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Nitrosamines in saliva
92
Preservatives--FL/ Cos~~rr. Toxicol.
(Kramers, Mutation Res. 1975, 32, 81). Now, however, comes another report of mutagenicity, both in oivo and in vitro. The saccharin preparations tested consisted of both a retail (A) and a wholesale (B) pharmaceutical material, a sample (C) from the lot used in a Canadian rat-feeding study in which bladder tumours were found (Food Chemical News 1977, 19(15) 39). a highly purified version (D) of the last type, a powder (E) containing 40 g saccharin and 1 g lactose-potassium tartrate filler in a widely used convenience package, and a second sample (F) from a different lot used in the Canadian study. Samples E and F were used in the in vitro studies only, but the sweeteners xylitol and neohesperidin were included in all the tests. The test materials (25 g/kg) were given to mice by stomach tube and mutagenicity was determined with Sahone/la typhimurium strains TA98 and TAlOO (used respectively to detect frameshift mutations and base-pair substitutions). These bacterial strains were incubated either with the 24-hr urine or in the peritoneal cavity of the mice (host-mediated assay), and they were also used to detect mutagenicity in vitro. using sweetener concentrations about 20% below those producing 10% growth inhibition.
Vol. 17. No.
I
In the urine assay, saccharin samples A-C were mutagenic to both strains, and activity towards strain TA98 was markedly enhanced after incubation of the urine with fi-glucuronidase. The addition of a rat-liver microsomal preparation (S,) increased mutagenicity to this strain but inhibited activity towards strain TAlOO, suggesting that two different mutagens were present. The urines of mice given sample D demonstrated low but significant activity to strain TAlOO; this was increased by Sg after P-glucuronidase treatment. The excreted product(s) of this highly purified saccharin showed no mutagenic activity against TA98. however. In the host-mediated assay. samples A-C displayed a lower activity, and D was inactive. Similarly. of the saccharin samples tested in c-ho, only D failed to display any significant activity. the other saccharin samples being weakly mutagenic to both strains. The activities of samples A and B were increased by Sg but those of C and E were not. while sample F was activated by Ss, in tests with TAIOO but not in those with TA98. No mutagenicity was demonstrated with xylitol or NHDC under any conditions.
PRESERVATIVES Nitrited
pork
for
rats
and
mice
Knudsen, 1. & Meyer, 0. A. (1977). Mutagenicity studies on rats and mice given canned, heated, nitritetreated pork. Mutation Res. 56, 177. The possible induction of human cancer by consumption of nitrosamines formed from food amines and the nitrite used in curing and preserving food has received a great deal of attention (Cited in F.C.T. 1978, 16, 69 & 389). The study reported below examines the possibility of a dose relationship between nitrite added to pork and genetic damage in germ cells, leading to dominant lethality, sterility or semisterility in the F, generation. Lean salted pork was treated before canning and heating with 200, 1000 or 40 ppm sodium nitrite and fed to rats and mice as their sole protein source throughout the period of spermatogenesis (to rats for 12 wk and to mice for 8 wk). Just after the end of treatment the males were allowed to mate (three virgin females were introduced each week) over a period of 3 wk for mice and 5 wk for rats. The females were killed and dissected on day 15 (mice) and day 17 (rats) after introduction to the males. At no feed level of nitrited pork did treated rats differ from the controls in pregnancy rate, number of implantation sites or percentage of dead implantations. In mice fed pork treated with 4000ppm nitrite there was a significant decrease in the number of implantations from the wk 3 matings, but no associated increase in post-implantation loss. The F, males for the heritable translocation test were derived from male and female mice both given nitrite-treated pork, at the levels indicated above. Females mated to the F, males were killed and dissected 15 days after introduction to the males. Fertility was considered to be normal if two out of
three females mated to the same male had ten or more implants and the average live litter size for the three females was at least eight. The appearance of one semi-sterile male among 50 in the maximally exposed group of mice in the heritable translocation test could not be regarded as significant; nor could the one completely sterile mouse found among the 90 fed pork containing 1000 ppm nitrite in the dominant lethal test. Experiments with more animals are needed to reach a firmer conclusion in the heritable translocation test. The absence of any doserelated response in dead implantations in the dominant lethal test indicates that the levels of sodium nitrite used in the treated pork do not increase dominant lethal mutations. Nitrosamines
in saliva
Tannenbaum, S. R., Archer, M. C., Wishnok, J. S. Kc Bishop, W. W. (1978). Nitrosamine formation in human saliva. J. narn. Cancer Ins. 60, 251. The role of nitrosamines in carcinogenesis and their possible formation in the human gut from the reaction between amines in foods and added nitrites were recently reviewed (Cooper, Fd Cosmer. Toxicol. 1978, 16, 69). Nitrites are also found in normal human saliva, probably as a product of microbial reduction of nitrate, which circulates through the salivary glands. The study cited above examined the nitrosation reaction in saliva and in saliva mixed with acid to simulate conditions in the stomach. Human saliva in 5-ml aliquots was incubated with an amine and in some cases with additional nitrite for 30min at 37°C. Resulting nitrosamine concentrations were determined using gas chromatography
Agricultural
chemicals-Fd
with a thermal energy analyser. In whole saliva from several individuals a low but consistent amount of nitrosamine, around 30 nmol/litre, was synthesized in all samples tested, even in saliva containing relatively low nitrite concentrations. Saliva from one individual was used to determine the effects on nitrosamine formation of adding 1000 mg morpholine/litre (a) alone, (b) with 100mg sodium nitrite/litre or (c) with HCI to achieve a pH of 3. Yields of nitrosomorpholine (NMOR) were determined after incubating whole saliva (containing living cells), cell-free supernatant, resuspended cells, heated whole saliva and a buffer control under these three regimes. After incubation with morpholine alone, NMOR was detected (12 nmol/litre) only with unheated whole saliva. When sodium nitrite was also added to the incubation mixture, NMOR was found in all five systems ranging from 188 nmol/litre with the resuspended cells to 5 nmol/litre with the buffer control. Whole saliva produced about a third as much NMOR as did the resuspended cells and very little was produced by either the cell-free supernatant or the heated whole saliva. At the lowered pH, whole saliva, cell-free supernatant and heated whole saliva produced respectively 3176, 1676 and 1857 nmol NMOR/litre. No NMOR was produced by the acidified resuspended cells or the buffer control. Significant amounts of nitrosamines
AGRICULTURAL Dieldrin,
DMSO
and the pregnant
mouse
Dix, K. M., van der Pauw, C. L. & McCarthy, W. V. (1977). Toxicity studies with dieldrin: teratological studies in mice dosed orally with HEOD. Teratology 16, 57.
Cleft palate and webbed foot have been described in the offspring of mice and hamsters exposed to dieldrin during pregnancy, but these effects may have been relegated to inhibition of growth (Cited in F.C.T. 1975, 13, 397) and the design of the study excluded any possibility of detecting a dose-response relationship. The mouse study now reported was carried out on 1,8,9.10,11,1 I-hexachloro-4,5-exo-epoxy-2,3,7,6endo-2,l,7,8-exo-tetracyclo[6.2.l.l3~6.02~7]dodec-9ene (HEOD), which constitutes about 85% of dieldrin. The HEOD used was over 99% pure. Female mice were dosed orally with 1.5 or 4,Omg HEOD/kg/day in corn oil from day 6 to day 14 of gestation and were examined on day 18. Additional groups received 0.25.0.5 or 1.0 mg/kg/day in dimethylsulphoxide (DMSO) for the same period. Some mice given HEOD in DMSO, or DMSO alone, became dyspnoeic and hypothermic and showed decreased motor activity. One animal given DMSO, one given 0.25 mg HEOD/kg in DMSO, and four given 1.0 mg/ kg in DMSO died during the study. Significantly fewer mice survived to term in the groups given 0.5 or l.Omg/kg in DMSO than in the other groups. but none of the treated groups showed consistent differences in the mean numbers of male and female foetuses in the litters. mean litter size, numbers of
Cosrwt.
Toxicol.
Vol. 17. No. I
93
were also formed on addition of di-n-propylamine, di-n-butylamine and di-n-hexylamine to whole saliva, while addition to dimethylamine, diethylamine, piperidine and pyrrolidine produced trace amounts. The results indicate that nitrosamine formation is possible in saliva even at neutral pH. Cells in whole saliva accelerate nitrosation, although the saliva contains components that both accelerate and retard nitrosamine formation. The investigators also showed that various amines may be nitrosated in saliva and that the rate of the reaction was independent of the basicity of the amine but dependent on the length of the alkyl chain. The results support their hypotheses that (1) nitrosamine formation in the mouth is catalysed by the oral microflora, (2) nitrite formed in the oral cavity contributes to nitrosamine formation in the normal stomach, (3) nitrosamines can be formed in the weakly acidic or even neutral stomachs of patients with intestinal metaplasia and (4) nitrosamines can be formed at other sites that normally contain bacteria or become infected. [It seems surprising that the authors used saliva from one individual for the bulk of this experiment, especially since the saliva chosen had a much lower nitrite concentration (2.5 mg/litre) than the saliva samples used in the preliminary experiment (mean. 7.0 mg nitrite/litre).]
CHEMICALS resorptions or numbers of foetal deaths. In the DMSO study, the test groups had foetuses of significantly lower weight than the controls, but this did not occur in the corn oil study. Most of the skeletal abnormalities observed in both experiments involved minor changes in the sternum. None of the treated groups showed any significant increase in the number of litters containing foetuses with structural abnormalities, but delayed ossification showed a higher incidence in litters of the group given 0.5 mg HEOD/kg in DMSO and of that given DMSO alone. These two groups also showed a significant increase in the proportion of total foetuses showing minor skeletal changes. These results provide no evidence of any compound-related teratogenic effects and suggest that the effects observed were largely a reflection of the toxicity of the DMSO used as a vehicle in the second experiment. Liver nodules in mirex study Ulland, B. M., Page, N. P., Squire, R. A., Weisburger, E. K. & Cypher, R. L. (1977). A carcinogenicity assay of Mirex in Charles River CD rats. J. narn. Cancer Insr. 58, 133. Reuber, M. D. (1977). Acute and chronic renal hepatic disease in rats fed Mirex. J. mtn. Cancer Inst. 59, 1051. Ulland, mtn.
B. M. (1977). [Reply Inst. 59, 1052.
Cancer
to M. D. Reuber.]
J.
Preservatives--FL/ Cos~~rr. Toxicol.
(Kramers, Mutation Res. 1975, 32, 81). Now, however, comes another report of mutagenicity, both in oivo and in vitro. The saccharin preparations tested consisted of both a retail (A) and a wholesale (B) pharmaceutical material, a sample (C) from the lot used in a Canadian rat-feeding study in which bladder tumours were found (Food Chemical News 1977, 19(15) 39). a highly purified version (D) of the last type, a powder (E) containing 40 g saccharin and 1 g lactose-potassium tartrate filler in a widely used convenience package, and a second sample (F) from a different lot used in the Canadian study. Samples E and F were used in the in vitro studies only, but the sweeteners xylitol and neohesperidin were included in all the tests. The test materials (25 g/kg) were given to mice by stomach tube and mutagenicity was determined with Sahone/la typhimurium strains TA98 and TAlOO (used respectively to detect frameshift mutations and base-pair substitutions). These bacterial strains were incubated either with the 24-hr urine or in the peritoneal cavity of the mice (host-mediated assay), and they were also used to detect mutagenicity in vitro. using sweetener concentrations about 20% below those producing 10% growth inhibition.
Vol. 17. No.
I
In the urine assay, saccharin samples A-C were mutagenic to both strains, and activity towards strain TA98 was markedly enhanced after incubation of the urine with fi-glucuronidase. The addition of a rat-liver microsomal preparation (S,) increased mutagenicity to this strain but inhibited activity towards strain TAlOO, suggesting that two different mutagens were present. The urines of mice given sample D demonstrated low but significant activity to strain TAlOO; this was increased by Sg after P-glucuronidase treatment. The excreted product(s) of this highly purified saccharin showed no mutagenic activity against TA98. however. In the host-mediated assay. samples A-C displayed a lower activity, and D was inactive. Similarly. of the saccharin samples tested in c-ho, only D failed to display any significant activity. the other saccharin samples being weakly mutagenic to both strains. The activities of samples A and B were increased by Sg but those of C and E were not. while sample F was activated by Ss, in tests with TAIOO but not in those with TA98. No mutagenicity was demonstrated with xylitol or NHDC under any conditions.
PRESERVATIVES Nitrited
pork
for
rats
and
mice
Knudsen, 1. & Meyer, 0. A. (1977). Mutagenicity studies on rats and mice given canned, heated, nitritetreated pork. Mutation Res. 56, 177. The possible induction of human cancer by consumption of nitrosamines formed from food amines and the nitrite used in curing and preserving food has received a great deal of attention (Cited in F.C.T. 1978, 16, 69 & 389). The study reported below examines the possibility of a dose relationship between nitrite added to pork and genetic damage in germ cells, leading to dominant lethality, sterility or semisterility in the F, generation. Lean salted pork was treated before canning and heating with 200, 1000 or 40 ppm sodium nitrite and fed to rats and mice as their sole protein source throughout the period of spermatogenesis (to rats for 12 wk and to mice for 8 wk). Just after the end of treatment the males were allowed to mate (three virgin females were introduced each week) over a period of 3 wk for mice and 5 wk for rats. The females were killed and dissected on day 15 (mice) and day 17 (rats) after introduction to the males. At no feed level of nitrited pork did treated rats differ from the controls in pregnancy rate, number of implantation sites or percentage of dead implantations. In mice fed pork treated with 4000ppm nitrite there was a significant decrease in the number of implantations from the wk 3 matings, but no associated increase in post-implantation loss. The F, males for the heritable translocation test were derived from male and female mice both given nitrite-treated pork, at the levels indicated above. Females mated to the F, males were killed and dissected 15 days after introduction to the males. Fertility was considered to be normal if two out of
three females mated to the same male had ten or more implants and the average live litter size for the three females was at least eight. The appearance of one semi-sterile male among 50 in the maximally exposed group of mice in the heritable translocation test could not be regarded as significant; nor could the one completely sterile mouse found among the 90 fed pork containing 1000 ppm nitrite in the dominant lethal test. Experiments with more animals are needed to reach a firmer conclusion in the heritable translocation test. The absence of any doserelated response in dead implantations in the dominant lethal test indicates that the levels of sodium nitrite used in the treated pork do not increase dominant lethal mutations. Nitrosamines
in saliva
Tannenbaum, S. R., Archer, M. C., Wishnok, J. S. Kc Bishop, W. W. (1978). Nitrosamine formation in human saliva. J. narn. Cancer Ins. 60, 251. The role of nitrosamines in carcinogenesis and their possible formation in the human gut from the reaction between amines in foods and added nitrites were recently reviewed (Cooper, Fd Cosmer. Toxicol. 1978, 16, 69). Nitrites are also found in normal human saliva, probably as a product of microbial reduction of nitrate, which circulates through the salivary glands. The study cited above examined the nitrosation reaction in saliva and in saliva mixed with acid to simulate conditions in the stomach. Human saliva in 5-ml aliquots was incubated with an amine and in some cases with additional nitrite for 30min at 37°C. Resulting nitrosamine concentrations were determined using gas chromatography
Agricultural
chemicals-Fd
with a thermal energy analyser. In whole saliva from several individuals a low but consistent amount of nitrosamine, around 30 nmol/litre, was synthesized in all samples tested, even in saliva containing relatively low nitrite concentrations. Saliva from one individual was used to determine the effects on nitrosamine formation of adding 1000 mg morpholine/litre (a) alone, (b) with 100mg sodium nitrite/litre or (c) with HCI to achieve a pH of 3. Yields of nitrosomorpholine (NMOR) were determined after incubating whole saliva (containing living cells), cell-free supernatant, resuspended cells, heated whole saliva and a buffer control under these three regimes. After incubation with morpholine alone, NMOR was detected (12 nmol/litre) only with unheated whole saliva. When sodium nitrite was also added to the incubation mixture, NMOR was found in all five systems ranging from 188 nmol/litre with the resuspended cells to 5 nmol/litre with the buffer control. Whole saliva produced about a third as much NMOR as did the resuspended cells and very little was produced by either the cell-free supernatant or the heated whole saliva. At the lowered pH, whole saliva, cell-free supernatant and heated whole saliva produced respectively 3176, 1676 and 1857 nmol NMOR/litre. No NMOR was produced by the acidified resuspended cells or the buffer control. Significant amounts of nitrosamines
AGRICULTURAL Dieldrin,
DMSO
and the pregnant
mouse
Dix, K. M., van der Pauw, C. L. & McCarthy, W. V. (1977). Toxicity studies with dieldrin: teratological studies in mice dosed orally with HEOD. Teratology 16, 57.
Cleft palate and webbed foot have been described in the offspring of mice and hamsters exposed to dieldrin during pregnancy, but these effects may have been relegated to inhibition of growth (Cited in F.C.T. 1975, 13, 397) and the design of the study excluded any possibility of detecting a dose-response relationship. The mouse study now reported was carried out on 1,8,9.10,11,1 I-hexachloro-4,5-exo-epoxy-2,3,7,6endo-2,l,7,8-exo-tetracyclo[6.2.l.l3~6.02~7]dodec-9ene (HEOD), which constitutes about 85% of dieldrin. The HEOD used was over 99% pure. Female mice were dosed orally with 1.5 or 4,Omg HEOD/kg/day in corn oil from day 6 to day 14 of gestation and were examined on day 18. Additional groups received 0.25.0.5 or 1.0 mg/kg/day in dimethylsulphoxide (DMSO) for the same period. Some mice given HEOD in DMSO, or DMSO alone, became dyspnoeic and hypothermic and showed decreased motor activity. One animal given DMSO, one given 0.25 mg HEOD/kg in DMSO, and four given 1.0 mg/ kg in DMSO died during the study. Significantly fewer mice survived to term in the groups given 0.5 or l.Omg/kg in DMSO than in the other groups. but none of the treated groups showed consistent differences in the mean numbers of male and female foetuses in the litters. mean litter size, numbers of
Cosrwt.
Toxicol.
Vol. 17. No. I
93
were also formed on addition of di-n-propylamine, di-n-butylamine and di-n-hexylamine to whole saliva, while addition to dimethylamine, diethylamine, piperidine and pyrrolidine produced trace amounts. The results indicate that nitrosamine formation is possible in saliva even at neutral pH. Cells in whole saliva accelerate nitrosation, although the saliva contains components that both accelerate and retard nitrosamine formation. The investigators also showed that various amines may be nitrosated in saliva and that the rate of the reaction was independent of the basicity of the amine but dependent on the length of the alkyl chain. The results support their hypotheses that (1) nitrosamine formation in the mouth is catalysed by the oral microflora, (2) nitrite formed in the oral cavity contributes to nitrosamine formation in the normal stomach, (3) nitrosamines can be formed in the weakly acidic or even neutral stomachs of patients with intestinal metaplasia and (4) nitrosamines can be formed at other sites that normally contain bacteria or become infected. [It seems surprising that the authors used saliva from one individual for the bulk of this experiment, especially since the saliva chosen had a much lower nitrite concentration (2.5 mg/litre) than the saliva samples used in the preliminary experiment (mean. 7.0 mg nitrite/litre).]
CHEMICALS resorptions or numbers of foetal deaths. In the DMSO study, the test groups had foetuses of significantly lower weight than the controls, but this did not occur in the corn oil study. Most of the skeletal abnormalities observed in both experiments involved minor changes in the sternum. None of the treated groups showed any significant increase in the number of litters containing foetuses with structural abnormalities, but delayed ossification showed a higher incidence in litters of the group given 0.5 mg HEOD/kg in DMSO and of that given DMSO alone. These two groups also showed a significant increase in the proportion of total foetuses showing minor skeletal changes. These results provide no evidence of any compound-related teratogenic effects and suggest that the effects observed were largely a reflection of the toxicity of the DMSO used as a vehicle in the second experiment. Liver nodules in mirex study Ulland, B. M., Page, N. P., Squire, R. A., Weisburger, E. K. & Cypher, R. L. (1977). A carcinogenicity assay of Mirex in Charles River CD rats. J. narn. Cancer Insr. 58, 133. Reuber, M. D. (1977). Acute and chronic renal hepatic disease in rats fed Mirex. J. mtn. Cancer Inst. 59, 1051. Ulland, mtn.
B. M. (1977). [Reply Inst. 59, 1052.
Cancer
to M. D. Reuber.]
J.