’s test (Steel and Tonie, 1960) for adult body weights, litter size, and pup body weights; Fisher’s exact probability test (Siegel, 1956) for mortality and fertility data; Mann-Whitney U test (Siegel, 1956) for fetal body weights; and x2 test with Yates’ correction or Fisher’s exact probability test (Siegel, 1956) for litters with anomalies. In all instances, p < 0.05 was selected as the level of significance.

RESULTS

Teratology Study The general appearance and behavior of rats were not altered by treatment. While one adult died in each of the experimental groups, the overall survival in these groups was 96% (Table 1). Dams in the experimental groups either failed to gain weight or lost weight between Days 6 and 9 of gestation (Table 1). However, this effect was reversible, and there were no statistically significant effects on body weight at any of the times examined. Dams from all test groups produced normal-sized litters, and there was no evidence of postimplantation loss (Table 1). Compared with concurrent controls, fetal body weights were slightly reduced for all test groups, but the reductions were statistically significant only in the low- and high-dose groups, However, this is not considered to be compound-related, because fetal weights for

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IN RATS

TABLE 1 MATERNAL AND FETAL DATA FROM RATS TREATED WITH MALEIC ANHYDR~DE DURING GESTATION Maleic anhydride (mg/kg/day)

Number treated Pregnant Alive Nonpregnant Alive Body weight(g) Day 0 Day 6 Day 9 Day 12 Day 15 Day 20 Implants/dam Viable fetuses/dam Resorptions/dam Fetal weight(g)

0

30

90

140

25 23 23 2 2

25 24 23 1 1

25 20 19 5 5

25 21 21 4 3

242 261 272 293 315 397 13.3 (14) 12.6(13) 0.8 (0) 4.0 (3.8)

244 264 264 286 302 377 13.4(14) 12.7(13) 0.7 (0) 3.7 (3.8)’

242 266 264 283 303 385 13.2(13) 12.5 (13) 0.7 (1) 3.8 (3.8)

243 264 258 282 298 378 13.5 (14) 12.7 (13) 0.8 (0) 3.1 (3.6)b

’ Mean (median). b Mean significantly different from control. The historical control value for fetal weight was 3.6 g for 1774 fetuses.

concurrent control and all treated groups were slightly greater than the values for historical controls. The malformations and variations observed during the fetal examinations are summarized in Table 2. Malformations were observed in one fetus (one litter) from the control group, two fetuses (two litters) from the low-dose group, and three fetuses (three litters) from the high-dose group. Since each malformation was a single occurrence and the malformations differed among the various groups, there was no evidence of a doserelated increase in any specific malformation. The fetal variations were comparable both in type and frequency in the control and treated groups.

Multigeneration

Study

With the exception of a few cases of respiratory rales, the clinical appearance and behavior of these Fo rats were not remarkably

different from those of their controls. Respiratory rales also occurred in Fl rats, and the incidence and severity appeared to increase with dose. In addition, these rats often vigorously resisted handling at the time of dosing. This behavior may be related to irritating effects of maleic anhydride or a taste aversion to the test material. As a result, some animals were difficult to treat. In the Fo generation, significant mortality occurred in adults of both sexes from the high-dose group (Table 3). The Fl generation had a greater number of deaths, many of which were attributed to gavage-related injuries. If these traumatic deaths are omitted, mortality in the Fl generation tended to parallel that of the Fo generation except for the increase recorded for low-dose males. One animal in the latter group died of interstitial pneumonia. While no cause could be identified, the three other deaths in this group are not believed to be compound-related because no deaths among mid-dose males were attrib-

362

SHORT ET AL. TABLE 2

Maleic anhydride (mg/kg/day)

Number examined Litters Fetuses External Skeletal Soft tissue Malformations Short tail Omphalocele Spherical enlargement on ribs Fused stemebrae Bent ribs Multiple anomalies Total with above malformations Variations Rudimentary 14th rib(s) Full 14th rib(s) 27 presacral vertebrae Stemebrae 5 and/or 6 unossified 7th cervical rib Misaligned centra Reduced ossification of skull Hyoid unossified Pubis unossified Renal papillar not developed

0

30

23

23

19

21

289 189 100

292 190 102

237 155 82

267 174 93

l(l) l(1)

l(lY l(l) -

0 (0)

l(l) l(l) 1 (I)* 3 (3)

46(16) 3 (3)

37(13) l(l)

51(17) l(l) 12~9) 3 (2) -

2(2)

2 (2) 62 (20)

2 (2) 5 (4) 16 (7) l(l) l(l) 5 (3) l(l) -

90

2(l) 12(6) l(l) -

140

2 (2) 20(12) l(l) l(l) l(l)

o Number of fetuses (number of litters). * Includes malformed humerus, stemoschisis, and ossification defects of the pubis, cervical arches, and skull.

uted to the test material and characteristic compound-induced lesions discussed subsequently were not noted. Adult body weights were not affected in the low-dose groups (Table 3). While there were some differences in mean body weights between control animals and those of the middose group, none of the differences was statistically significant. In the high-dose group, mean body weights of both sexes of the Fo generation were significantly reduced by Week 11, and this reduction persisted for the remainder of the test. The Fl generation showed a pattern that was roughly similar, except that the only significantly depressed mean body weight occurred in high-dose males at 30 weeks.

Fertility was significantly reduced in the experimental groups at several times (Table 4). However, there was neither a dose-related reduction nor a pattern within a generation that suggested the presence of a treatment-related effect. Treatment with maleic anhydride had no effect on litter size or on pup survival at doses up to 150 mg/kg/day in the F 1a and Fl b litters and at doses up to 5 5 mg/ kg/day in the F2a and F2b litters (Table 5). Pup body weights in the experimental groups were significantly reduced at a few of the observation periods (Table 6). In the low- and mid-dose groups, pup weights were significantly reduced only at weaning of the F2b litters. However, these effects were observed only in one litter of a generation, and there

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TABLE 3 MORTALI~ANDBODYWEIGHTOFADULTRATSTREATEDWITHMALEICANHYDR~DEDURINGA MULTIGENERATIONREPRODUCTIONSTUDY Maleic anhydride (mg/kg/day)

Fo generation Males Mortality” Body weight’ Females Mortaiitf Body weight’ Fl generation Males Mortality” Body weightd Females Mortalitf Body weightd

0

20

55

150

0 (0) 143 502 700

lO(10) 142 524 697

10 (0) 143 506 666

70’ (60)b 143 431b 562’

0 (f-9 129 289 368

0 (0) 130 281 345

5 (0) 130 280 337

65b (65)b 127 259’ 3176

40 (Wb 107 500 703

40 (0) 103 445 683

75b(58)b 85’ 431 -

30 (5) loo 272 343

52’(10) 95 265 347

20(0) 113 495 722

20(0) 96 265 334

loob(l4) 84 247 -

DTotal dead/total treated X 100 (% mortality minus gavage-related deaths). Groups contained 10 to 12 males and 20 to 2 1 females. b Significantly different from the appropriate control. ’ Body weight (g/rat) at Weeks 0, 1 1, and 32 of the study. d Body weights (g/rat) at Weeks 30,4 1, and 6 1 of the study.

was no pattern beween litters of a generation or between generations that suggested the presence of a treatment-related effect. Microscopic examination of tissues from Fo adults revealed compound - related changes in the kidneys of rats from the highdose group. Renal cortical necrosis, present in 60% -ofthe males and in 15% of the females from this group, was not observed in any other groups. In the Fl generation, the absolute kidney weights of adult females in the low- and mid-dose groups were significantly increased to 108 and 1 1 l%, respectively, of the control value (2.3 1 g). There were no microscopic changes in these kidneys. The eval-

uation of tissues from F2a pups revealed no compound-related changes in organ weights. In addition, the examination of tissues from F2b pups revealed no compound-related changes in organ weights or in incidences of microscopic lesions. DISCUSSION Teratology Study In a pilot study, conducted to identify doses for the teratology study, three of five and five of five dams died after doses of 192

364

SHORT ET AL. TABLE 4

FERTILITY

OF RATS TREATED

WITH MALEIC

ANHYDRIDE

DURING

A MULTIGENERATION

REPRODUCTION

STUDY

Maleic anhydride (mg/kg/day)

Females Fla Flb F2a F2b Males Fla Flb F2a F2b

0

20

55

150

14120 (7O)a 10/20 (50) 14/20 (70) 12/16 (75)

7120 (35)b 812 (40) 13/15 (87) 12/14 (86)

14/20 (70) I l/19 (58) 9/l 1 (82) S/IO (80)

l/20 (35)b 6/10 (60) -

8/10 (80) 6/ 10 (60) 9110 (90) v3 (loo)

5/10 (50) 519 (56) 6/b (1W 7/7 (100)

9110 (90) 7/10 (70) 6/6 (100) 5/5 (100)

4/ 10 (40)b 519 (56) -

’ Number pregnant/number mated X 100 (% pregnant). ’ Significantly different from control. ’ Number fertile/number mated X 100 (% fertile).

and 256 mg/kg, respectively, of maleic anhydride. In the present study, maternal toxicity was demonstrated by the failure of dams to gain weight between Days 6 and 9 of gestation. Larger doses of maleic anhydride would

have had more severe effects on weight gain and probably would have produced mortality, thus compromising the present study. In the teratology study, dams were treated orally with up to 140 mg/kg/day from Day 6

TABLE 5 LITER

SIZE OF RATS TREATED

WITH MALEIC

ANHYDRIDE

DURING

A MULTIGENERATION

REPRODUCTION

STUDY

Maleic anhydride (mg/kg/day) Litter Fla Flb F2a F2b

Days after birth

0

20

55

150

0 4 21 0 4 21 0 4 21 0 4 21

12.2” 12.0 (9.9)b 9.9 13.3 13.0 (9.8) 9.8 13.4 13.1 (9.9) 9.9 10.5 10.4 (8.2) 8.2

11.0 10.5 (9.3) 9.3 10.3 9.6 (9.0) 8.9 12.2 11.6(10.0) 9.9 13.6 13.3 (9.8) 9.7

11.6 11.2 (9.3) 8.8 13.4 13.2 (9.9) 9.8 12.0 11.8 (9.8) 9.8 14.0 13.8 (10.0) 9.0

13.1 13.4(10.0) 10.0 11.3 10.8 (9.7) 9.3 -

-

a Mean live pups/litter on indicated day. The number of pregnant females that gave birth on Day 0 is presented in Table 4. b Mean live pups/litter before- litter reduction (mean live pups/litter after reduction to five pups/sex/litter when possible).

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IN RATS

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TABLE 6 BODYWEIGHTOFPUPSFROMRATSTREATEDWITHMALEICANHYDRIDEDURING AMULTIGENERATIONREPRODUCTIONSTUDY Maleic anhydride (mg/kg/day) Litter Fla Flb F2a F2b

Days after birth

0

20

55

150

0 4 21 0 4 21 0 4 21 0 4 21

6.7’ 12(ll)c 58 (56) 6.4 11 (10) 53 (50) 6.6 ll(l0) 47 (45) 6.8 ll(l1) 50 (57)

6.6 ll(l1) 54 (53) 7.lb 12 (12) 54 (54) 6.4 lO(10) 47 (46) 6.4 10 (9) 45 (44)b

6.7 12(11) 58 (55) 6.2 ll(10) 5 1 (50) 6.7 lO(10) 46 (45) 6.1 9 (9) 43 (44)”

Mb 10 (9) 46b (44)b 6.3 10 (10) 47 (46) -

LIMean weight (g) of both males and females. b Significantly different from control. ’ Mean weight (g) of males (mean weight (g) of females).

through Day 15 of gestation. An examination of the fetuses did not reveal any effects that were attributed to maleic anhydride. No increase in fetal malformations was observed, and the variations detected were similar in control and treated groups. Under the conditions of this study, it is concluded that maleic anhydride is not teratogenic.

Multigeneration Study In addition to treatment-related deaths, there were also gavage-related deaths in this study. If allowance is made for the latter, it appears that treatment-related mortality in the Fo generation occurred primarily in the high-dose group. Although mortality was observed at lower doses in the Fl generation, there was no dose-related pattern at these levels that suggested an effect related to treatment. Therefore, in the Fl generation, mortality that can reasonably be attributed to treatment also occurred primarily at high dose. As a result of increased mortality, this

dose group was terminated during the F 1 generation. Since there was a significant reduction in neither the percentage of pregnant females nor the percentage of fertile males, it is concluded that no adverse effects on fertility were observed with maleic anhydride at doses up to 55 mg/kg/day administered over two generations. At 150 mg/kg/day, maleic anhydride was toxic to parental animals. No adverse effects on litter size and on pup survival were observed with maleic anhydride at doses up to 150 mg/kg/day in Fl a and F 1b litters or 55 mg/kg/day in F2a and F2b litters. Although a few statistically significant changes in pup body weights were observed, they were too inconsistent to be considered treatment-related. Microscopic examination of tissues from pups in the F2b litters revealed no treatment-related changes. Therefore, treatment with up to 55 mg/kg/day for two generations had no adverse effects on pups.

General Conclusion If during a working day, a 50-kg worker inhaling 10 m3 of air containing maleic anhy-

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dride at its TLV (1 mg/m3) and absorbing all of the maleic anhydride would receive 0.2 mg/kg/day. In the present study no teratogenie effects were observed at dosages up to 140 mg/kg/day, and no adverse effects on reproduction and fertility were detected at daily dosages up to 55 mg/kg/day of maleic anhydride. Thus, in relation to the endpoints evaluated in the present study, maleic anhydride does not appear to present a teratogenic or reproductive hazard in the workplace.

REFERENCES ACGIH (AMERICANCONFERENCEOFGOVERNMENTAL INDUSTRIAL HYGIENISTS) (1982a). Threshold limit values of air-borne contaminants. Cincinnati, Ohio.

ACGIH (AMERICANCONFERENCEOFGOVERNMENTAL INDUSTRIALHYGIENIST$ ( 1982b). Documentation of the threshold limit values, 4th ed. Cincinnati, Ohio. DAWSON, A. B. (1926). A note on the staining ofthe skeleton of cleared specimens with Alizarin Red S. Stain Technol. 1, 123-124. CIIT (CHEMICAL INDUSTRY INSTITUTE OF TOXICOLOGY) (1983). Chronic dietary administration of maleic anhydride-final report, CIIT Docket No. 114N3. Research Trianale Park. N.C. SIEGEL, S. (1956). Nonparametric Statistics for the Behavioral Sciences. McGraw-Hill, New York. STEEL, R. G. D., AND TORRIE, J. H. (1960). Principles and Procedures of Statistics. McGraw-Hill, New York. U.S. INTERNATIONALTRADECOMMISSION(~~~~). Synthetic organic chemicals, U.S. production and sales. U.S. Govt. Printing Office, Washington, D.C. WILSON, J. G. (1965). Embryological considerations in teratology. In Teratology-Principles and Techniques (J. G. Wilson and Warkany, e&x), pp. 25 l-277. Univ. of Chicago Press, Chicago.