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Effect of large daily doses of ascorbic acid on pregnancy in guinea pigs, rats, and hamsters
TOXICOLOGYAND
APPLIEDPHARMACOLOGY35,393-395(1976)
SHORT Effect
of Large
Daily Guinea
COMMUNICATIONS Doses of Ascorbic Acid on Pregnancy Pigs, Rats, and Hamsters
in
Effect of Large Daily Doses of Ascorbic Acid on Pregnancy in Guinea Pigs, Rats, and Hamsters. ALLEVA, F. R., ALLEVA, J. J. AND BALAZS, T. (1976). Toxicol. Appl. Pharmacol. 35, 393-395. L-Ascorbic acid was administered orally during pregnancy in doses of 400 mg/kg/day to guinea pigs and 50, 150, and 450 mg/kg/day to rats and hamsters. No increase in abortion or mortality of offspring was observed. Large daily doses of ascorbic acid are taken by women of childbearing age for minor ailments such as the common cold. Neuweiler (1951), Mouriquand and Edel (1954), and Samborskaya (1964) reported that such treatment increased abortion in guinea pigs. Samborskaya and Ferdman (1966) and Fahim et al. (1972) reported a similar effect in rats. We tried to confirm these findings and tested a third species, the golden hamster. METHODS The three species were exposed to controlled temperature (25”C), relative humidity (40x), and lighting (LD 16: 8 for guinea pigs and rats; LD 14: 10 for hamsters). Purina guinea pig chow (1 mg of ascorbic acid/g) and tap water were freely available to the guinea pigs, and Purina laboratory chow and tap water were available to the rats and hamsters. Fifty-three female guinea pigs (4 months old, 626-958 g) were housed three per cage with a male. Beginning 6 days later, 11 females received twice daily SCinjections of sodium L-ascorbate dissolved in saline (225 mg/ml) at a dose of 200 mg of free acid/kg (400 mg/kg/day). Thirteen controls received saline. Because the ascorbate injections caused inflammation, L-ascorbic acid dissolved in deionized water (200 mg/ml) was given orally after the fifth day of treatment. In the remaining 29 guinea pigs, the daily oral treatments (ascorbic acid or water) were initiated after pregnancy was established, as evidenced by palpation (about Day 23 of pregnancy). Females were isolated at this time. Dosing was discontinued before birth to allow normal delivery. Abortion, mortality of offspring, and live pup weights were determined. The day of conception was estimated by counting back 63 days (average length of pregnancy) from birth. Data from guinea pigs treated for similar periods were grouped for analysis. Three-month-old female Holtzman rats (233-304 g) were exposed to males on the night of estrus and those with vaginal sperm on the following morning (Day I of the 23-day pregnancy period) were given a single oral dose of 0 (deionized water), 50, 150, or 450 mg of L-ascorbic acid/kg (2 ml/kg) daily from Day 1 to 19 of pregnancy. Twomonth-old Lakeview hamsters (76-99 g) with fresh vaginal sperm (Day 1 of the 16Copyright 0 1976byAcademicPress,Inc. All rights of reproduction in any form reserved. Printed in Great Britain
393
394
SHORTCOMMUNICATIONS
day pregnancy period) were given L-ascorbic acid (5 ml/kg), in the samedosesgiven the rats, daily from Day 1 to 15 of pregnancy. Abortion, mortality of offspring, and live litter weights were determined. RESULTS AND DISCUSSION No increasein abortion or mortality of offspring was observed in guinea pigs, rats, or hamstersgiven high oral dosesof ascorbic acid (Tables 1 and 2). A slight increasein TABLE 1 EFFECT OF ASCORBIC ACID ON MATING AND PREGNANCY IN GUINEA PIGS
Number Dose of animals hx/Wdw)
58 z
13 16
400 0 400 0 400 0
Number of Number Number PUPS of Days of Live pup weight treated” abortions births Dead Alive (mean+ SE(g)) -21-45 -26-47 -1-57 -6-53 23-50 23-50
1 2 0 1 2 3
4 6 6 4 11 13
3 1 4 2 50 22
11 15 17 :::
108f 7 109+5 112+4 111+6 117+ 3’ 103+ 3
27
’ Day 0 = estimateddayof conception(mating).The meandayis given.SE= 1 or 2 for all means except-21 (+ 4) and-26 (* 5). bSignificantlydifferentfrom pairedcontrolvalue(p < 0.05),accordingto x2 analysis(percentage of deadpups)andStudent’st test (deadpups/litter). c Significantlydifferentfrom pairedcontrol value(p <:0.05),accordingto Student’sI test. TABLE 2 EFFECT OF ASCORBIC ACID ON PREGNANCY IN RATS AND HAMSTERS
Pups/litter Group Rats 1 2 3 4 Hamsters 1 2 3 4
Dose bx!kgY’
Nb
Dead’
Alive’
Live litter weight” (23
0 50 150 450
14 11 11 11
0.36 + 0.20 0.40 + 0.31 0.60 + 0.27 1.20+ 0.53
11.1+ 0.6 11.7+ 0.8 11.0+0.5 10.5+ 0.9
74.6+_3.9 75.3+ 4.3 73.3I? 3.3 68.1+ 5.6
0 50 150 450
11 12 10 14
0.09L-0.09 0.08 f 0.08 0.10+ 0.10 0.29 310.16
10.1+ 0.5 9.9 + 0.6 10.5+ 0.7 11.0+ 0.5
22.3i- 0.9 22.5 + 1.2 24.3+ 1.2 25.6+ 1.3d
’ Singleoral doseof ascorbicacidgivendailyfrom Day 1to 19(rats)or 15(hamsters) of pregnancy. Controls(0 mg/kg)receivedwater. bEveryanimalgavebirth; no abortionswereobserved;threeratsin groups2-4 atelittersat birth. c Mean+ SE. dSignificantlygreaterthancontrolvalue(p < 0.05),accordingto Student’st test.
SHORT
COMMUNICATIONS
395
pup weight was observed in one group of treated guinea pigs and hamsters. Neuweiler (19 51), Mouriquand and Edel (19 54), and Samborskaya (1964) reported increased abortion in guinea pigs given daily doses of 25,200, and 150 or 1500 mg/kg, respectively. Our dose of 400 mg/kg/day is within this range. We cannot explain these conflicting results. Similarly, Samborskaya and Ferdman (1966) and Fahim et al. (1972) reported increased abortion in rats given daily doses of 833 mg/kg SCand 250 mg/kg po, respectively. We found no effects with oral doses of 50-450 mg/kg/day. Our results are in agreement with those of Frohberg et al. (1973) who reported, while the present investigation was in progress, that ascorbic acid had no effects on pregnancy in rats given oral doses of 150-1000 mg/kg/day. These workers also found no effects in mice given 250-1000 mg/kg/day. Thus, the present investigation together with that of Frohberg et al. (1973) revealed no adverse effects of large daily doses of ascorbic acid on pregnancy in four animal species. REFERENCES M. S., HILDERBRAND, D., WILSON, R. HARMAN, J. M. AND HALL, D. G. (1972).Effect of high dosesof ascorbicacid on femalereproduction. In Fifth International Congress on Pharmacology, Abstracts of Volunteer Papers, p. 66, San Francisco. FROHBERG, H., GLEICH, J. AND KIESER, H. (1973).ReproduktionstoxikologischeStudien mit Ascorbinsaurean Mausenund Ratten. Arzneim. Forsch. 23, 1081-1082. MOURIQUAND, G. AND EDEL, V. (1954).HypervitaminoseC et gestation.C. R. Sot. Biol. 148, 1422-1423. NEUWEILER, W. (1951).Die Hypervitaminoseund ihre Beziehungzur Schwangerschaft.Znt. FAHIM,
Z. Vitaminforsch.
22, 392-396.
E. P. (1964).The effect of high ascorbicacid doseson the courseof pregnancy on the guineapig and on the progeny (Translation). Byull. Eksp. Biol. Med. 57, 105-108. SAMBORSKAYA, E. P. AND FERDMAN, T. D. (1966).The mechanismof termination of pregnancy by ascorbicacid (Translation). Byull. Eksp. Biol. Med. 62, 96-98. SAMBORSKAYA,
FREDERIC R. ALLEVA JOHN J. ALLEVA TIB~R BALAZS
Division of Drug Biology, Food and Drug Administration Department of Health, Education, and Welfare Washington, D.C. 20204 Received June 26; accepted September 26 1975
APPLIEDPHARMACOLOGY35,393-395(1976)
SHORT Effect
of Large
Daily Guinea
COMMUNICATIONS Doses of Ascorbic Acid on Pregnancy Pigs, Rats, and Hamsters
in
Effect of Large Daily Doses of Ascorbic Acid on Pregnancy in Guinea Pigs, Rats, and Hamsters. ALLEVA, F. R., ALLEVA, J. J. AND BALAZS, T. (1976). Toxicol. Appl. Pharmacol. 35, 393-395. L-Ascorbic acid was administered orally during pregnancy in doses of 400 mg/kg/day to guinea pigs and 50, 150, and 450 mg/kg/day to rats and hamsters. No increase in abortion or mortality of offspring was observed. Large daily doses of ascorbic acid are taken by women of childbearing age for minor ailments such as the common cold. Neuweiler (1951), Mouriquand and Edel (1954), and Samborskaya (1964) reported that such treatment increased abortion in guinea pigs. Samborskaya and Ferdman (1966) and Fahim et al. (1972) reported a similar effect in rats. We tried to confirm these findings and tested a third species, the golden hamster. METHODS The three species were exposed to controlled temperature (25”C), relative humidity (40x), and lighting (LD 16: 8 for guinea pigs and rats; LD 14: 10 for hamsters). Purina guinea pig chow (1 mg of ascorbic acid/g) and tap water were freely available to the guinea pigs, and Purina laboratory chow and tap water were available to the rats and hamsters. Fifty-three female guinea pigs (4 months old, 626-958 g) were housed three per cage with a male. Beginning 6 days later, 11 females received twice daily SCinjections of sodium L-ascorbate dissolved in saline (225 mg/ml) at a dose of 200 mg of free acid/kg (400 mg/kg/day). Thirteen controls received saline. Because the ascorbate injections caused inflammation, L-ascorbic acid dissolved in deionized water (200 mg/ml) was given orally after the fifth day of treatment. In the remaining 29 guinea pigs, the daily oral treatments (ascorbic acid or water) were initiated after pregnancy was established, as evidenced by palpation (about Day 23 of pregnancy). Females were isolated at this time. Dosing was discontinued before birth to allow normal delivery. Abortion, mortality of offspring, and live pup weights were determined. The day of conception was estimated by counting back 63 days (average length of pregnancy) from birth. Data from guinea pigs treated for similar periods were grouped for analysis. Three-month-old female Holtzman rats (233-304 g) were exposed to males on the night of estrus and those with vaginal sperm on the following morning (Day I of the 23-day pregnancy period) were given a single oral dose of 0 (deionized water), 50, 150, or 450 mg of L-ascorbic acid/kg (2 ml/kg) daily from Day 1 to 19 of pregnancy. Twomonth-old Lakeview hamsters (76-99 g) with fresh vaginal sperm (Day 1 of the 16Copyright 0 1976byAcademicPress,Inc. All rights of reproduction in any form reserved. Printed in Great Britain
393
394
SHORTCOMMUNICATIONS
day pregnancy period) were given L-ascorbic acid (5 ml/kg), in the samedosesgiven the rats, daily from Day 1 to 15 of pregnancy. Abortion, mortality of offspring, and live litter weights were determined. RESULTS AND DISCUSSION No increasein abortion or mortality of offspring was observed in guinea pigs, rats, or hamstersgiven high oral dosesof ascorbic acid (Tables 1 and 2). A slight increasein TABLE 1 EFFECT OF ASCORBIC ACID ON MATING AND PREGNANCY IN GUINEA PIGS
Number Dose of animals hx/Wdw)
58 z
13 16
400 0 400 0 400 0
Number of Number Number PUPS of Days of Live pup weight treated” abortions births Dead Alive (mean+ SE(g)) -21-45 -26-47 -1-57 -6-53 23-50 23-50
1 2 0 1 2 3
4 6 6 4 11 13
3 1 4 2 50 22
11 15 17 :::
108f 7 109+5 112+4 111+6 117+ 3’ 103+ 3
27
’ Day 0 = estimateddayof conception(mating).The meandayis given.SE= 1 or 2 for all means except-21 (+ 4) and-26 (* 5). bSignificantlydifferentfrom pairedcontrolvalue(p < 0.05),accordingto x2 analysis(percentage of deadpups)andStudent’st test (deadpups/litter). c Significantlydifferentfrom pairedcontrol value(p <:0.05),accordingto Student’sI test. TABLE 2 EFFECT OF ASCORBIC ACID ON PREGNANCY IN RATS AND HAMSTERS
Pups/litter Group Rats 1 2 3 4 Hamsters 1 2 3 4
Dose bx!kgY’
Nb
Dead’
Alive’
Live litter weight” (23
0 50 150 450
14 11 11 11
0.36 + 0.20 0.40 + 0.31 0.60 + 0.27 1.20+ 0.53
11.1+ 0.6 11.7+ 0.8 11.0+0.5 10.5+ 0.9
74.6+_3.9 75.3+ 4.3 73.3I? 3.3 68.1+ 5.6
0 50 150 450
11 12 10 14
0.09L-0.09 0.08 f 0.08 0.10+ 0.10 0.29 310.16
10.1+ 0.5 9.9 + 0.6 10.5+ 0.7 11.0+ 0.5
22.3i- 0.9 22.5 + 1.2 24.3+ 1.2 25.6+ 1.3d
’ Singleoral doseof ascorbicacidgivendailyfrom Day 1to 19(rats)or 15(hamsters) of pregnancy. Controls(0 mg/kg)receivedwater. bEveryanimalgavebirth; no abortionswereobserved;threeratsin groups2-4 atelittersat birth. c Mean+ SE. dSignificantlygreaterthancontrolvalue(p < 0.05),accordingto Student’st test.
SHORT
COMMUNICATIONS
395
pup weight was observed in one group of treated guinea pigs and hamsters. Neuweiler (19 51), Mouriquand and Edel (19 54), and Samborskaya (1964) reported increased abortion in guinea pigs given daily doses of 25,200, and 150 or 1500 mg/kg, respectively. Our dose of 400 mg/kg/day is within this range. We cannot explain these conflicting results. Similarly, Samborskaya and Ferdman (1966) and Fahim et al. (1972) reported increased abortion in rats given daily doses of 833 mg/kg SCand 250 mg/kg po, respectively. We found no effects with oral doses of 50-450 mg/kg/day. Our results are in agreement with those of Frohberg et al. (1973) who reported, while the present investigation was in progress, that ascorbic acid had no effects on pregnancy in rats given oral doses of 150-1000 mg/kg/day. These workers also found no effects in mice given 250-1000 mg/kg/day. Thus, the present investigation together with that of Frohberg et al. (1973) revealed no adverse effects of large daily doses of ascorbic acid on pregnancy in four animal species. REFERENCES M. S., HILDERBRAND, D., WILSON, R. HARMAN, J. M. AND HALL, D. G. (1972).Effect of high dosesof ascorbicacid on femalereproduction. In Fifth International Congress on Pharmacology, Abstracts of Volunteer Papers, p. 66, San Francisco. FROHBERG, H., GLEICH, J. AND KIESER, H. (1973).ReproduktionstoxikologischeStudien mit Ascorbinsaurean Mausenund Ratten. Arzneim. Forsch. 23, 1081-1082. MOURIQUAND, G. AND EDEL, V. (1954).HypervitaminoseC et gestation.C. R. Sot. Biol. 148, 1422-1423. NEUWEILER, W. (1951).Die Hypervitaminoseund ihre Beziehungzur Schwangerschaft.Znt. FAHIM,
Z. Vitaminforsch.
22, 392-396.
E. P. (1964).The effect of high ascorbicacid doseson the courseof pregnancy on the guineapig and on the progeny (Translation). Byull. Eksp. Biol. Med. 57, 105-108. SAMBORSKAYA, E. P. AND FERDMAN, T. D. (1966).The mechanismof termination of pregnancy by ascorbicacid (Translation). Byull. Eksp. Biol. Med. 62, 96-98. SAMBORSKAYA,
FREDERIC R. ALLEVA JOHN J. ALLEVA TIB~R BALAZS
Division of Drug Biology, Food and Drug Administration Department of Health, Education, and Welfare Washington, D.C. 20204 Received June 26; accepted September 26 1975