Registration Dossier

Administrative data

Endpoint:
toxic effects on livestock and pets
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Exposure to BADGE in isolation was not performed and therefore causality of the effect to BADGE cannot be determined in this study.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2014

Materials and methods

Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Plastic bag samples
Details on test material:
Different batches of plastic bags for semen storage were bought from an international supplier and provided by the company that participated in this
study. Plastic bags were classified as ‘‘suspicious bags’’, those related to reproduction problems, and ‘‘control bags’’, those without any influence on reproductive results. For the characterization of the plastic bags, more than 500 suspicious bags were analyzed. They were new and empty and were recovered from 5 different farms (A, B, C, D and E), which reported different rates of reproductive failure.

Test animals

Species:
other: Pig
Sex:
male
Details on test animals and environmental conditions:
Semen samples were obtained from 5 boars of proven fertility that produced one heterospermic sample with appropriate sperm motility, morphology and concentration. Animals were currently in use at Cinse SL AI station (Zaragoza, Spain). Boars were held in individual pens with surface areas of 6 m2 and concrete slatted floor and sides that allowed physical and visual contact between animals, consistent with European Directive 91/630/CEE.
Boars were exposed to both, natural and artificial light for a total of 14 h/day. They were fed with 2.5–3 kg/day of standard feed, and they had access to fresh water ad libitum.

Administration / exposure

Route of exposure:
other: Semen stored in bags
Vehicle:
other: Water or long-term extender (VitasemH, Magapor SL)
Details on exposure:
Migration study of the plastic bags. Migration studies were performed on the suspicious bags. Bags were filled with 20 mL of Milli-Q water and also with 80 mL of a long-term extender (VitasemLD, Magapor SL) and placed in a horizontal position so that the liquid was in contact with the entire surface of the bags. Each experiment was performed in triplicate. Bags were kept in the oven at 38uC for 48 hours and then, the aqueous phase was analyzed by UPLC-MS(QTOF) and UPLC-MS(TQ).

Methodology for spiking semen with toxic compounds. Sperm from several boars was diluted in long-term extender (VitasemH, Magapor SL) and introduced into control bags; then half of the doses were spiked with the compounds previously identified and at the same concentration levels found in the migration tests (2.5 mg/L of cyclic lactone, 1.5 mg/L of cyclic phthalate, 0.4 mg/L BADGE, 0.4 mg/L of BADGE-H2O and 0.2 mg/L of BADGE-2H2O). Pure standards of these compounds were prepared in aqueous solutions, and several replicates were tested. The same replicates of seminal doses without toxic compounds were used as a control group.

Results and discussion

Details on results:
Chemical analysis of the suspicious bags identified unexpected compounds such as BADGE, a cyclic lactone and an unknown phthalate that leached into the semen at concentrations of 0.2 to 2.5 mg/L. Spermatozoa preserved in these bags passed all of the routine quality control tests, and no differences were observed between storage in the control and suspicious bags (p . 0.05). In vitro fecundation tests and endocrine profiler panel analysis (EPP) did not show any alterations, whereas the in vivo tests confirmed the described failure.

The migration data demonstrates the highest migration levels with cyclic lactone (~2.5 ug/ml) followed by the cyclic phthalate (~1.2 ug/ml). The BADGE species migrated at lower levels: BADGE (~0.4 ug/ml), BADGE-H2O (~0.3 ug/ml), and BADGE-2H2O (~0.2 ug/ml)

Applicant's summary and conclusion

Conclusions:
The migration data demonstrates the highest migration levels with cyclic lactone (~2.5 ug/ml) followed by the cyclic phthalate (~1.2 ug/ml). The BADGE species migrated at lower levels: BADGE (~0.4 ug/ml), BADGE-H2O (~0.3 ug/ml), and BADGE-2H2O (~0.2 ug/ml). In vitro fecundation tests and endocrine profiler panel analysis (EPP) did not show any alterations, whereas the in vivo tests confirmed the described failure. BADGE has not been demonstrated to have any effect on fertility or development in guideline studies including: one and two-generation reproductive toxicity studies and rat and rabbit developmental toxicity studies. The experiments conducted in this study cannot causally link BADGE with the effects because BADGE was not tested in isolation.