<p style="text-align: justify;"><b>Dietary exposure</b></p>

<p style="text-align: justify;">The genetic modification in J101 and J163 will not result in changes in the use of alfalfa as an animal feed. Any cultivars resulting from crosses with either of events J101 or J163 are expected to be used in similar applications as conventional alfalfa cultivars, with the sole exception that these cultivars can only be cultivated as pure stands. In mixed grass-alfalfa stands, the grass portion would be eliminated by the use of glyphosate. The nutritional quality analysis of these alfalfa lines did not reveal any novel compositional characteristics compared to conventional cultivars, and the nutritional composition was within the normal range of variability observed in conventional alfalfa. Furthermore, due to the availability of many alfalfa cultivars adapted to different environmental and management conditions, the dietary exposure of livestock animals to forage from either J101 or J163 alfalfa cultivars is therefore expected to be similar to that of conventional alfalfa.</p>

<p style="text-align: justify;"><b>Nutritional and Compositional Data</b></p>

<p style="text-align: justify;">The nutritional composition of lines J101 and J163 was determined analytically and compared to that of a null segregant control population (from backcrosses with parental populations), and to twelve conventional alfalfa cultivars. Samples were obtained from second cut forage, harvested at early to late bloom, from five locations in the United States. Forage samples were analyzed for 35 nutritional components including proximates (crude protein, fat, ash, and moisture), acid detergent fibre, neutral detergent fibre, amino acids, and nine minerals, including calcium, magnesium, phosphorus and potassium. A 99% tolerance interval for the compositional constituents was developed using the twelve conventional alfalfa cultivars.</p>

<p style="text-align: justify;">Statistically significant differences were observed in the levels of cystine, histidine, lysine, tyrosine, acid detergent fibre, neutral detergent fibre, and lignin in either J101 or J163, compared to the control population. While some of these differences were observed within and among locations, all of the compositional values of J101 and J163 were within the tolerance interval established for each constituent, and were also comparable to published literature values.<br />
Coumestrol is an antinutritional estrogenic compound found naturally in alfalfa. Forage samples from trials conducted at four locations were analyzed for coumestrol. No significant differences in the levels of coumestrol were observed among lines J101, J163, the control line, and the commercial cultivars.</p>

<p style="text-align: justify;">Results from the nutritional and compositional analyses supported the conclusion that forage from events J101 and J163 is nutritionally comparable to that from currently available commercial cultivars of alfalfa.</p>

<p style="text-align: justify;"><b>Toxicity and Allergenicity</b></p>

<p style="text-align: justify;">The potential for toxicity and allergenicity of the alfalfa events J101 and J163 was assessed from data and information obtained from previous studies and investigations conducted in support of the safety of CP4 EPSPS, and the gene donor <i>A. tumefaciens</i> strain CP4. Results from these studies and investigations have demonstrated that CP4 EPSPS is functionally equivalent to native EPSPS, except for a reduced affinity for glyphosate; the <i>cp4 epsps</i> gene donor has a history of safe use; CP4 EPSPS is not homologous to known toxins and allergens; the protein it is rapidly digested in simulated gastric fluids; and that no treatment-related adverse effects were observed in acute toxicity tests where mice were fed up to 572 mg of CP4 EPSPS per kg of body weight. The no-observed adverse effects level of 572 mg/kg is approximately 20 times the amount of CP4 EPSPS to which a ruminant animal would be exposed when consuming a typical daily ration of alfalfa forage. The results of these studies and investigations, and data demonstrating the relatively low expression levels of CP4 EPSPS, led to the conclusion that the alfalfa events J101 and J163 are no more toxic or allergenic than conventional alfalfa cultivars.</p>
