Background 
Application A1239 seeks amendment to the Australia New Zealand Food Standards Code to permit the sale and use of food derived from genetically modified (GM) canola line LBFLFK. The canola line produces omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the seed and has tolerance to imidazolinone herbicides. 
 The production of EPA and DHA in LBFLFK is achieved by the introduction of ten genes encoding various desaturase and elongase enzymes involved in the fatty acid (FA) biosynthesis pathway. The genes were derived from microalgae, water moulds and moss. 
 Tolerance to imidazolinone herbicides is achieved through the expression of the acetohydroxy acid synthase (AHAS) gene from Arabidopsis thaliana. Imidazolinone tolerance was used for selection of putative transformants during the transformation stage as well as for selective post-emergence weed control during field production. The AHAS protein has been previously assessed by FSANZ. 
 This safety assessment addresses food safety and nutritional issues associated with the GM food. It does not address: 
• risks related to the environmental release of GM plants used in food production 
• risks to animals that may consume feed derived from GM plants 
• the safety of food derived from the non-GM (conventional) plant. 
 Further assessment of n-3 LC-PUFAs in canola line LBFLFK was undertaken as part of a nutrition risk assessment, and is described in Supporting Document 2 .

 History of use 
Canola is the second largest oilseed crop in the world behind soybean. It has a long history of safe use in the food supply. Canola oil is routinely used directly for cooking and as an ingredient in a variety of manufactured food products including salad and cooking oil, margarine, shortening and a range of prepared foods such as mayonnaise, sandwich spreads, creamers and coffee whiteners. Whole canola seeds are being used increasingly in products such as breads. Canola meal provides a good source of protein in stock feed for a variety of animals, primarily pigs, poultry and dairy cattle. 

 Molecular characterisation 
Canola line LBFLFK was generated through Agrobacterium-mediated transformation with a single T-DNA containing thirteen expression cassettes. Detailed molecular analyses indicate there are two insertion sites on different chromosomes and a single fully functional T-DNA exists at each insertion site. There are no extraneous plasmid sequences or antibiotic resistance genes present in this line. 
 The introduced genetic elements were shown by molecular techniques and phenotypic analyses to be stably inherited across multiple generations. The pattern of inheritance supports the conclusion that the introduced traits occur in two loci in the LBFLFK genome and are inherited according to Mendelian principles. 

 Characterisation and safety assessment of new substances 
The LBFLFK expresses eleven new proteins – 10 desaturase and elongase proteins as well as the AHAS protein. 
 Eight of the ten proteins in the FA biosynthesis pathway were only detected in seeds of LBFLFK. This is expected since the genes encoding the proteins all have seed-specific promoters. The levels of all eight proteins were low. The remaining two proteins were below the levels of detection and quantification in LBFLFK seeds. The newly expressed AHAS protein, driven by a constitutive promoter, was quantifiable in every tissue except mature seed. 
 Bioinformatic studies confirmed the lack of any significant amino acid sequence similarity to known protein toxins or allergens. Laboratory studies demonstrated the newly expressed proteins would be thoroughly degraded before being absorbed in the gastrointestinal tract. The proteins are also susceptible to heat denaturation and degradation at the high temperatures typically used in food processing. 

 Herbicide metabolites 
The spraying of LBFLFK with imidazolinone herbicides produces the same metabolites that arise when non-GM canola is sprayed with the same herbicide.

 Compositional analyses 
Detailed compositional analyses were undertaken on seed, crude oil and refined, bleached and deodorized (RBD) oil from LBFLFK. Seed samples were harvested for seed composition analysis from 12 field trials across two growing seasons: winter 2014/2015 and spring 2015. Seeds from a further five field trials conducted in 2016 were used for oil compositional analysis. Seed and oil samples were analysed for proximates, FAs, amino acids, minerals, vitamins, phytosterols, and anti-nutrients. The levels of each analyte in canola line LBFLFK were compared to levels in: a) the non-GM parental line, Kumily; b) six non-GM commercial reference lines grown at the same locations; and c) levels published in the literature. 
 In seeds, a total of 28 analytes (including individual FAs) differed significantly between LBFLFK and Kumily. In oil, 24 (crude oil) and 22 (RBD oil) individual analytes (including FAs) differed significantly between LBFLFK and Kumily. The changes in FA profile in both seed and oil were consistent with those expected as a result of the introduction of the EPA and DHA producing trait. 
 Apart from the intended change to the FA profile and a slightly higher content of trans fatty acids (TFAs) in seed and crude oil, canola line LBFLFK is otherwise compositionally equivalent to conventional canola varieties. 

 Conclusion 
No potential public health and safety concerns have been identified in the assessment of canola line LBFLFK. On the basis of the data provided in the present application, and other available information, food derived from LBFLFK is considered to be as safe for human consumption as food derived from conventional canola varieties.