Reported seasonal dependence of herbicide developmental toxicity in mice.

In their paper published in the November 2002 issue of EHP, Cavieres et al. (2002) exposed pregnant mice to a commercial herbicide mixture and determined pregnancy outcomes. Separate experiments were conducted for each season of the year; the spring exposure occurred between gestation days (GD) 0 and 15, whereas in the other seasons, exposure was postimplantation (GDs 5-15). The authors concluded that, although apparently influenced by season, the results showed an inverted or U-shaped dose-response pattern for reduced litter size and reduced implantation sites. These decreases were reported to occur "only at very low environmentally relevant doses of the herbicide mixture."

These findings are of interest to us, but we are concerned about several inconsistencies in the reporting and also about the merging of different groups of data. As a result, it is not possible to reconstitute the original data for independent analysis. For example, the numbers of observations per group are different in Cavieres et al.'s Figure 1 and Table 2, and among Tables 2, 3, and 4. It is also unclear why Cavieres et al. (2002) reported a higher number of implantation sites than the number of litter size recordings (e.g., the summer high-dose group), especially when the authors stated in the text that implantations were only recorded for a subset of litters. The authors did not explain why they analyzed implantations on the basis of covariance with final litter size. The control litter-size data [Figure 1B (Cavieres et al. 2002)] has an unusual distribution, with the mode being the most frequent. The authors tentatively rationalized their findings in terms of the chemical treatment causing either preimplantation loss or fetal death. However, preimplantation exposure occurred only in the spring group, and no significant increases in resorptions were observed in any group. Thus, in the fall, winter, and summer groups, herbicide-induced preimplantation loss could not have occurred; therefore, the reported reduced implantations and reduced litter sizes in the absence of an increase in resorptions was an effect that simply could not be the result of herbicide exposure. The situation was further confused by the imperfect correlation between litter size and implantation sites. For example, in the very low-dose summer group, a significant (23%) reduction in litter size was associated with a significant (12%) reduction in implantation sites. However, in the high-dose summer group, the nonsignificant (7%) reduction in litter size was associated with a larger, but nonsignificant (15%) reduction in implantation sites.

[FIGURE 1 OMITTED]

Although the test data were tabulated according to season and a seasonal influence on test outcome was noted, the data were merged for all cases where implantation data existed [Figure 2 (Cavieres et al. 2002)], ignoring the individual seasonal data. These seasonal data are shown in Figure 1, in the format of the Cavieres et al.'s Figure 2 (Cavieres et al. 2002). Significant reductions in litter size are distributed across all the dose groups, with the fall data following a normal monotonic decrease in litter size (Figure 1). However, Cavieres et al. excluded these monotonic fall data from their Figure 2 because of the absence of implantation data. We suggest that a primary decision should be made regarding whether or not the data are seasonally related. If they are not, the data could be merged and analyzed as such. If they are, the merging of data (as presented by Cavieres et al. in their Figure 2) is invalid, and an explanation for the seasonal influence must be sought. In the extreme, such a seasonal influence would lead to an observed normal monotonic response in the fall and an inverted U response in the summer for the same chemical. The animals used in these experiments (Cavieres et al. 2002) were purchased and maintained for 2 weeks before the experiments began to allow them to adjust to the light-dark cycle and temperature of the animal rooms. Similar conditions would have applied in the commercial animal-breeding unit; therefore, the seasonal perceptions of the mice must have derived from the journey between the supplier and the laboratory--an unlikely proposition.

We conclude that substantial uncertainty exists regarding the origin of the litter size effects reported and that the conclusion of a low-dose inverted U-shaped dose-response curve cannot be made at this stage.

The authors declare a real, apparent, or potential conflict of interest as defined in EHP's Instructions to Authors.

REFERENCES

Cavieres MF, Jaeger J, Porter W. 2002. Developmental toxicity of a commercial herbicide mixture in mice: 1. Effects on embryo implantation and litter size. Environ Health Perspect 110:1081-1085.

Editor's note: In accordance with journal policy, Cavieres et al. were asked whether they wanted to respond to this letter, but they did not provide a response.

John Ashby

Ian Pate

Helen Tinwell

Syngenta Central Toxicology Laboratory

Alderley Park, Cheshire, United, Kingdom

E-mail: [e-mail]