BL6 mice: 2,4,5-T administered on days 6-14 or days 9-17, and mice sacrified on day 18 of pregnancy.

BL6/AK/mice: 2,4,5-T administered on days 6-14, and mice sacrificed on day 18 of pregnancy.

AK mice: 2,4,5-T administered from days 6-15, and mice sacrificed on day 19 of pregnancy.

Sprague Dawley rats: 2,4,5-T administered from days 10-15 and rats sacrificed on day 20 of pregnancy.

In the 4 tables that follow dealing with a wide dose range, the results are expressed as percentages of abnormal fetuses. I would point out those occasions where statistically significant incidence of results was noted.

Major abnormalitities in mice were cleft palates and cystic kidneys, and in rats, cystic kidneys and gastrointestinal hemorrhages. Increased fetal mortality was generally concomitant with these abnormalities. It is of particular interest that 39 percent abnormal embryos with cystic kidneys were seen in rats even at the lowest dose tested. Thus the no effect level was not reached even at 4.6 mg./kg.

Teratogenicity data on 2,4,5-T, as summarized in the bionetics report (appendix III) are quoted in extenso below. Some critical sentences are italicized:

This compound was given by the oral route to BL6 mice at dosages of 46.6 and 113 mg/kg and to AKR mice at 113 mg/kg. It was given by subcutaneous injection to BL6 mice at dosages of 21.5 and 113 mg/kg and to AKR mice and B6AK hybrids at 113 mg/kg. It was also given subcutaneously to CH mice at 215 mg/kg, but there were too few of these to merit inclusion in the discussion which follows.

Administration was for eight days (6th through 14th) in most cases; for nine days (6th through 15th) in some; and for five days (10th through 14th) in one case the details are indicated in the tabulated results. Subcutaneous administration used DMSO as a vehicle; oral used 50 percent honey.

With the single exception of the lowest dosage used (21.5 mg/kg to BL6 subcutaneously) all dosages, routes and strains resulted in increased incidence of abnormal fetuses. The incidence of cleft palate was high at the 113 mg/kg dosage, but not at lower levels. The incidence of cystic kidney was also high except in the AKR strain and in the BL6 mice which received 46.4 mg/kg orally. Fetal mortality was increased in all groups given 113 mg/kg for eight or nine days, but not in mice (BL6) given this dosage for only five days nor in the two groups of BL6 mice given lesser dosages (46.4 mg/kg orally and 21.5 mg/kg subcutaneously.

Most fetal and maternal measurements showed inconsistent changes from which no conclusions can be drawn. In contrast, there was a highly consistent decrease in maternal weight gain in BL6 mice given 113 mg/kg by either route. Lower dosages and the AKR strain showed either no change or a slight increase. All dosages, strains, and routes showed an increase in the maternal liver weight and this led to a further study discussed separately below.

These results imply a hazard of teratogenesis in the use of this compound. The problems of extrapolation preclude definition of the hazard on the basis of these studies, but its existence seems clear.

The observed influence of 2,4,5-T on maternal liver weight as mentioned above raised a question as to its effect on the fetal liver. This was answered by a study carried out in BL6 mice using subcutaneous injections of DMSO solutions at a dosage of 113 mg/kg only. The period of administration was lengthened to cover the period from the 9th through 17th day of gestation. Separate control groups were used concurrently. Except for the inclusion of fetal liver weight, measurements were made as previously described.

The fetal livers of the 2,4,5-T treated mice weighed significantly more than those of controls given DMSO only and the weights of the whole fetuses were

significantly less. Correspondingly, there was an increase in the fetal liver weight expressed as percent of body weight.

Other observations were consistent with those reported above. The incidence of abnormal fetuses was unusually high as were those of cleft palate and cystic kidney.

Because of the potential importance of the findings in mice, an additional study was carried out in rats of the Sprague-Dawley strain. Using dosages of 21.5 and 46.4 mg/kg suspended in 50 percent honey and given by the oral route on the 6th through 15th days of gestation, we observed excessive fetal mortality (almost 80 percent) and a high incidence of abnormalities in the survivors. When the beginning of administration was delayed until the 10th day, fetal mortality was somewhat less, but still quite high even when dosage was reduced to 4.6 mg/kg.

The incidence of abnormal fetuses was threefold that in controls even with the smallest dosage and shortest period used. Fetal and maternal measurements showed only occasional instances of significant differences from controls except in the case of maternal liver weight which was consistently increased in all 2,4,5-T treated animals.

It seems inescapable that 2,4,5-T is teratogenic in this strain of rats when given orally at the dosage schedules used here. These findings lend emphasis to the hazard implied by the results of studies on mice.

D. Recent reanalysis of the Bionetic data on teratogenicity of 2,4,5-T. More refined and more appropriate additional statistical analyses of these data were presented and discussed in the report of the Advisory Panel on Teratogenicity of Pesticides (appendix II). These are clearly confirmatory of the original conclusions of the Bionetics report on the teratogenicity of 2,4,5-T. Some relevant portions of the HEW panel report are quoted in extenso below:

Tested more extensively than other pesticides, 2,4,5-T was clearly teratogenic as evidenced by production of statistically increased proportions of litters affected, and increased proportions of abnormal fetuses within litters in both DMSO and honey for both C57BL/6 and AKR mice. In particular, cleft palate and cystic kidneys were significantly more prevalent. In addition, a hybrid strain resulting from a C57BL/6 female and AKR male showed significant increases in anomalies, in particular cystic kidney, when administered at 113 mg/kg of body weight in DMSA.

Additionally, 2,4,5-T was tested in Sprague-Dawley rats. When given orally at dosages of 4.6, 10.0, and 46.4 mg/kg on days 10 through 15 of gestation, an excessive fetal mortality, up to 60 percent at the highest dose, and high incidence of abnormalities in the survivors was obtained. The incidence of fetuses with kidney anomalies was threefold that of the controls, even with the smallest dosage tested.

E. Recent studies on teratogenicity testing of relatively pure 2,4,5-T. In view of the fact that the Bionetics study was conducted with a sample of 2,4,5-T which was subsequently shown to contain a relatively high concentration, 27 ppm, of a tetrachloro dioxin contaminant, testing has been recently repeated with relatively pure samples containing less than 1 ppm of this particular dioxin.

The results of these studies were presented by the FDA and NIEHS at a recent conference of February 24, 1970, at the FDA; the Dow Chemical Co. data were presented at the 9th annual meeting of the Society of Toxicology, Atlanta, March 17, 1970.

As can be seen from the data summarized below, purified 2,4,5-T is teratogenic in three species-rats, mice and hamsters. These data should be regarded as preliminary. Confirmatory data on chick eggs are not presented here.

1. Dow Chemical Co. studies (Emerson et al., 1970). 2,4,5-T with 0.5 ppm dioxins, as a probable contaminant, was tested in pregnant

rats by repeated oral administration at doses of 1, 3, 6, 12, and 24 mg./kg.; the maximal dose tested was 24 mg./kg. No embryo deaths or weight losses were noted within the dose range tested. However, at 24 mg./kg. there was a sevenfold increase in the incidence of fetuses with defective ossification of the fifth sternebra; poor sternebral ossification was noted in four out of 103 control fetuses, and in 29 out of 103 fetuses of 2,4,5-T treated groups.

Defective sternebral ossification has been described in the rat as an expression of the teratogenic effects of drugs such as protamine zinc insulin and tolbutamide (Lichtenstein et al., 1951; Dawson, 1954).

2. NIEHS studies: Using the purest sample of 2,4,5-T, made available by Dow Chemical Co., teratogenic effects were induced in Swiss-Webster mice. Cleft palates were noted at dose levels of 150 mg./kg. and scattered abnormalities at 100 mg./kg.; the cleft palate incidence in control mice was essentially zero.

3. FDA studies: Hamsters were injected with five doses of 100 mg./kg./day of various batches of purified 2,4,5-T between days 6-10 of pregnancy. In one of these studies, there was a 66-percent incidence of mortality in 50 fetuses. Of the surviving fetuses, 17 percent had congenital abnormalities crooked tail, missing limb, and defect in skull fusion. No data was presented on possible effects induced by doses less than 100 mg./kg.

Of additional interest was a report also presented at the same conference on purified 2,4-D, which produced a 22-percent incidence of congenital abnormalities in hamsters at a dose level of 100 mg./kg./day.

I should like now to address myself to the toxicity of dioxins.

Toxicity of dioxins: Rabbit ear skin is highly sensitive to dioxins, repeated application of which can produce chloracne, as a cumulative manifestation of local toxicity. Approximately 0.3 micrograms of the tetra isomer will produce a positive response; "more than 10 micrograms on a surface wipe sample indicates acute hazard" (to man) (Silverstein, 1970).

The acute oral LD-50 dose of tetra dioxin in male guinea pigs is 0.5-1.0 micrograms/kg., and in male and female rats, 22.5 and 45 microgram/kg., respectively. Feeding chicken edema factor diets, containing dioxins, produced cumulative toxicity in monkeys (Allen and Carstein, 1967). Storage of hexa, hepta and octa isomers, as identified by GLC, has been reported in chickens and rats fed chicken edema factor diets (FDA, unpublished). Chronic administration of 2,4,5-T or 2,4-D to dogs produces cumulative toxicity with gastrointestinal haemorrhage, suggestive of cumulative dioxin effects (Drill and Hiratzka, 1953).

TERATOGENICITY OF DIOXINS

1. FDA studies (FDA Conference, Feb. 24, 1970): A mixture of dioxins, 21 percent trichloro and 53 percent tetrachloro isomers, were injected in hamsters between days 6-10 of pregnancy over a dose range from 0.5 to 9.1 microgram/kg. per day. At the highest dose, the incidence of fetal mortality was 82 percent and the incidence of congenital abnormalities, 82 percent. At the 0.5 microgram/kg. dose, there was a 5 percent incidence of abnormalities. The no-effect level was thus not reached at 0.5 micrograms per kg.

2. Dow studies (Sparschu et al., 1970): The tetra dioxin isomer was fed to Sprague Dawley rats between days 6-15 of pregnancy, over a dose range from 0.03 to 8.0 micrograms/kg. per day. There was a marked increase in resorption sites at the 2 microgram level. Gastro-intestinal hemorrhages occurred over a range from 0.125 to 8 micrograms, dose-dependently. Additionally, at the 0.125 microgram/kg. level there was a decrease in male fetal weights.

It should be emphasized that cystic kidneys were not seen at the 0.125 microgram/kg. dose of the tetra isomer or even higher levels. In the Bionetics study, 2,4,5-T at 4.6 mg./kg., containing 25 ppm of the tetra dioxin isomer equivalent to 0.124 microgram/kg., produced a 39 percent incidence of congenital abnormalities with cystic kid

neys.

There is thus a clear discrepancy between the teratogenic effects of 2,4,5-T containing 25 ppm of dioxin, and the effects of the equivalent concentration of the same dioxin. It is, however, conceivable that this discrepancy may reflect synergistic interactions between dioxin and 2,4,5-T.

SOME UNRESOLVED PROBLEMS RELATING TO 2,4,5-T AND DIOXINS

1. Chemical composition of 2,4,5-T formulations: Currently used 2,4,5-T formulations contain about 5 percent of known impurities, largely polychlorophenols. Analytic data on a sample of 2,4,5-T (Dow data, on production batch 120449) in the following table substantiates the approximate 5 percent of polychlorophenol impurities in 2,4,5-T formulations as currently used.

There are no available data on the presence and concentration of the more than 60 positional isomers of dioxin, other than the 2,3,7,8-tetrachloro dioxin isomer, in this batch of 2,4,5-T, or in other batches produced for food crop or other purposes in the United States and abroad.

In view of the relatively high concentration of polychlorophenol impurities in 2,4,5-T, it is likely that a wide range of dioxins are also present. 2,4-D and other phenoxy herbicides are similarly chemically uncharacterized.

The higher positional dioxin isomers, hexa, hepta, and octa, have been identified in 2,4-dichlorophenol, a precursor of 2,4-D. Apart from the presence of dioxins in polychlorophenols, heating of polychlorophenols will produce additional and very high yields of dioxin.

Illustratively, heating 5 g. of pentachlorophenol at 300° C. for 12 hours yielded 1.5 g. of the octa-dioxin isomer (Cowan, 1970). There are no available data on the possible production of dioxins from combustion of 2,4,5-T or 2,4-D. While improved production techniques may well reduce the levels of polychlorophenols and the levels of the 2,3,7,8-dioxin isomer, apart from other isomers, in 2,4,5-T and other phenoxy herbicides, the degree to which this is practical does not yet appear to have been clearly defined.

2. Stability and persistence of dioxins: The extent of usage of 2,4,5-T and other phenoxy herbicides on food crops and for other purposes in the United States and abroad dictates the scale of resulting environmental contamination with 2,3,7,8-dioxin and other

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isomers. The following data are illustrative and (Agricultural Economic Report No. 131, USDA, 1968).

These data reflect deliberate applications of phenoxy herbicides on crops, and do not reflect unintentional crop contamination following the more extensive application of herbicides for brush control or other purposes. There are no available data on the extent of such unintentional contamination. It is, however, well known that phenoxy herbicide dusts may drift for miles, even on nonwindy days, following routine application (Federal Register, 1969). The concentration of phenoxy herbicides in the air in Washington in 1964 reached a maximum of 3.4 microgram/m.3, with an average of 0.045 microgram/m3 (Bamesberger and Adams, 1966).

These figures probably underestimate the proportional concentration of atmospheric dioxins, in view of their high stability relative to phenoxy herbicides.

I now present a table on calculated dioxin contamination of an acre of soil, following 2,4,5-T application.

CALCULATED DIOXIN CONTAMINATION PER ACRE FOLLOWING 2,4,5-T APPLICATION

Now, these calculations are based on the 2,3,7,8-dioxin isomer alone, and ignore additional contamination due to other dioxin isomers. The figures for export use should be adjusted to reflect varying concentrations of 2,4,5-T in different formulations.

sources of

The high concentration of polychlorophenolic impurities in 2,4,5T, approximately 5 percent, apart from other polychlorophenols, may result in extremely high yields of dioxins. As mentioned previously, heating of 5 grams of pentachlorophenol at 300° C. for 12 hours results in a yield of 1.5 grams of the octadioxin isomer. Combustion of shrub, brush, timber, or other materials exposed to phenoxy herbicides or other polychlorophenols, may thus liberate high concentrations of dioxins in the atmosphere.

It is thus of interest to examine the data on stability and persistence of dioxins in the environment. The 2,3,7,8-tetra isomer is known to be heat stable up to 800° C. There are, however, no available data on the heat stability of other dioxin isomers. There are also no available data on the stability and persistence of the 2,3,7,8- and other dioxin isomers in soil, water, crops, milk, and animal or human tissues.

Most importantly, there are no available data on the possible accumulation and transmission of 2,3,7,8- and other dioxins in the food chain-air, soil, and water-to plants, brush and crops-to fish, birds and cattle to man, with attendant accumulation in man.

The heat stability of the tetra isomer, the general lipid solubility of the dioxins, and their cumulative toxicity in experimental ani