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2402. Japanese work on furylfuramide toxicity
EMULSIFIERS AND STABILIZERS, PRESERVATIVES
717
containing [14C]cyclamat e for 2 hr, no metabolites of cyclamate could be detected in blood, liver or bile. This finding was taken as evidence of the extrahepatic metabolism of cyclamate to cyclohexylamine.
EMULSIFIERS AND STABILIZERS 2401. Alginates survive the threat of colon ulceration
Watt, J. & Marcus, R. (1971). Effect of degraded and undegraded alginates on the colon of guinea-pigs. Proc. Nutr. Soc. 30, 81A. The discovery that the food stabilizer, carrageenan, can cause ulcerative colitis when incorporated into the drinking-water of rabbits and guinea-pigs (Cited in F.C.T. 1971, 9, 561) inevitably meant that the finger of suspicion would be pointed at the alginates, another group of stabilizers also derived from seaweed. The paper cited above, from the authors who presented the original work on carrageenan, does just this. Adult male albino guinea-pigs were used throughout the study. Two groups of five animals were used in a short 10-wk study in which 1% sodium alginate was incorporated into the drinking-water of the treated group, while four groups of six animals were used in a longer study, lasting 7 months. In the latter test, the two experimental groups were given drinkingwater containing 1% solutions of food-grade sodium alginate, in one case a high-viscosity sodium alginate and in the other a degraded low-viscosity preparation. The consumption of alginate in the two tests was in the range of 1.3-2.2 g/kg/day. The animals appeared healthy and gained weight satisfactorily, although the weight gain of the group receiving degraded low-viscosity sodium alginate for 7 months was considerably greater than that of either the controls or the other treated groups. No diarrhoea occurred and no occult blood appeared in the faeces of any of the animals. Ulceration was absent from the large bowel of all the alginate-treated guinea-pigs. [Without further details or a discussion by the authors it is impossible to estimate the significance of the increased weight gain in the group given the low-viscosity product. It is possible, however, that the animals in this group were younger and therefore weighed less than the other animals at the beginning of the experiment. Of much greater importance were the negative findings in respect of diarrhoea, occult blood in the faeces and ulceration in the colon.]
PRESERVATIVES 2402. Japanese work on furylfuramide toxicity
Miyaji, T. (1971). Acute and chronic toxicity of furylfuramide in rats and mice. Tohoku J. exp. Med. 103, 331.
Miyaji, T. (1971). The effect of simultaneous and alternate feeding of furylfuramide and 4-dimethylaminoazobenzene on rats. TohokuJ. exp. Med. 103, 371. Miyaji, T. (1971). Effect of furylfuramide on reproduction and malformation. Tohoku J. exp. Med. 103, 381.
718
PRESERVATIVES
Furylfuramide (2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide;FNFA) has been used as a food preservative in Japan since 1965. The efficacy of this preservative was established earlier (Cited in F.C.T. 1966, 4, 75) but no evidence was given at the time to support the claim of low chronic toxicity. This and other evidence has now been published. The acute oral LDsos of FNFA in rats and mice were 1554 and 475 mg/kg respectively (first paper cited above). In a short-term feeding study in rats lasting 15 wk and employing dietary levels of 0.0125, 0.05 and 0.2 ~, the only significant finding was liver enlargement at the 0.2 ~ dietary level, unaccompanied by histological, histochemical or serum-transaminase change. Liver enlargement was also induced by feeding 0 . 2 ~ FNFA for 1-4 wk. The effect was reversed following transfer of the animals to a control diet. Following 18- or 24-month feeding of 0.0125, 0-05 and 0 . 2 ~ in rats, liver enlargement at 0 . 2 ~ without pathological damage represented the only treatment-related finding. This liver enlargement was not reversed by 3 months on a control diet following the feeding of 0.2 ~ FNFA for 12 months. The weights and histological picture of other organs were normal. A high mortality rate encountered in control and test groups was attributable to respiratory disease. A 2-year feeding study in mice given 0.0125, 0.05 and 0"2~o FNFA confirmed liver enlargement at 0.2 ~o, but no histological damage to the liver or other organs was seen. No treatment-related effects were found, but the finding of a lower incidence of hepatocellular carcinoma in the test groups than in the controls was unexpected. Again the test was partly marred by heavy mortality. The author also compared the acute and subacute toxicity of FNFA with that of two other permitted preservatives, nitrofurazone (NF) and sorbic acid (SA). Acute oral LDsos were, for NF, 590 mg/kg in rats and 640 mg/kg in mice and, for SA, 10,500 mg/kg in rats. In 15-wk feeding studies in rats fed 0 . 2 ~ NF or 8 ~ SA, the main findings were growth retardation, liver enlargement and testicular atrophy with NF and liver enlargement with SA. The liver enlargement was less marked but was less readily reversed on cessation of treatment than that induced by FNFA. The second paper cited above explores the possibility that FNFA might accentuate liver cancer induced by 4-dimethylaminoazobenzene (DAB) in rats. This was examined by simultaneous, prior or subsequent feeding of FNFA in three experiments terminating after 20-24 wk, during which period protein-bound azo dye in the liver was determined and liver histology was studied. In one experiment, in which three groups were fed 0.045 ~ DAB, 0.2 ~o FNFA or the two together, it was found that FNFA counteracted the hepatocarcinogenic effect of DAB. However 4-wk feeding of 0.2 ~ FNFA prior to the feeding of 0.045 ~o DAB for 16 wk, or addition of 0.2 ~ FNFA for 6-18 wk to the diet of rats fed previously on 0"045~o DAB for 4-16 wk and a control diet for 2 wk, failed to affect the outcome of DAB-induced liver carcinogenesis. The FNFA suppression of DAB hepatocarcinogenesis was associated with a reduction in the quantity of protein-bound azo dye in the liver, possibly reflecting an increase in the capacity of the liver to metabolize DAB. The third paper cited above reports reproduction and teratogenic studies on FNFA involving four generations of mice and rats. Males and females were fed continuously on 0.0125~ FNFA, the former from weaning to mating at 3.5 months of age and the latter from weaning right through to lactation. The experiment was terminated when the fourth generation was weaned. Treated males were also mated with untreated females and the foetuses were examined for malformations. No significant effect on fertility, on litter size or weight or on weanling survival was seen in any generation of either species and no malformations were found in foetuses or offspring. Negative results were also obtained in one experiment in which females were fed 0.2 or 0.0125~ FNFA for 7 days from day 7 after
PRESERVATIVES
719
mating and in another in which males were fed 0.2 % FNFA for 5-7 wk before mating with untreated females. In a comparative study, in which 0.2 % NF was fed to males for 5-7 wk before mating with untreated females, reductions in male fertility and foetal weight were seen. [Although testicular atrophy was not seen in rats and mice fed furylfuramide (FNFA) in these studies, in monkeys fed a diet providing 8.5 mg FNFA/kg body weight for 8 months or in dogs fed a similar level for 10 months (unpublished observations by the same group), it is pertinent to point out that other workers are noted to have reported that the SpragueDawley strain of rat developed testicular atrophy following FNFA treatment. Despite the ability of nitrofurantoin and other nitrofuran derivatives to produce peripheral neuropathy, the tissues submitted to histological examination in the current tests did not include nervous tissue. Although neurological signs did not appear following FNFA treatment, the chance to look out for the possibility of subclinical pathological damage was not taken.]
2403. A look at ethylene chlorohydrin and ethylene glycol
McDonald, T. O., Roberts, D. M. & Borgmann, A. R. (1972). Ocular toxicity of ethylene chlorohydrin and ethylene glycol in rabbit eyes. Toxic. appl. Pharmac. 21, 143. Ethylene glycol (EG) and ethylene chlorohydrin (EC) are potential residual reaction products of ethylene oxide sterilization. EC causes severe local reactions in rabbits following intramuscular or subcutaneous administration or mucosal application but not following topical skin application (Cited in F.C.T. 1971, 9, 150). Repeated inhalation exposure of rabbits or rats to EG (12 mg/m 3 for 8 hr/day) caused eye damage within 8 days, but instillation of a 10% aqueous solution of EG into the rabbit eye did not inflict corneal damage (ibid 1971,9, 597). As an extension of previous work (Guess, Toxic. appl. Pharmac. 1969, 14, 659), both of these compounds have now been tested for toxicity and irritancy to the rabbit eye following repeated topical or intraocular administration, the latter by instillation into the anterior chamber. Balanced salt solutions containing 0-20 % EC or 0-40 % EG were administered intraocularly in 0.5 mi doses daily for 5 days or topically by instillation of 36 × 0.05 ml drops over a 6-hr period. Eyes were examined biomicroscopically for iritis, corneal opacity and flare before the test, at intervals between days 2 and 14 after the first intraocular dose and immediately after the last topical dose, and a graded scoring system was used to assess ocular toxicity. In addition, eyes were examined grossly for conjunctival irritation at 2, 4 and 6 hr after topical treatment and daily thereafter, and irritation was assessed by the Draize scoring system. In the case of EG, intraocular toxicity was seen with 4% but not with 0.4 or 0.04% concentrations, while irritation unaccompanied by toxicity followed topical application of 4 or 40 % but not of 0.04 or 0.4 % concentrations. With EC, intraocular toxicity developed with concentrations of 1-20 % but not of 0-06-0.5 %, while after topical application irritation developed with 2-20% but not with 0.06-1.0% solutions. Toxic effects accompanied the irritation caused by 5-20 % solutions. Thus the maximum concentrations found to be nontoxic and non-irritating following topical and intraocular application were 0.4% for EG and 10//o(topical) and 0-5 % (intraocular) for EC.
717
containing [14C]cyclamat e for 2 hr, no metabolites of cyclamate could be detected in blood, liver or bile. This finding was taken as evidence of the extrahepatic metabolism of cyclamate to cyclohexylamine.
EMULSIFIERS AND STABILIZERS 2401. Alginates survive the threat of colon ulceration
Watt, J. & Marcus, R. (1971). Effect of degraded and undegraded alginates on the colon of guinea-pigs. Proc. Nutr. Soc. 30, 81A. The discovery that the food stabilizer, carrageenan, can cause ulcerative colitis when incorporated into the drinking-water of rabbits and guinea-pigs (Cited in F.C.T. 1971, 9, 561) inevitably meant that the finger of suspicion would be pointed at the alginates, another group of stabilizers also derived from seaweed. The paper cited above, from the authors who presented the original work on carrageenan, does just this. Adult male albino guinea-pigs were used throughout the study. Two groups of five animals were used in a short 10-wk study in which 1% sodium alginate was incorporated into the drinking-water of the treated group, while four groups of six animals were used in a longer study, lasting 7 months. In the latter test, the two experimental groups were given drinkingwater containing 1% solutions of food-grade sodium alginate, in one case a high-viscosity sodium alginate and in the other a degraded low-viscosity preparation. The consumption of alginate in the two tests was in the range of 1.3-2.2 g/kg/day. The animals appeared healthy and gained weight satisfactorily, although the weight gain of the group receiving degraded low-viscosity sodium alginate for 7 months was considerably greater than that of either the controls or the other treated groups. No diarrhoea occurred and no occult blood appeared in the faeces of any of the animals. Ulceration was absent from the large bowel of all the alginate-treated guinea-pigs. [Without further details or a discussion by the authors it is impossible to estimate the significance of the increased weight gain in the group given the low-viscosity product. It is possible, however, that the animals in this group were younger and therefore weighed less than the other animals at the beginning of the experiment. Of much greater importance were the negative findings in respect of diarrhoea, occult blood in the faeces and ulceration in the colon.]
PRESERVATIVES 2402. Japanese work on furylfuramide toxicity
Miyaji, T. (1971). Acute and chronic toxicity of furylfuramide in rats and mice. Tohoku J. exp. Med. 103, 331.
Miyaji, T. (1971). The effect of simultaneous and alternate feeding of furylfuramide and 4-dimethylaminoazobenzene on rats. TohokuJ. exp. Med. 103, 371. Miyaji, T. (1971). Effect of furylfuramide on reproduction and malformation. Tohoku J. exp. Med. 103, 381.
718
PRESERVATIVES
Furylfuramide (2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide;FNFA) has been used as a food preservative in Japan since 1965. The efficacy of this preservative was established earlier (Cited in F.C.T. 1966, 4, 75) but no evidence was given at the time to support the claim of low chronic toxicity. This and other evidence has now been published. The acute oral LDsos of FNFA in rats and mice were 1554 and 475 mg/kg respectively (first paper cited above). In a short-term feeding study in rats lasting 15 wk and employing dietary levels of 0.0125, 0.05 and 0.2 ~, the only significant finding was liver enlargement at the 0.2 ~ dietary level, unaccompanied by histological, histochemical or serum-transaminase change. Liver enlargement was also induced by feeding 0 . 2 ~ FNFA for 1-4 wk. The effect was reversed following transfer of the animals to a control diet. Following 18- or 24-month feeding of 0.0125, 0-05 and 0 . 2 ~ in rats, liver enlargement at 0 . 2 ~ without pathological damage represented the only treatment-related finding. This liver enlargement was not reversed by 3 months on a control diet following the feeding of 0.2 ~ FNFA for 12 months. The weights and histological picture of other organs were normal. A high mortality rate encountered in control and test groups was attributable to respiratory disease. A 2-year feeding study in mice given 0.0125, 0.05 and 0"2~o FNFA confirmed liver enlargement at 0.2 ~o, but no histological damage to the liver or other organs was seen. No treatment-related effects were found, but the finding of a lower incidence of hepatocellular carcinoma in the test groups than in the controls was unexpected. Again the test was partly marred by heavy mortality. The author also compared the acute and subacute toxicity of FNFA with that of two other permitted preservatives, nitrofurazone (NF) and sorbic acid (SA). Acute oral LDsos were, for NF, 590 mg/kg in rats and 640 mg/kg in mice and, for SA, 10,500 mg/kg in rats. In 15-wk feeding studies in rats fed 0 . 2 ~ NF or 8 ~ SA, the main findings were growth retardation, liver enlargement and testicular atrophy with NF and liver enlargement with SA. The liver enlargement was less marked but was less readily reversed on cessation of treatment than that induced by FNFA. The second paper cited above explores the possibility that FNFA might accentuate liver cancer induced by 4-dimethylaminoazobenzene (DAB) in rats. This was examined by simultaneous, prior or subsequent feeding of FNFA in three experiments terminating after 20-24 wk, during which period protein-bound azo dye in the liver was determined and liver histology was studied. In one experiment, in which three groups were fed 0.045 ~ DAB, 0.2 ~o FNFA or the two together, it was found that FNFA counteracted the hepatocarcinogenic effect of DAB. However 4-wk feeding of 0.2 ~ FNFA prior to the feeding of 0.045 ~o DAB for 16 wk, or addition of 0.2 ~ FNFA for 6-18 wk to the diet of rats fed previously on 0"045~o DAB for 4-16 wk and a control diet for 2 wk, failed to affect the outcome of DAB-induced liver carcinogenesis. The FNFA suppression of DAB hepatocarcinogenesis was associated with a reduction in the quantity of protein-bound azo dye in the liver, possibly reflecting an increase in the capacity of the liver to metabolize DAB. The third paper cited above reports reproduction and teratogenic studies on FNFA involving four generations of mice and rats. Males and females were fed continuously on 0.0125~ FNFA, the former from weaning to mating at 3.5 months of age and the latter from weaning right through to lactation. The experiment was terminated when the fourth generation was weaned. Treated males were also mated with untreated females and the foetuses were examined for malformations. No significant effect on fertility, on litter size or weight or on weanling survival was seen in any generation of either species and no malformations were found in foetuses or offspring. Negative results were also obtained in one experiment in which females were fed 0.2 or 0.0125~ FNFA for 7 days from day 7 after
PRESERVATIVES
719
mating and in another in which males were fed 0.2 % FNFA for 5-7 wk before mating with untreated females. In a comparative study, in which 0.2 % NF was fed to males for 5-7 wk before mating with untreated females, reductions in male fertility and foetal weight were seen. [Although testicular atrophy was not seen in rats and mice fed furylfuramide (FNFA) in these studies, in monkeys fed a diet providing 8.5 mg FNFA/kg body weight for 8 months or in dogs fed a similar level for 10 months (unpublished observations by the same group), it is pertinent to point out that other workers are noted to have reported that the SpragueDawley strain of rat developed testicular atrophy following FNFA treatment. Despite the ability of nitrofurantoin and other nitrofuran derivatives to produce peripheral neuropathy, the tissues submitted to histological examination in the current tests did not include nervous tissue. Although neurological signs did not appear following FNFA treatment, the chance to look out for the possibility of subclinical pathological damage was not taken.]
2403. A look at ethylene chlorohydrin and ethylene glycol
McDonald, T. O., Roberts, D. M. & Borgmann, A. R. (1972). Ocular toxicity of ethylene chlorohydrin and ethylene glycol in rabbit eyes. Toxic. appl. Pharmac. 21, 143. Ethylene glycol (EG) and ethylene chlorohydrin (EC) are potential residual reaction products of ethylene oxide sterilization. EC causes severe local reactions in rabbits following intramuscular or subcutaneous administration or mucosal application but not following topical skin application (Cited in F.C.T. 1971, 9, 150). Repeated inhalation exposure of rabbits or rats to EG (12 mg/m 3 for 8 hr/day) caused eye damage within 8 days, but instillation of a 10% aqueous solution of EG into the rabbit eye did not inflict corneal damage (ibid 1971,9, 597). As an extension of previous work (Guess, Toxic. appl. Pharmac. 1969, 14, 659), both of these compounds have now been tested for toxicity and irritancy to the rabbit eye following repeated topical or intraocular administration, the latter by instillation into the anterior chamber. Balanced salt solutions containing 0-20 % EC or 0-40 % EG were administered intraocularly in 0.5 mi doses daily for 5 days or topically by instillation of 36 × 0.05 ml drops over a 6-hr period. Eyes were examined biomicroscopically for iritis, corneal opacity and flare before the test, at intervals between days 2 and 14 after the first intraocular dose and immediately after the last topical dose, and a graded scoring system was used to assess ocular toxicity. In addition, eyes were examined grossly for conjunctival irritation at 2, 4 and 6 hr after topical treatment and daily thereafter, and irritation was assessed by the Draize scoring system. In the case of EG, intraocular toxicity was seen with 4% but not with 0.4 or 0.04% concentrations, while irritation unaccompanied by toxicity followed topical application of 4 or 40 % but not of 0.04 or 0.4 % concentrations. With EC, intraocular toxicity developed with concentrations of 1-20 % but not of 0-06-0.5 %, while after topical application irritation developed with 2-20% but not with 0.06-1.0% solutions. Toxic effects accompanied the irritation caused by 5-20 % solutions. Thus the maximum concentrations found to be nontoxic and non-irritating following topical and intraocular application were 0.4% for EG and 10//o(topical) and 0-5 % (intraocular) for EC.