TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin)

COM/99/S2 - May 1999

The following statement was agreed under matters arising at the May 1999 meeting of the Committee.

Introduction

1. There are 75 possible chlorinated polychlorinated-para-dioxins (PCDDs) and 135 possible polychlorinated-para-dibenzofurans (PCDFs). A number of these substances (congeners), predominantly the tetra- through to octa-congeners may be found as trace contaminants in food. The toxicological properties and potencies of PCDDs and PCDFs mixtures have been estimated from the available information on TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) through the use of toxic equivalency factors (TEFs). TCDD thus serves as a model compound for the risk assessment of other PCDD/PCDF mixtures.(1)

2. PCDDs are produced as a trace contaminant during the manufacture of many organochlorine compounds such as 2,4,5-trichlorophenol (TCP), chlorophenoxy herbicides (2,4,5-T), hexachlorophene, chlorodiphenyl ether herbicides), and during certain industrial processes such as pulp bleaching using chlorine gas. They are also produced in a number of thermal reactions such as the incineration of municipal waste, sewage sludge, PVC as well as from automobile emissions. The highest exposures have predominantly occurred under occupational settings, often following accidental release, the exception being the large widespread release of TCDD to the environment with population exposure following the Seveso incident in 1976.

3. The IARC (International Agency for Research on Cancer) concluded in 1997 that TCDD should be regarded as a known human carcinogen and thus classified as Group 1.(2) Previously it had been classified as Group 2A (probable human carcinogen). This change prompted the Department of Health to request COC to review their earlier conclusion. The COC concluded in 1998 that there were insufficient epidemiological and toxicological data on TCDD to conclude a causal link with cancer in humans, but it would be prudent to consider TCDD as a 'probable weak human carcinogen'. The IARC working group concluded that the "experimental data indicated that 2,3,7,8-TCDD and probably other PCDDs and PCDFs are not direct-acting genotoxic agents and that TCDD is considered a non-genotoxic substance." The previous COM evaluation of TCDD was completed in 1987 and it is therefore timely for the committee to reconsider its previous conclusions.

COM conclusions from 1987

4. The COM considered all the available information in 1987 and drew the following conclusions.

5. TCDD does not produce gene-mutations in Salmonella assays, but there is some evidence from higher eukaryotes (yeasts) and a mammalian cell assay that the compound has some potential for producing gene mutations although the mammalian cell assay has not been repeated. However negative results were obtained in the sex linked recessive lethal assay in Drosophila.

6. There is no convincing evidence that TCDD has clastogenic properties. Negative results were also obtained in studies to investigate the compound's ability to produce sister chromatid exchanges (SCEs) in vitro and in vivo, and also unscheduled DNA synthesis (UDS) in rat hepatocytes, indicating that TCDD does not produce DNA damage in these systems.

7. These results suggest that although TCDD may have some mutagenic potential in yeast systems, on balance negative results were obtained, and it is unlikely that the carcinogenicity observed in the rodent assays is due to a mutagenic mechanism. There is limited evidence from cell transformation assays that TCDD has tumour promotor activity.

8. The very limited data available on the other chlorinated dibenzo dioxins and dibenzo furans precludes any conclusions being drawn about the mutagenic potential of these compounds.

1999 COM review

9. The Committee needed to update its advice to enable a COC/COM statement on the current position to be agreed. In this regard, the Department of Health Toxicology Unit at Imperial College of Science, Technology and Medicine (ICSTM) was asked to provide a review of the literature published since 1990 on the TCDD regarding mutagenicity data. The authors noted that the majority of studies conducted since 1987 had yielded negative results. The report highlighted a number of investigations where further consideration might be required.

10. The Committee agreed that negative results had been obtained in the in-vitro mutagenicity tests conducted in Salmonella and in L5178Y tk+/tk- cells mouse lymphoma cells.(3, 4) However TCDD induced micronuclei formation had been reported in one study using human lymphocytes and the cytochalasin B technique.(5) The same research group had also noted sister chromatid exchange (SCE) in a subsequent publication.(6) Members commented that it was not possible to draw any conclusions based on these results particularly in view of the unusually long incubation period of 71 hours prior to harvesting the cells. The Committee agreed that a repeat test would be desirable in order to validate the method used in these investigations.

11. The Committee agreed that negative results had been obtained in mouse hepatocytes following dosing of animals with up to 150 µg/kg (i.p).(7) No increase in SCEs in peripheral blood lymphocytes was noted in Rhesus monkeys, 2 years post administration of a diet containing 25 ppt TCDD for 4 years.(8) However, a small, but statistically significant increase in SCEs in peripheral blood lymphocytes had been documented in a limited study in rats given weekly gavage doses of 5 µg/kg for 2 weeks but not at 0.5 µg/kg.(9) Evidence of TCDD induced single stand DNA breaks in peritoneal lavage cells had been documented in rats given a single oral dose of 25 µg/kg bw up to 100µg/kg.(10) In addition, a positive result had been reported in a deletion recombination spot test (11) but not in a separate study which used a similar dosing regime.(12) Members considered that no weight could be attached to this investigation in view of the limited study design, the concerns previously expressed by Members regarding these tests and the negative findings reported in a mouse spot test by a separate research group.

COM Conclusions

12. The Committee agreed the following conclusions.

(a) "In studies published since 1987, TCDD has continued to give largely negative results in tests for several different genetic endpoints, such as DNA damage, gene mutations, sister chromatid exchange and cell transformation. In a few studies TCDD has given a positive or equivocal result, often using assays with either highly non-standard or sub-optimal design. Currently the weight of the available experimental data continue to indicate that TCDD is not a genotoxic agent."

(b) The Committee wished to continue to monitor any further genotoxicity publications on TCDD.

References

1. Safe S (1990). Polychlorinated biphenyls, dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs) and related compounds: environmental and mechanistic considerations which support the development of toxic equivalency factors (TEFs). CRC Critical Reviews in Toxicology, 21, 51-88.

2. IARC (1997). IARC Monographs on the evaluation of carcinogenic risks to humans. Polychlorinated dibenzo-para- dioxins and polychlorinated-para-dibenzofurans. Volume 69, pp1-666, WHO, Lyons, France.

3. Blevis RD (1991). 2,3,7,8-tetrachlorodibenzo-p-dioxin in fish from Pigeon River of Eastern Tennessee, USA, its toxicity and mutagenicity as revealed by the Ames test. Arch Environ Contam Toxicol, 20, 366-370.

4. McGreegor DB et al (1991). Responses of the L5178Y mouse lymphoma cell forward mutation assay. 527 coded chemicals. Environ Mol Mut, 17, 196-219.

5. Nagayama J and Masuda Y (1993). Effects of 3-methylsulphonyl-4,5,3,4- tetrachlorobiphenyl and 7,8-benzoflavone on aryl hydrocarbon hydroxylase activities of murine hepatic microsomes prepared from inbred strains with different Ah responsiveness. Fukuoka Acta Med, 84, 195-202.

6. Nagayama J et al (1995). Effect of 2,3,4,7,8-pentachlorodibenzofuran and its analogues on induction of sister chromatid exchanges in cultured lymphocytes. Fukuoka Acta Med, 86, 184-189.

7. Brooks Al et al (1988). The cytogenetic and hepatotoxic effects of dioxin on mouse liver cells. Cell Biol Toxicol, 4, 31-40.

8. Lim M et al (1987). Effect of chronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin on sister chromatid exchange levels in peripheral lymphocytes in Rhesus Monkey. Cell Biol Toxicol, 3, 279-284.

9. Mustonen J et al (1989). Effects of commercial chlophenate, 2,3,7,8-TCDD, and pure phenoxyacetic acids on hepatic peroxysome proliferation, xenobiotic metabolism and sister chromatid exchange in the rat. Arch Toxicol, 63, 203-208.

10. Alsharif NZ et al (1994). Stimulation of NADPH-dependent reactive oxygen species formation and DNA damage by 2,3,7,8-tetrachlorodibenzo-p-dioxin in rat peritoneal lavage cells. Arch Environ Contam Toxicol, 26, 392-397.

11. Schiestl RH et al (1997). Polychlorinated biphenyls and 2,3,7,8-tetrachlorodibenzo-p-dioxin induce intrachromosomal recombination in-vitro and in-vivo. Cancer Research,57, 4378-4383.

12. Fahrig R (1993). Genetic effects of dioxins in the spot test with mice. Environ Health Perspect ,101, 257-261.