**Appendix A**

The animals were allocated to different test groups by a manual randomization method. Individual body weights of allocated animals were within 20% of the group means, and the body weights of the animals were analysed statistically to rule out significant differences between the groups before administration of the HDM extract. Following allocation to the experimental groups, each animal was uniquely identified by turmeric fur marking/microtoe pad tattooing and colour-coded cage card labelled with project number, project type, test system, sex, dose, group, animal number, date of treatment, and experiment start and completion dates.

The mice were housed in groups of five animals per individually ventilated cages. Corn cob, prepared from pure corn and dried, freed of dust and sterilized, was procured from an approved vendor and used as bedding material. The bedding was replaced as often as necessary to keep the animals and their surroundings clean and dry. The room temperature was maintained between 21 ◦C to 22.5 ◦C and relative humidity at 50.9% to 66.8% for the duration of the study. Artificial light was set to give a cycle of 12 h light and 12 h dark. Adequately filtered air was provided with at least 12 changes per hour. The animals were offered a conventional laboratory rodent diet ad libitum supplied by Nutrivet Life Sciences, and Aquaguard filtered drinking water was provided ad libitum.

For the differential leukocyte counts, including eosinophil count, 80 μL of resuspended cell pellets were stained using the HEMA3 stain set (Fisher Scientific), and centrifuged using a StatSpin Cytofuge 2 (Beckman Coulter).

Ninety-six well plates coated with 5 μg HDM in 100 μL coating buffer and incubated for 12 h at 4 ◦C were blocked with 200 μL/well of assay diluent. Precleared serum samples (Protein G Sepharose beads) were washed with PBS, and a 50 μL aliquot of undiluted serum was added to each well and incubated at 4 ◦C overnight. The wells were washed with PBS; 100 μL of biotin-anti-mouse IgE (Sigma, Virginia Beach, VA, USA) was added and incubated for 1 h, followed by a 30 min incubation with avidin-horse radish peroxidase. TMB substrate solution (100 μL) was added to each well and incubated in the dark for 30 min. The reaction was stopped with 2 N sulphuric acid. Optical densities, normalized to saline controls, were read at 450 nm using a Varian Cary 50 spectrophotometer.
