Assessment of genetically modified RF3 Canola Quality (CQ) Brassica juncea (application EFSA‐GMO‐NL‐2019‐158)

Abstract

Genetically modified RF3 Canola Quality (CQ) Brassica juncea was developed to confer tolerance to glufosinate‐based herbicides and to restore fertility in Barnase‐expressing plants. The RF3 event was originally developed in Brassica napus and has previously been assessed by EFSA in application EFSA‐GMO‐RX‐MS8‐RF3. The GMO Panel assessment of the molecular characterisation data and bioinformatics analyses did not raise issues that would require specific food and feed safety assessments. None of the identified differences in the agronomic, phenotypic and compositional characteristics tested between RF3 CQ B. juncea and its comparator need further assessment, except for yield, crude fat, aspartic acid and behenic acid (C22:0), which underwent additional evaluation and were found to not raise any environmental impact and safety or nutritional concerns. The GMO Panel does not identify safety concerns regarding the toxicity and allergenicity of the PAT/bar and Barstar proteins as expressed in RF3 CQ B. juncea and finds no evidence that the genetic modification would change the overall allergenicity of RF3 CQ B. juncea. In the context of this application, the consumption of food and feed from RF3 CQ B. juncea does not represent a nutritional concern in humans and animals. The GMO Panel concludes that RF3 CQ B. juncea is as safe as the comparator and CQ non‐GM B. juncea varieties tested, and no post‐market monitoring of food and feed is considered necessary. In the case of release of RF3 CQ B. juncea material, including viable seeds, into the environment, this would not raise environmental safety concerns. The post‐market environmental monitoring plan and reporting intervals are in line with the intended uses of RF3 CQ B. juncea. The GMO Panel concludes that RF3 CQ B. juncea is as safe as its comparator and the tested CQ non‐GM B. juncea varieties with respect to potential effects on human and animal health and the environment.

SUMMARY

Following the submission of application EFSA‐GMO‐NL‐2019‐158 under Regulation (EC) No 1829/2003 from BASF Agricultural Solutions US LCC (referred to hereafter as ‘the applicant’), the Panel on Genetically Modified Organisms of the European Food Safety Authority (referred to hereafter as the ‘GMO Panel’) was asked to deliver a Scientific Opinion on the safety of genetically modified (GM) herbicide tolerant RF3 Canola Quality (CQ) Brassica juncea according to Regulation (EU) No 503/2013. The scope of application EFSA‐GMO‐NL‐2019‐158 is for import, processing, and food and feed uses within the European Union (EU) of RF3 CQ B. juncea and does not include cultivation in the EU. RF3 CQ B. juncea, as defined by various sectorial associations, is characterised by defined low‐erucic acid and low‐glucosinolate levels in the oilseed.

In this scientific opinion, the GMO Panel reports on the outcome of its risk assessment of RF3 CQ B. juncea according to the scope of the application EFSA‐GMO‐NL‐2019‐158. The GMO Panel conducted the assessment of RF3 CQ B. juncea in line with the principles described in Regulation (EU) No 503/2013 and its applicable guidelines for the risk assessment of GM plants. The molecular characterisation data establish that RF3 CQ B. juncea contains a single insert consisting of one copy of each of the PAT/bar and Barstar expression cassettes. The quality of the sequencing methodology and datasets was assessed by the EFSA GMO Panel and complies with the requirements listed in the EFSA Technical Note. Bioinformatics analyses of the sequences encoding the newly expressed proteins (NEPs) and the sequences corresponding to open reading frames (ORFs) predicted within the introgressed event do not raise safety concerns. The stability of the introgressed insert DNA and of the introduced trait has been confirmed over several generations. The methodology used to quantify the levels of the PAT/bar and Barstar proteins is considered adequate. The protein characterisation data, comparing the biochemical, structural and functional properties of plant‐ and microbe‐produced PAT/bar and Barstar proteins, indicate that these proteins are equivalent and the microbial‐derived proteins can be used in the safety studies.

Considering the selection of test materials, the field trial sites and the associated management practices, as well as the agronomic‐phenotypic characterisation as an indicator of the overall field trial quality, the GMO Panel concludes that the field trials are appropriate to support the comparative analysis. None of the identified differences in the agronomic, phenotypic, or compositional characteristics tested between RF3 CQ B. juncea and its comparator needs further assessment, except for yield, crude fat, aspartic acid and behenic acid (C22:0), which underwent additional evaluation and were found to not raise any environmental impact and safety or nutritional concerns. The GMO Panel does not identify safety concerns regarding the toxicity and allergenicity of the PAT/bar and Barstar proteins as expressed in RF3 CQ B. juncea and finds no evidence that the genetic modification would change the overall allergenicity of RF3 CQ B. juncea. In the context of this application, the consumption of food and feed from RF3 CQ B. juncea does not represent a nutritional concern in humans or animals. The GMO Panel concludes that RF3 CQ B. juncea is as safe as the comparator and non‐GM B. juncea varieties tested, and no post‐market monitoring of food and feed is considered necessary.

Considering the introduced traits, the outcome of the agronomic and phenotypic analysis and the routes and levels of exposure, RF3 CQ B. juncea would not raise safety concerns in the case of release of GM B. juncea material, including viable seeds, into the environment. The post‐market environmental monitoring (PMEM) plan and reporting intervals are in line with the intended uses of RF3 CQ B. juncea.

The GMO Panel considered the overall quality of the performed literature searches acceptable. Based on the relevant publications identified through the literature searches, the GMO Panel does not identify any safety issues pertaining to the intended uses of RF3 CQ B. juncea.

The GMO Panel concludes that RF3 CQ B. juncea is as safe as its comparator and the tested non‐GM B. juncea reference varieties with respect to potential effects on human and animal health and the environment.

1. INTRODUCTION

The scope of application EFSA‐GMO‐NL‐2019‐158 is for food and feed uses, import and processing of RF3 Canola Quality (CQ) Brassica juncea and does not include cultivation in the European Union (EU). The RF3 event was originally developed in B. napus and has previously been assessed by EFSA in application EFSA‐GMO‐RX‐MS8‐RF3 (EFSA GMO Panel, 2009). B. napus RF3 was developed to confer tolerance to glufosinate‐based herbicides, as well as to express Barstar to restore fertility in Barnase expressing plants by neutralising the Barnase protein. The event RF3 was then transferred by conventional breeding from B. napus into B. juncea via a series of crosses and backcrosses with different B. juncea varieties.

B. juncea has several agronomic advantages over B. napus (Burton et al., 2004; Oram et al., 1999). Consequently, in the early 2000s, low‐erucic acid and low‐glucosinolate B. juncea varieties were released (Azhar et al., 2025). As defined by the applicant, the scope of this application is specific for RF3 CQ B. juncea. The name ‘Canola’ was adopted in late 1970s in Canada (OECD, 2015). Canola quality B. juncea, as defined by different sectorial associations is characterised by defined low‐erucic acid and low‐glucosinolate levels in the oilseed. ¹

1.1. Background and Terms of Reference as provided by the requestor

On 25 June 2019, the European Food Safety Authority (EFSA) received from the Competent Authority of the Netherlands application EFSA‐GMO‐NL‐2019‐158 for authorisation of RF3 CQ B. juncea (Unique Identifier ACS‐BNØØ3‐6), submitted by BASF Agricultural Solutions US LLC (hereafter referred to as ‘the applicant’) according to Regulation (EC) No 1829/2003. ² Following receipt of application EFSA‐GMO‐NL‐2019‐158, EFSA informed EU Member States (MS) and the European Commission (EC) and made the application available to them. Simultaneously, EFSA published a summary of the application. ³

EFSA checked the application for compliance with the relevant requirements of Regulation (EC) No 1829/2003 and Regulation (EU) No 503/2013, ⁴ with the EFSA guidance documents, and, when needed, asked the applicant to supplement the initial application. On 14 September 2020, EFSA declared the application valid.

From the validity date, EFSA and the GMO Panel of the European Food Safety Authority (referred to hereafter as ‘GMO Panel’) endeavoured to respect a time limit of 6 months to issue a scientific opinion on application EFSA‐GMO‐NL‐2019‐158. This time limit was extended whenever EFSA and/or GMO Panel requested supplementary information from the applicant. Consistent with Regulation (EC) No 1829/2003, any supplementary information provided by the applicant during the risk assessment was made available to the EU Member States and European Commission (for further details, see the Section 5). The EU Member States had 3 months to make their opinion known on application EFSA‐GMO‐NL‐2019‐158 as of the date of validity.

According to Articles 6 and 18 of Regulation (EC) No 1829/2003, EFSA and its GMO Panel were requested to carry out a scientific risk assessment of RF3 CQ B. juncea in the context of its scope as defined in application EFSA‐GMO‐NL‐2019‐158. According to Regulation (EC) No 1829/2003, this scientific opinion is to be seen as the report requested under Articles 6(6) and 18(6) of that Regulation and thus will be part of the EFSA overall opinion in accordance with Articles 6(5) and 18(5). In addition to the present scientific opinion, EFSA was also asked to report on the particulars listed under Articles 6(5) and 18(5) of Regulation (EC) No 1829/2003, but not to give an opinion on them because they pertain to risk management.

2. DATA AND METHODOLOGIES

2.1. Data

The GMO Panel based its scientific assessment of RF3 CQ B. juncea on the valid application EFSA‐GMO‐NL‐2019‐158, additional information provided by the applicant during the risk assessment, relevant scientific comments submitted by EU MS, and relevant peer‐reviewed scientific publications. As part of this comprehensive information package, the GMO Panel received an additional unpublished study submitted by the applicant to comply with the specific provisions of Regulation (EU) No 503/2013 (see Appendix A).

2.2. Methodologies

The GMO Panel conducted its assessment in line with the principles described in Regulation (EU) No 1829/2003, the applicable guidelines (i.e. EFSA GMO Panel, 2010a, 2011a, 2011b, 2015; EFSA Scientific Committee, 2011) and explanatory notes and statements (i.e. EFSA, 2010, 2014, 2017, 2018,2019a, 2019b; EFSA GMO Panel, 2010b, 2018) for the risk assessment of GM plants.

For this application, in the context of the contracts OC/EFSA/GMO/2018/04, OC/EFSA/AMU/2020/02 ‐SC 4 and OC/EFSA/GMO/2021/06, the contractors performed preparatory work for the evaluation of the applicant's literature search, the statistical analysis of field trial data and the bioinformatics analyses.

3. ASSESSMENT ⁵

3.1. Systematic literature review

The GMO Panel assessed the applicant's literature searches on RF3 CQ B. juncea, which included a scoping review, according to the guidelines given in EFSA (2010, 2019b). A systematic review as referred to in Regulation (EU) No 503/2013 has not been provided in support to the risk assessment of application EFSA‐GMO‐NL‐2019‐158. Based on the outcome of the scoping review, the GMO Panel agrees that there is limited value in undertaking a systematic review for RF3 CQ B. juncea at present.

The GMO Panel considered the overall quality of the performed literature searches acceptable. The literature searches identified two relevant publications on RF3 CQ B. juncea RF3. Based on the relevant publications identified through the literature searches (Appendix B), the GMO Panel does not identify any safety issues pertaining to the intended uses of RF3 CQ B. juncea.

3.2. Molecular characterisation ⁶

3.2.1. Production of the RF3 event and the introgression process

B. juncea RF3 was developed by conventional breeding, crossing a CQ B. juncea line with Brassica napus RF3. The RF3 CQ B. juncea was then obtained through a series of backcrosses. The RF3 transformation event for the parental RF3 B. napus line has already been characterised in application EFSA‐GMO‐RX‐MS8‐RF3 (EFSA GMO Panel, 2009).

The plasmid used to obtain the parental B. napus RF3 contained two expression cassettes within the T‐DNA: bar and barstar. The bar expression cassette contains the following genetic elements: the promoter region of the ribulose‐1,5‐biphosphate carboxylase small subunit gene from Arabidopsis thaliana (PssuAt), the coding sequence of the phosphinothricin acetyltransferase gene from Streptomyces hygroscopicus (bar), and the 3′ untranslated region of the TL‐DNA gene 7 from the A. tumefaciens octopine Ti plasmid (3′g7).The barstar expression cassette contains the following genetic elements: the promoter of the anther‐specific gene TA29 of Nicotiana tabacum, the coding sequence of the barstar gene from Bacillus amyloliquefaciens, and the 3′ untranslated region of the nopaline synthase gene from Agrobacterium tumefaciens (3′nos) TL‐DNA gene 7 from the A. tumefaciens octopine Ti plasmid (3′g7).

3.2.2. Transgene constructs in the GM plant

Molecular characterisation of RF3 CQ B. juncea was performed by Southern analysis, polymerase chain reaction (PCR) and DNA sequence analysis, in order to determine insert copy number and size and to verify that the organisation of the introgressed event was the same as in B. napus and to confirm the absence of plasmid backbone sequences. The approach used was acceptable both in terms of coverage and sensitivity. Overall, the quality of the sequencing methodology and datasets was assessed by the EFSA GMO Panel and complies with the requirements listed in the EFSA Technical Note (2018).

The Southern analyses confirmed that RF3 CQ B. juncea contains a single insert, which consists of a single copy of the T‐DNA in the same configuration as in the B. napus RF3. The insert and copy number were confirmed by multiple restriction enzyme/probe combinations covering the T‐DNA region and flanking regions. PCR and DNA sequences analyses confirmed the results obtained by the Southern analyses. The absence of vector backbone sequences was confirmed by Southern analysis using five backbone‐specific overlapping probes.

The nucleotide sequence of the entire insert of RF3 CQ B. juncea, together with 1 Kb of the 5′ and 1 Kb of the 3′ flanking regions, was determined. The sequenced fragment of 9635 bp (which includes the 5′ and 3′ flanking regions) is identical to the corresponding sequence of RF3 in B. napus. Since the event was introduced in B. juncea by introgression from B. napus, the flanking regions of the RF3 in B. juncea correspond to the event in B. napus. Therefore, the bioinformatics analyses of the pre‐insertion locus and of the genomic sequences flanking the insert, to investigate possible interruption of known endogenous genes by the insertion of event RF3 as previously conducted in B. napus, is not relevant in B. juncea. The results of segregation analysis (see Section 3.2.5) establish that the insert is in the nuclear genome.

Bioinformatics analyses of the amino acid sequences of the newly expressed PAT/bar and Barstar proteins revealed no significant similarities to known toxins and allergens. In addition, bioinformatics analyses of the newly created open reading frames (ORFs) within the introgressed sequence of the event did not indicate significant similarities to known toxins and allergens.

To assess the possibility for horizontal gene transfer (HGT) by homologous recombination (HR), the applicant performed a sequence identity analysis of the introgressed sequence of the event, which consists of two expression cassettes containing plant and bacteria‐derived regulatory sequences and bacteria‐derived coding DNA sequences, with microbial DNA. The likelihood and potential consequences of plant‐to‐bacteria transfer are described in Section 3.5.1.2.1.

3.2.3. Protein characterisation and equivalence

RF3 CQ B. juncea expresses two new proteins: Barstar protein for inhibition of the Barnase protein and phosphinothricin acetyltransferase (PAT/bar) protein for tolerance to glufosinate herbicides. The EFSA GMO Panel has previously assessed these proteins expressed in the same event in B. napus (EFSA GMO Panel, 2009). Given the technical constraints in producing large enough quantities from plants, these proteins were recombinantly produced in Escherichia coli. Prior to safety studies, a set of biochemical methods was employed to demonstrate the equivalence between the B. juncea and E. coli derived proteins. Purified proteins from these two sources were characterised and compared in terms of their biochemical, structural and functional properties.

• Barstar characterisation and equivalence

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and western blot (WB) analysis showed that both plant and E. coli‐produced Barstar proteins had the expected molecular weight of ~10 kDa. The antibodies specific to Barstar protein were comparably immunoreactive. Glycosylation detection analysis demonstrated that none of the Barstar proteins were glycosylated. N‐terminal sequencing of the plant‐derived Barstar protein showed that the protein matched the deduced sequence as defined by the barstar gene. The sequence analysis data were consistent with the previously analysed microbe‐produced Barstar (EFSA GMO Panel, 2020). Functional equivalence was demonstrated by an in vitro assay, which showed that plant and E. coli‐derived proteins had comparable activity.

• PAT/bar protein characterisation and equivalence

SDS–PAGE and WB analysis showed that both plant and E. coli‐produced PAT/bar proteins had the expected molecular weight of ~21 kDa. The antibodies specific to PAT/bar protein were comparably immunoreactive. Glycosylation detection analysis demonstrated that none of the PAT/bar proteins were glycosylated. Amino acid sequence analysis of the plant‐derived PAT/bar protein by mass spectrometry (MS) and N‐terminal sequencing methods showed that the protein matched the deduced sequence as defined by the pat gene. These data also showed that the N‐terminal methionine of the plant‐derived PAT/bar protein was acetylated. Such modifications are common in eukaryotic proteins (e.g. Polevoda & Sherman, 2000). Functional equivalence was demonstrated by an in vitro assay, which showed that plant and E. coli‐derived proteins had comparable activity for the intended herbicide.

The protein characterisation data comparing the biochemical, structural and functional properties of plant‐ and E.coli‐produced Barstar and PAT/bar proteins indicate that these proteins are equivalent, and the microbial derived proteins can be used in the safety studies.

3.2.4. Information on the expression of the insert

Protein levels of Barstar and PAT/bar were analysed by an enzyme‐linked immunosorbent assay (ELISA) in material harvested in a field trial across three locations in the USA and Canada during the 2017 growing season. Samples analysed included whole plant (BBCH stages 14–16, 30–39 and 57–65), root (BBCH stages 30–39 and 57–65), raceme (BBCH stage 57–65) and seed (BBCH stage 87–99), both treated and non‐treated with glufosinate‐ammonium. The mean values and standard deviations of protein expression levels in seed (n = 12), of the Barstar and PAT/bar proteins used to estimate human and animal dietary exposure (see Section 3.4.4) are reported in Table 1.

TABLE 1.

Mean values (n = 12) and standard deviation of newly expressed protein in seeds [μg/g dry weight (dw) and μg/g fresh weight (fw)] from RF3 CQ B. juncea.

Proteins	Glufosinate ammonium treatment
	Not treated		Treated
	μg/g dry weight (dw)	μg/g fresh weight (fw)	μg/g dry weight (dw)	μg/g fresh weight (fw)
PAT/bar	3.23 a  ± 2.68 b (1.30–8.20) c	3.03 ± 2.49 (1.22–7.61)	2.43 ± 1.67 (1.17–6.22)	2.30 ± 1.57 (1.12–5.84)
Barstar	ND d (< LLOQ e )	ND d (< LLOQ)	ND d (< LLOQ)	ND d (< LLOQ)

^a Mean value.

^b Standard deviation.

^c Range.

^d Not determined. Extraction efficiency not determined since Barstar values were below the limit of quantification (LOQ = 0.063 μg/g).

^e For dietary exposure estimations (see Section 3.4.4), the LOQ (0.063 μg/g) was used to estimate the mean values when samples were reported as below LOQ.

3.2.5. Inheritance and stability of introgressed insert DNA

Genetic stability of the RF3 CQ B. juncea introgressed insert was assessed by Southern analysis of genomic DNA from five generations (F1, BC1, BC2, BC3, BC3S2). The restriction enzyme and probe combinations used were sufficient to conclude that all the plants tested retained a single copy of the insert and flanking regions, which was stably inherited in subsequent generations.

Phenotypic stability was assessed by means of lateral flow strip analysis. The expression of the PAT/bar protein was confirmed in all tested plants. The inheritance pattern was investigated by an RF3 event‐specific PCR method. The results support the presence of a single insertion, segregating in a Mendelian fashion.

3.2.6. Conclusions on molecular characterisation

B. juncea CQ RF3 was developed by conventional breeding, starting by a cross of a CQ B. juncea line with RF3 B. napus followed by a series of backcrosses with a CQ B. juncea line. The RF3 transformation event for the parental B. napus RF3 line has been already characterised in application EFSA‐GMO‐RX‐MS8‐RF3 (EFSA GMO Panel, 2009). The stability of the introgressed event and of the introduced herbicide tolerance trait has been confirmed over several generations. Bioinformatics analyses of the sequences encoding the newly expressed proteins and the sequences corresponding to ORFs predicted within the introgressed sequence of the event do not raise any safety concerns.

The methodology used to quantify the levels of the Barstar and PAT/bar is considered adequate.

The protein characterisation data comparing the structural, biochemical and functional properties of plant and microbe‐produced Barstar and PAT/bar proteins indicate that these proteins are equivalent and the microbial derived proteins can be used in the safety studies.

3.3. Comparative analysis ⁷

3.3.1. Overview of studies conducted for the comparative analysis

Application EFSA‐GMO‐NL‐2019‐158 presents data on agronomic and phenotypic characteristics, as well as on seed composition of RF3 CQ B. juncea (Table 2).

TABLE 2.

Overview of the comparative analysis studies to characterise RF3 CQ B. juncea provided in application EFSA‐GMO‐NL‐2019‐158.

Study focus	Study details	Comparator	Non‐GM reference varieties
Agronomic and phenotypic analysis	Field study, US and Canada, 2023, 11 SITES a	10CJ28‐094	8 b
Compositional analysis	Field study, US and Canada, 2023, 8 SITES a

Abbreviation: GM, genetically modified.

^a Eight field trials were used for both the compositional and the agronomic/phenotypic analysis: five in Canada (two field trials in Manitoba, two in Saskatchewan and one in Ontario) and three in the United States (in Idaho, North Dakota and South Dakota). Three field trials were used only for the agronomic/phenotypic analysis: one in Canada (Manitoba) and two in the United States (in Washington and Minnesota). An additional site was excluded from the statistical analysis due to poor emergence.

^b Non‐GM B. juncea varieties were 45J10, Dahinda, Arid, VR07, 8571, Amulet, Davin and Oasis CL.

3.3.2. Experimental field trial design and statistical analysis

At each field trial site, the following materials were grown in a randomised complete block design with four replicates: RF3 CQ B. juncea not exposed to the intended herbicide (not treated), RF3 CQ B. juncea exposed to the intended herbicide (treated), the comparator 10CJ28‐094, and three non‐GM reference varieties.

The agronomic, phenotypic and compositional data were analysed as specified by EFSA GMO Panel (2010b, 2011a). These include, for each of the two treatments of RF3 CQ B. juncea, the application of a difference test (between the GM B. juncea and the non‐GM comparator) and an equivalence test (between the GM B. juncea and the set of non‐GM commercial reference varieties). The results of the equivalence test are categorised into four possible outcomes (I‐IV, ranging from equivalence to non‐equivalence). ⁸

3.3.3. Suitability of selected test materials

3.3.3.1. Selection of the test materials

Event RF3 was originally developed in B. napus and then by conventional breeding transferred into CQ B. juncea using the CQ non‐GM variety 10CJ28‐094 as the recurrent parent (as documented by the pedigree). B. juncea 10CJ28‐094 was used in the field trials and is considered to be an appropriate comparator.

RF3 CQ B. juncea and the comparator were considered appropriate for growing in environments across North America, where the comparative field trials were conducted. Eight commercial non‐GM reference varieties were selected by the applicant (see Table 2) and, at each selected site, three reference varieties were tested. On the basis of the information provided, the GMO Panel considers the selected non‐GM reference varieties appropriate for the comparative assessment.

3.3.3.2. Seed production and quality

Seeds of RF3 CQ B. juncea and the comparator used in the 2023 field trials were produced, harvested and stored under similar conditions, before being sown in the field trial sites. The seed lots were verified for their identity via event specific quantitative polymerase chain reaction analysis.

The seeds were tested for their germination capacity under warm conditions. ⁹ The germination capacity of the RF3 CQ B. juncea was compared with that of its comparator using the AOSA/ISTA standards and the results ¹⁰ of these studies indicate that the seed germination of RF3 CQ B. juncea and the comparator were similar.

3.3.3.3. Conclusion on suitability

The GMO Panel is of the opinion that the RF3 CQ B. juncea, the comparator and the non‐GM B. juncea reference varieties were properly selected and are of adequate quality. Therefore, the test materials are considered suitable for the comparative analysis.

3.3.4. Representativeness of the receiving environments

3.3.4.1. Selection of field trial sites

The selected field trial sites were located in commercial B. juncea cultivation regions of Canada and the United States of America. The soil and climatic characteristics of the selected fields ¹¹ represent regions of diverse environmental conditions for B. juncea growth. The GMO Panel considers that the selected sites, including the subset chosen for the compositional analysis, reflect commercial B. juncea regions in which the test materials are likely to be grown.

3.3.4.2. Meteorological conditions

Maximum and minimum mean temperatures and sums of precipitation were provided for each site on a weekly basis. Some exceptional weather conditions were reported at two of the selected sites. ¹² However, due to the lack of major impacts on plant growth at these sites, the GMO Panel considers that the exceptional weather conditions did not invalidate the selection of the field trial sites for the comparative analysis.

3.3.4.3. Management practices

The field trial sites included plots containing RF3 CQ B. juncea, plots with the comparator and plots with non‐GM B. juncea reference varieties, managed according to local agricultural practices. In addition, the field trial included plots containing RF3 CQ B. juncea that were managed following the same agricultural practices and also exposed to the intended glufosinate‐ammonium‐based herbicide, applied at the BBCH 12–14 growth stage. ¹³ The GMO Panel considers that the management practices, including sowing, harvesting, and application of plant protection products, were appropriate for the selected receiving environments. Practices, including sowing, harvesting and application of plant protection products were acceptable for the selected receiving environments.

3.3.4.4. Conclusion on representativeness

The GMO Panel concludes that the geographical locations, soil and climatic characteristics, meteorological conditions and management practices of the field trial sites are typical for receiving environments where the tested materials could be grown.

3.3.5. Agronomic and phenotypic analysis

Thirteen agronomic and phenotypic endpoints ¹⁴ plus information on abiotic stressors, disease incidence and arthropod damage were collected from the field trials (see Table 2). The endpoint lodging was not analysed with formal statistical methods because of lack of variability in the data.

The statistical analysis (Section 3.3.2) was applied to 12 endpoints, with the following results:

• For RF3 CQ B. juncea (not treated), the test of difference identified statistically significant differences with the comparator for eight endpoints (early stand count, crop development, days to flowering, plant height, days to maturity, fruit count, seed loss and seed moisture). All these endpoints fell into equivalence category I.

• For RF3 CQ B. juncea (treated), the test of difference identified statistically significant differences with the comparator for seven endpoints (early stand count, crop development, days to flowering, final stand count, days to maturity, yield and seed moisture.). All these endpoints fell under equivalence category I except for yield, which fell into equivalence category III. ¹⁵

For RF3 CQ B. juncea (treated with the intended herbicide) an increase in yield was observed with respect to its comparator and the non‐GM reference varieties. As no similar results were observed for the other yield components, the GMO Panel considers that this result does not affect the use of the field trials for the comparative analysis. Whether the difference can lead to a potential environmental impact is considered in Section 3.5.1.1.

3.3.6. Compositional analysis

RF3 CQ B. juncea harvested from eight sites (Table 2) were analysed for 94 constituents, including those recommended by OECD (2012). The statistical analysis was not applied to 39 constituents because their concentration in at least one third of the samples were below the limit of quantification. ¹⁶

The statistical analysis was applied to a total of 55 constituents ¹⁷ ; a summary of the outcome of the test of difference and the test of equivalence is presented in Table 3:

• For RF3 CQ B. juncea not treated with the intended herbicide, statistically significant differences with the comparator were found for 34 endpoints. All these endpoints fell under equivalence category I or II, except for crude fat and aspartic acid which fell under equivalence category III and behenic acid (C22:0) which fell under equivalence category IV. Among the endpoints for which no significant difference was observed, total tocopherols fell under equivalence category III.

• For RF3 CQ B. juncea treated with the intended herbicide, statistically significant differences with the comparator were found for 34 endpoints. All these endpoints fell under equivalence category I or II, except for crude fat and aspartic acid which fell under equivalence category III and behenic acid (C22:0) which fell under equivalence category IV. Among the endpoints for which no significant difference was observed, total tocopherols fell under equivalence category III.

TABLE 3.

Outcome of the comparative compositional analysis in seeds of RF3 CQ B. juncea. The table shows the number of endpoints in each category.

		Test of difference a
		Treated c		Not‐treated c
		Not different	Significantly different	Not different	Significantly different
Test of equivalence b	Category I/II	20	31 d	20	31 d
	Category III/IV	1 e	3 f	1 e	3 f
	Not categorised	–	–	–	–
	Total endpoints	55		55

^a Comparison between RF3 CQ B. juncea and its comparator.

^b Four different outcomes: category I (indicating full equivalence to the non‐GM reference varieties); category II (equivalence is more likely than non‐equivalence); category III (non‐equivalence is more likely than equivalence); and category IV (indicating non‐equivalence). Not categorised means that the test of equivalence was not applied because of the lack of variation among the non‐GM reference varieties.

^c Treated/not treated with the intended herbicides.

^d Endpoints with significant differences between RF3 CQ B. juncea and its conventional counterpart falling in equivalence category I‐II (treated and not treated). Both treated and not treated: crude protein, calcium, magnesium, phosphorous, zinc, potassium, alanine, arginine, cystine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, a‐tocopherol, palmitic acid (C16:0), palmitoleic acid (C16:1), arachidic acid (C20:0), eicosenoic acid (C20:1), progoitrin; not treated only: eicosadienoic acid (C20:2), 4‐hydroxyglucobrassicin; treated only: linolenic acid (C18:3), total glucosinolates.

^e Endpoints in seeds and forage with no significant differences between RF3 CQ B. juncea and its comparator and falling under equivalence category III: total tocopherols (both treated and not treated).

^f Endpoints with significant differences between the RF3 CQ B. juncea and its comparator and falling in equivalence category III‐IV. Estimated means are reported for these endpoints in Table 4.

The GMO Panel assessed all significant differences between RF3 CQ B. juncea and its comparator, considering potential impact on plant metabolism and the natural variability observed for the set of non‐GM reference varieties. Quantitative results for the endpoints showing significant differences between RF3 CQ B. juncea and its comparator and falling under category III/IV are given in Table 4.

TABLE 4.

Quantitative results (estimated means and equivalence limits) for compositional endpoints in RF3 CQ B. juncea seeds that are further assessed based on the results of the statistical analysis.

Endpoint	RF3 CQ B. juncea a		Comparator 10CJ28‐094	Non‐GM reference varieties
	Not treated	Treated		Mean	Equivalence limits
Crude fat (% dw)	40.6*	40.6*	42.1	37.3	34.6–40.1
Aspartic acid (% dw)	1.88*	1.90*	1.81	2.18	1.93–2.46
Behenic acid (C22:0) (% FA)	0.235*	0.235*	0.225	0.320	0.257–0.393

^a For the RF3 CQ B. juncea, significantly different values are marked with an asterisk, while the outcomes of the test of equivalence are differentiated by greyscale backgrounds: white (equivalence category I or II), light grey (equivalence category III) and dark grey (equivalence category IV).

dw: dry weight; % FA: percentage total fatty acids. Treated: treated with the intended herbicide; not treated: treated only with conventional herbicides (see Section 3.3.4.3).

Means and equivalence limits were calculated on a log‐transformed scale; the values shown in the table are back‐transformed to the original scale.

3.3.7. Conclusion on comparative analysis

Considering the selection of test materials, the field trial sites and the associated management practices and the agronomic‐phenotypic characterisation as an indicator of the overall field trial quality, the GMO Panel concludes that the field trials are appropriate to support the comparative analysis.

Taking into account the natural variability observed for the set of non‐GM reference varieties, the GMO Panel concludes that:

• None of the differences identified in agronomic and phenotypic characteristics tested between RF3 CQ B. juncea and the comparator needs further assessment regarding their potential environmental impact except for yield, which is further assessed in Section 3.5.1.1.

• None of the differences identified in seed composition between the RF3 CQ B. juncea and the comparator needs further assessment regarding food and feed safety except for crude fat, aspartic acid and behenic acid (C22:0), which are further assessed in Section 3.4.5.

3.4. Food and feed safety assessment ¹⁸

3.4.1. Overview of overarching information for food and feed assessment

3.4.1.1. Compositional analysis

The compositional analysis of RF3 CQ B. juncea and the comparator provided by the applicant and assessed by the GMO Panel is described in Sections 3.3.6 and 3.3.7.

3.4.1.2. Newly expressed proteins

Two proteins, PAT/bar and Barstar, are present as NEPs in RF3 CQ B. juncea. The two proteins were previously assessed by the GMO Panel (EFSA GMO Panel, 2020) and no safety concerns were identified. The GMO Panel is not aware of any new information that would change previous conclusions on the safety of the PAT/bar and Barstar proteins.

The barstar cassette includes a tapetum‐specific promoter (Pta29), which restricts expression of the transcript for the barstar ribonuclease inhibitor to tapetum cells during another development. As a result, the Barstar protein is not translated in plant tissues outside the flower buds. Thus, the applicant considers the presence of barstar in food and feed materials such as seeds to be negligible, however without clearly excluding its presence (LOQ was considered in the exposure assessment see Section 3.4.4).

In the context of this assessment the weight‐of‐evidence approach followed in the context of EFSA GMO Panel (2020) remains valid.

Molecular characterisation

The characterisation of the PAT/bar and Barstar proteins provided by the applicant and assessed by the GMO Panel is described in Section 3.2.3. Furthermore, the equivalence between the RF3 CQ B. juncea and the Escherichia coli‐derived proteins was demonstrated.

Synergistic and antagonistic interactions among NEPs

Based on current scientific knowledge on the biological function of the two proteins (Table 5), no synergistic or antagonistic interactions between these two proteins are expected which could raise safety concerns for food and feed from RF3 CQ B. juncea.

TABLE 5.

Intended effects and modes of action of NEPs in RF3 CQ B. juncea.

Protein	Intended effect and mode of action in GM plant
PAT/bar	The PAT/bar protein confers tolerance to glufosinate‐based herbicides by acetylating L‐phosphinothricin, the active isomer of the glufosinate‐based herbicide
Barstar	Barstar is a highly specific inhibitor of the Barnase protein, forming a tight complex that neutralises Barnase activity, with no evidence of additional functions of relevance to food and feed safety

3.4.1.3. Effects on processing

RF3 CQ B. juncea as described in this application will undergo existing production processes used for conventional oilseed rape. No novel production process is envisaged. Based on the outcome of the comparative assessment, processing of the GM RF3 CQ B. juncea into food and feed products is not expected to result in products being different from those of conventional non‐GM oilseed rape varieties currently in the EU market.

3.4.2. Toxicology assessment

The strategies to assess the toxicological impact of any changes on the whole genetically modified food and feed resulting from the genetic modification focus on the assessment of (i) NEPs; (ii) new constituents other than NEPs; (iii) altered levels of food and feed constituents; and (iv) the whole genetically modified food and feed.

3.4.2.1. Assessment of NEPs

Updated bioinformatics analyses revealed no similarities of the Barstar and PAT/bar proteins with known toxins (Section 3.2.2), further supporting the previous GMO Panel assessments (see Section 3.4.1.2). The GMO Panel concludes that the newly expressed Barstar and PAT/bar proteins in RF3 CQ B. juncea do not pose toxicological concerns.

3.4.2.2. Assessment of new constituents other than NEPs

Based on the outcome of the studies considered in the comparative analysis and molecular characterisation, no new constituents other than NEPs have been identified in seed from RF3 CQ B. juncea. Therefore, no further food and feed safety assessment of components other than NEPs is required.

3.4.2.3. Information on altered levels of food and feed constituents

Based on the outcome of the studies considered in the comparative analysis and molecular characterisation, no altered levels of food and feed constituents have been identified in seed from RF3 CQ B. juncea, except for crude fat, aspartic acid and behenic acid (C22:0). These changes are considered not to represent a toxicological concern, considering the biological role of the affected constituent and the magnitude of the changes, therefore, no further toxicological assessment is needed. Further information on the relevance of these findings is provided in Sections 3.4.5.1 and 3.4.5.2.

3.4.2.4. Assessment of the whole genetically modified food and feed

The GMO Panel considers animal studies with food/feed derived from the RF3 CQ B. juncea unnecessary based on the outcome of molecular characterisation, comparative analysis and toxicological assessment (EFSA GMO Panel, 2011a). There were no concerns regarding the stability and expression of the insert or unintended effects relevant to food and feed safety, and no changes of toxicological concern in the composition of the RF3 CQ B. juncea were identified (Sections 3.2, 3.3.6 and 3.4.2).

In accordance with Regulation (EU) No 503/2013, the applicant provided a 90‐day feeding study in rats fed with diets containing oilseed rape meal (toasted and defatted). In the context of this application, the applicant referred to a 90‐day study with RF3 B. napus, submitted previously under application EFSA‐GMO‐RX024, and considered this study adequate for the assessment of food and feed containing RF3 CQ B. juncea.

The GMO Panel concludes that the 90‐day study with RF3 B. napus submitted as part of application EFSA‐GMO‐RX‐024 (EFSA GMO Panel, 2023a) is adequate for the assessment of food and feed containing RF3 CQ B. juncea event on the basis of the following rationale ¹⁹ :

1. RF3 CQ B. juncea was developed by conventional breeding, crossing a B. juncea line with RF3 B. napus. Molecular analysis confirms that the RF3 event in B. napus and in B. juncea is identical;

2. An ad hoc comparative compositional analysis between seeds of RF3 B. napus and RF3 CQ B. juncea indicated that the two species are compositionally similar, except for the neutral detergent fibre (NDF) and acid detergent fibre (ADF) contents. The GMO Panel considered that the observed differences in NDF and ADF contents between RF3 B. napus and RF3 CQ B. juncea seeds did not preclude the use of the study with RF3 B. napus to assess RF3 CQ B. juncea. In this regard, the Panel noted that the diets used in the 90‐day study were formulated and analysed to ensure comparable total dietary fibre and an overall balanced nutrient composition across control and test groups. Dietary fibre has a limited nutritional relevance in rats, as it is not an essential nutrient and moderate variations in fibre type or level do not adversely affect growth, feed intake, or nutrient utilisation. Therefore, EFSA concluded that the variation in ADF and NDF levels would not influence the outcome or interpretation of the 90‐day feeding study, for the scope of this application.

The GMO Panel had previously concluded that the 90‐day feeding study in rodents on whole food/feed from RF3 B. napus is in line with Regulation (EU) No 503/2013 and does not show adverse effects related to diets incorporating the respective single‐events (EFSA GMO Panel, 2023a).

3.4.3. Allergenicity

The strategies to assess the potential risk of allergenicity focus: (i) on the source of the recombinant protein; (ii) on the potential of NEPs to induce sensitisation or to elicit allergic reactions in already sensitised persons; and (iii) on whether the transformation may have altered the allergenic properties of the modified plant. Furthermore, the assessment also takes into account potential adjuvant properties of NEPs, which is defined as the ability to enhance an allergic reaction.

3.4.3.1. Assessment of allergenicity of NEPs

A weight‐of‐evidence approach was followed, taking into account all of the information obtained on the NEP, as no single piece of information or experimental method yielded sufficient evidence to predict allergenicity (Codex Alimentarius, 2009; EFSA GMO Panel, 2011a, 2017a; Regulation (EU)No 503/2013).

The barstar and pat genes originate from B. amyloliquefaciens and S. hygroscopicus, respectively, which are not considered allergenic sources. The Barstar and PAT/bar proteins were previously assessed by the GMO Panel in the context of other applications (EFSA GMO Panel, 2020) and no safety concerns for humans and animals were identified. The GMO Panel is not aware of any new information that would change previous conclusions on the safety of the Barstar and PAT/bar proteins. Updated bioinformatics analyses revealed no similarities of the Barstar and PAT/bar proteins with known allergens (Section 3.2.2).

The applicant also provided information on the safety of the Barstar and PAT/bar proteins regarding their potential to cause a celiac disease response. For such assessment, the applicant followed the principles described in the EFSA GMO Panel guidance document (2017a). No indications of safety concern were identified by the GMO Panel as the relevant peptides containing the motif fail to mimic gluten sequences.

The GMO Panel considers that there is no indication that the newly expressed Barstar and PAT/bar proteins in RF3 CQ B. juncea may be allergenic.

3.4.3.2. Assessment of allergenicity of the whole GM plant or crop

Mustard, derived from B. juncea, is recognised as a common allergenic food. ²⁰ The scope of this application is limited to RF3 CQ B. juncea, a variety in which mustard‐quality traits have been removed through conventional breeding. ²¹ It is noted that RF3 CQ B. juncea assessed in this application does not meet the specifications required for mustard production and is handled with canola supply chains, which differ from those used for mustard‐processing pathways. Moreover, the molecular characterisation and comparative assessment did not identify any unintended effects. The intended modification was specifically designed to confer tolerance to glufosinate‐based herbicides and inhibition to the Barnase protein. Based on all the evidence, the GMO Panel identified no indications of a potentially increased allergenicity of food and feed derived from RF3 CQ B. juncea with respect to that derived from the comparator.

3.4.4. Dietary exposure assessment to new constituents

In line with Regulation (EU) No 503/2013, the applicant provided dietary exposure estimates to PAT/bar and Barstar proteins newly expressed in RF3 CQ B. juncea. Dietary exposure was estimated based on protein expression levels reported in this application for RF3 CQ B. juncea treated with the intended herbicide, the current available consumption data and feed practices, the foods and feeds currently available in the market and the described processing conditions. For the purpose of estimating dietary exposure, the levels of PAT/bar and Barstar proteins in RF3 CQ B. juncea seeds were derived from a replicated field trial (12 analyses from three locations) in 2017 in USA and Canada. Table 1 in Section 3.2.4 shows the protein expression levels used to estimate both human and animal dietary exposure. ²²

3.4.4.1. Human dietary exposure

Currently, oil is almost the only food commodity derived from oilseed rape regularly consumed by the European population, although other food commodities derived from oilseed rape are approved as novel food, i.e. protein isolates (EFSA NDA Panel, 2013) and oilseed rape powder (EFSA NDA Panel, 2020). In fact, various preparations of protein isolates from oilseed rape meal are found under commercial names. However, as today, both protein isolates and oilseed rape powder are virtually absent in the European market as verified in Mintel's Global New Products Database. ²³ This was further confirmed by the absence of consumption data for protein isolates/meat imitates from oilseed rape in the EFSA Comprehensive European Food Consumption Database (EFSA consumption database). ²⁴ Still, following an overly conservative approach, the applicant provided estimates of the dietary exposure to PAT/bar and Barstar proteins, making use of the available consumption data on meat analogues and protein supplements in the EFSA consumption database assuming they are derived from RF3 CQ B. juncea. ²⁵

Human dietary exposure was estimated across various European countries on different population groups: young population (infants, toddlers, ‘other children’), adolescents, adult population (adults, elderly, and very elderly) and special populations (pregnant and lactating women).

Mean protein expression values on a fresh weight basis are considered as the most adequate to estimate human dietary exposure (both acute and chronic) when working with raw primary commodities that are commonly consumed as processed blended commodities (EFSA, 2019a). Different recipes and factors were considered to estimate the amount of seeds in the consumed commodities before assigning newly expressed protein levels to the relevant commodities. ²⁶ No losses in the newly expressed proteins during processing were considered.

The highest anticipated chronic exposure was identified in high consumers of ‘Protein and protein components for sports people’ with estimates of 1.0 and 37.8 μg/kg bw per day for Barstar and PAT/bar, respectively. As regards acute exposure, the highest estimates were also identified in high consumers of ‘Protein and protein components for sports people’, with 1.6 and 58.1 μg/kg bw per day for Barstar and PAT/bar, respectively.

Additional dietary exposure to PAT/bar and Barstar proteins might occur via the consumption of pollen supplements under the assumption that these supplements contain pollen from RF3 CQ B. juncea. Consumption data on pollen supplements are available for few consumers across seven different European countries.²⁴ However, since no data on the presence of newly expressed proteins in pollen were provided, the potential dietary exposure to PAT/bar and Barstar proteins from the consumption of pollen supplements could not be estimated.

3.4.4.2. Animal dietary exposure

Anticipated dietary exposure to PAT/bar and Barstar proteins in RF3 CQ B. juncea was estimated by the applicant across farmed and companion animals, assuming the consumption of oilseed rape products commonly entering the feed supply chain, as below described. Estimations were based on the recommendations provided in EFSA GMO Panel (2023b). A conservative scenario with 100% replacement of conventional oilseed rape products by RF3 CQ B. juncea products was considered.

Mean levels (dry weight) of the newly expressed proteins in full‐fat seeds from RF3 CQ B. juncea treated with the intended herbicide used for animal dietary exposure are listed in Table 1. Mean levels (dry weight) of the newly expressed proteins in meal were calculated to be 1.6‐fold higher than in seeds, based on conversion factors that take into account the protein content in these feed materials relative to oilseed rape seeds, and assuming that no protein is lost during their production or processing. Values are reported in tables of Appendix C (Tables C.1, C.2, C.3, C.4). All seed samples analysed in RF3 CQ B. juncea for the presence of Barstar protein were below the limit of quantification (LOQ = 0.063 μg/g dry weight). For the purpose of estimating daily dietary intake (DDE), the determined values of LOQ were used as substitutes to compute an average concentration of Barstar in seed to be used for the exposure calculations. ²⁷

The applicant estimated dietary exposure in ruminants (dairy cow, beef cattle, dairy sheep and dairy goat) and other herbivorous animals (rabbit and horse), in pigs (fattening pig, lactating sow, piglet), in poultry (broiler, laying hens, turkey), in fish (salmon, carp) and in companion animals (cat and dog). The dietary exposure was estimated via the consumption of full‐fat seeds and meal in standard diets or rations. Estimated dietary exposure to PAT/bar and Barstar proteins in the concerned animals is reported in Appendix C.

3.4.5. Nutritional assessment of endogenous constituents

The comparative compositional analysis revealed differences in seed composition between RF3 CQ B. juncea and its conventional counterpart that needs further assessment regarding food and feed safety. This refers in particular to the levels of crude fat, aspartic acid, and behenic acid (C22:0) (see Table 4). The biological relevance of these components, the role of oilseed rape as contributor to their total intake and the magnitude and direction of the observed changes were considered during the nutritional assessment.

3.4.5.1. Human nutrition

The levels of crude fat in seeds were ~4% lower in treated and not treated RF3 CQ B. juncea than in its comparator. The EFSA NDA Panel has not defined a lower threshold intake (LTI) for total fat, but proposed a Reference Intake range of 20%–35% of total dietary energy (E%) (EFSA NDA Panel, 2010). The main commodity for human consumption is rapeseed oil, which is obtained by mechanical pressing or solvent extraction from oilseed rape. The decrease in crude fat in RF3 CQ B. juncea will only impact the yield of oil production, and therefore, this change does not raise any nutritional concern.

Behenic acid (C22:0) is a saturated fatty acid (FA) present at very low levels in CQ B. juncea (< 0.3% of total FA). Taking into account the relevance of this FA in the total FA content of rapeseed, the increase of ~4% relative to the comparator is not considered nutritionally relevant.

Aspartic acid is a dispensable amino acid, as it can be synthesised in the human body at the rate needed to meet its metabolic requirement (EFSA NDA Panel, 2012). The increase of ~5% observed in treated and not treated B. juncea RF3 as compared to the conventional counterpart poses no nutritional concern.

3.4.5.2. Animal nutrition

Animal complete diets are balanced to energy content; crude fat from feed ingredients is taken into account to meet animal nutritional requirements. Considering the small magnitude of the decrease (~4%) of crude fat as compared to the conventional counterpart, the nutritional impact in feeds is considered negligible.

Behenic acid (C22:0) from feed ingredients is taken into account to meet animal nutritional requirements. Oilseed rape does not significantly contribute to behenic acid intake and is generally considered negligible for this fatty acid. Considering the very low levels of behenic acid in oilseed rape (< 0.3% total FA), the biological relevance of behenic acid, the small magnitude of the increase (~4%) as compared to the conventional counterpart, the nutritional impact in feeds is considered negligible.

Aspartic acid is a non‐essential amino acid in animals, as it can be synthesised endogenously and is not required as a dietary essential amino acid. Considering the biological relevance of aspartic acid, the small magnitude of the increase (~5%) as compared to the conventional counterpart, the nutritional impact in feeds is considered negligible.

3.4.6. Post‐market monitoring of GM food and feed

RF3 CQ B. juncea, as described in this application, does not raise any nutritional concern and is as safe as its comparator and the non‐GM reference varieties tested. The GMO Panel concludes that a post‐market monitoring plan for food and feed is not necessary.

3.4.7. Conclusions on the food and feed safety assessment

The newly expressed PAT/bar and Barstar proteins in RF3 CQ B. juncea do not raise safety concerns for human and animal health. No interactions between PAT/bar and Barstar proteins were identified. Moreover, the GMO Panel does not identify indications of safety concerns regarding allergenicity or adjuvanticity related to the presence of the newly expressed PAT/bar and Barstar. The GMO Panel finds no evidence that the genetic modification impacts the overall safety, as food and feed, of RF3 CQ B. juncea. Based on the outcome of the comparative assessment and the nutritional assessment, the GMO Panel concludes that the consumption of RF3 CQ B. juncea does not represent any nutritional concern, in the context of the scope of this application. The GMO Panel concludes that RF3 CQ B. juncea, as described in this application, is as safe as the comparator and the non‐GM reference varieties tested, and that no post‐market monitoring of food or feed is considered necessary.

3.5. Environmental risk assessment and monitoring plan ²⁸

3.5.1. Environmental risk assessment

Considering the scope of this application, which excludes cultivation, the environmental risk assessment (ERA) takes into account mainly: (1) the exposure of microorganisms to recombinant DNA in the gastrointestinal tract of animals fed with GM material and of microorganisms present in environments exposed to manure or faeces of these animals; and (2) the deliberate or accidental release of GM material into the environment, including spillage of viable RF3 CQ B. juncea seeds during transportation or processing (EFSA GMO Panel, 2010a).

3.5.1.1. Persistence and invasiveness of the GM plant

B. juncea is an annual allotetraploid species (2n = 36, genome constitution AABB), which probably evolved in China and the Middle East through hybridisation between the two diploid species Brassica rapa (2n = 20, AA) and Brassica nigra (2n = 16, BB) followed by polyploidisation (Chen et al., 2013; Kang et al., 2021; OECD, 2012; Oram et al., 2005). As reported for oilseed rape (B. napus), secondary dormancy can be triggered in B. juncea seeds under unfavourable environmental conditions, so that mature seeds can remain viable in the soil years after they are shed (OGTR, 2017). However, the higher resistance to pod shattering reported for B. juncea would reduce its contribution to the soil seedbank compared to that of oilseed rape (Kaur et al., 2020; McCaffery et al., 2009; OECD, 2012; Salisbury, 2002). B. juncea often occurs in agricultural fields as a weed or a volunteer that generally does not cause significant problems (CFIA, 2012; OGTR, 2017; Oram et al., 2005; Salisbury, 2002), although in some areas it has the potential to cause major yield losses (Groves et al., 2003; Huangfu et al., 2007). This Brassica species has become naturalised outside of agricultural areas in different countries across the globe as a poor competitor which does not thrive in the absence of disturbance or in areas with established vegetation (OGTR, 2017; Oram et al., 2005), and which forms commonly transient populations in disturbed and ruderal habitats such as roadsides, riverbanks and along railways (Eastham & Sweet, 2002; Groves et al., 2003; Liu et al., 2013; Nishizawa et al., 2016). Thus, in unmanaged non‐agricultural areas, the presence of B. juncea populations over consecutive years is considered to rely mainly on recruitment from the soil seedbank and on new spillage events (OGTR, 2017). Brassica juncea fitness is limited by both biotic and abiotic stressors, including feeding by a range of herbivore arthropods, susceptibility to several pathogens, and sensitivity to high temperatures and frosts (McCaffery et al., 2009; OGTR, 2017; Oram et al., 2005; Rai et al., 2022). It is important to consider that CQ varieties have low concentrations of glucosinolates, which could render B. juncea RF3 more susceptible to herbivores. Generalist insect pests have been reported to feed less and have a lower relative growth rate on B. juncea lines with a high concentration of these antinutritional compounds (Kumar et al., 2013; Li et al., 2000).

The intended trait of RF3 CQ B. juncea and the observed increase in yield (see Section 3.3.5) would only provide a selective advantage to B. juncea plants when they are exposed to glufosinate‐based herbicides. Should these plants be exposed to such herbicides, their abundance may increase locally, allowing the establishment of transient populations. However, the likelihood of the establishment of transient populations will be restricted to managed environments, which may occasionally be treated with such herbicides. Moreover, this fitness advantage will not allow RF3 CQ B. juncea to overcome other biological and abiotic factors (described above) limiting a plant's persistence and invasiveness.

The data reported in an additional study on seed germination provided by the applicant do not point to an increased risk of persistence and invasiveness of RF3 CQ B. juncea as compared to its conventional counterpart.

The GMO Panel concludes that it is very unlikely that RF3 CQ B. juncea will differ from conventional CQ B. juncea varieties in their ability to survive until subsequent seasons, or to establish occasional feral plants under European environmental conditions in case of accidental release into the environment of viable RF3 CQ B. juncea seeds.

3.5.1.2. Potential for gene transfer

A prerequisite for any gene transfer is the availability of pathways for the transfer of genetic material, either through HGT of DNA, or through vertical gene flow via cross‐pollination from feral plants originating from spilled grains.

3.5.1.2.1. Plant‐to‐microorganism gene transfer

Genomic DNA can be a component of food and feed products derived from B. juncea. It is well documented that such DNA becomes substantially degraded during processing and digestion in the human or animal gastrointestinal tract. However, bacteria in the digestive tract of humans and animals, and in other environments, may be exposed to fragments of DNA, including the recombinant fraction of such DNA. Current scientific knowledge of recombination processes in bacteria suggests that horizontal transfer of non‐mobile, chromosomally located DNA fragments between unrelated organisms (such as from plants to bacteria) is not likely to occur at detectable frequencies under natural conditions (for further details, see EFSA, 2009).

Homologous recombination is known to facilitate horizontal transfer of non‐mobile, chromosomal DNA fragments to bacterial genomes. This requires the presence of at least two stretches of DNA sequences that are similar in the recombining DNA molecules. In the case of sequence identity with the transgene itself, recombination would result in gene replacement. In the case of identity with two or more regions flanking recombinant DNA, recombination could result in the insertion of additional DNA sequences in bacteria and thus confer the potential for new properties.

In addition to homology‐based recombination processes, at a lower transformation rate, the non‐homologous end joining and microhomology‐mediated end joining are theoretically possible (EFSA, 2009; Hülter & Wackernagel, 2008). Independently of the transfer mechanism, the GMO Panel did not identify a selective advantage that a theoretical HGT would provide to bacterial recipients in the environment.

The applicant submitted an updated bioinformatics analysis for event RF3 to assess the possibility for HGT by homologous recombination. This information confirms the assessments provided in the context of previous Scientific Opinions (EFSA GMO Panel, 2017b, 2023a).

In summary, there is no indication for an increased likelihood of horizontal transfer of DNA from RF3 CQ B. juncea to bacteria. Given the nature of the recombinant DNA, the GMO Panel identified no safety concern linked to an unlikely but theoretically possible HGT.

3.5.1.2.2. Plant‐to‐plant gene transfer

The potential of feral GM B. juncea plants originating from seed import spills to transfer recombinant DNA to sexually cross‐compatible plants and the environmental consequences thereof were considered.

For plant‐to‐plant gene transfer to occur, spilled seeds of RF3 CQ B. juncea would need to germinate and develop into plants in areas containing compatible wild or cultivated relatives or B. juncea plants with overlapping flowering periods and under environmental conditions favouring cross‐pollination. For the transgene to get stabilised into the recipient species, viable fertile progeny must result from the cross and the transgene needs to be retained for several generations of backcrossing (de Jong & Rong, 2013).

B. juncea is self‐compatible and mainly self‐pollinated (Badenes‐Pérez, 2022; OECD, 2012; Salisbury et al., 2017). As in other Brassica species, B. juncea pollen is dispersed generally within very short distances from the donor plant with the assistance of wind and pollinators, although it can also occur by physical contact between neighbouring plants in fields with high sowing densities (CFIA, 2012; Challa et al., 2019; Ghosh Dastidar et al., 2000; Hoyle et al., 2007; OGTR, 2017).

Successful hybridisation between B. juncea and several Brassica species has been reported in the literature (reviewed in FitzJohn et al., 2007; Warwick et al., 2009), albeit at low levels under natural pollination conditions, given the low outcrossing rates of this species (Tsuda, Ohsawa, & Tabei, 2014). A number of studies have described the production of viable hybrids between B. juncea and oilseed rape in greenhouse and field experiments, as well as transgene introgression from oilseed rape into B. juncea, with higher seed production and germination rates when oilseed rape was the pollen donor (Bing et al., 1996; Frello et al., 1995; Heenan et al., 2007; Liu et al., 2010; Séguin‐Swartz et al., 2013; Tang et al., 2018; Tsuda, Okuzaki, et al., 2012). Lower levels of hybridisation have been reported between B. juncea and its parental species B. rapa (Bing et al., 1996; Choudhary et al., 2002), whereas co‐cultivation experiments with B. nigra, its other parental species, yielded no or very few hybrid progeny (Bing et al., 1996; Olsson, 1960). A few examples of successful natural outcrossing between B. juncea and wild relatives are available in the literature (see references in FitzJohn et al., 2007), including extremely low hybridisation levels with Sinapis arvensis, Raphanus raphanistrum and Erucastrum gallicum, indicative of a very low likelihood of transgene introgression from B. juncea into these weedy relatives (Lefol et al., 1997; Warwick & Martin, 2013). Taking together the low levels of cross pollination and hybridisation, the potential of transgene introgression from B. juncea to its cultivated and weedy relatives can be considered rare under environmental conditions.

The GMO Panel does not consider the occurrence of feral RF3 CQ B. juncea plants, pollen dispersal and consequent cross‐pollination as environmental harms in themselves, as there is no evidence that the traits will enhance its vertical gene flow potential, or fitness, persistence or invasiveness, or that of cross‐compatible plants such as hybridising wild relatives. However, when exposed to glufosinate‐containing herbicides, occasional cross‐compatible plants that acquired the herbicide tolerance traits through vertical gene flow are likely to exhibit a selective advantage, which may lead to their increased abundance. The likelihood of such an event to happen will be restricted to managed environments, which may occasionally be treated with such herbicides, so that environmental impacts will be minimal. Therefore, the GMO Panel considers that the acquisition of the herbicide tolerance traits by cross‐compatible plants would not create additional environmental impacts.

In conclusion, the GMO Panel considers that the likelihood of environmental effects as a consequence of the spread of genes from RF3 CQ B. juncea in Europe will not differ from that of conventional CQ B. juncea varieties.

3.5.1.3. Interactions of the GM plant with target organisms

Taking the scope of application EFSA‐GMO‐NL‐2019‐158 (no cultivation) and the absence of target organisms into account, potential interactions of occasional feral RF3 CQ B. juncea plants arising from seed import spills with target organisms are not considered a relevant issue.

3.5.1.4. Interactions of the GM plant with non‐target organisms

The environmental risk assessment considers potential effects of the GM plant on populations of non‐target organisms, defined as all those species directly or indirectly exposed to the GM plant and which are not targets of the newly expressed metabolite(s). The GMO Panel evaluated the potential hazards of the NEPs and considered that the environmental exposure of non‐target organisms to spilled GM B. juncea material or occasional feral RF3 CQ B. juncea plants arising from spilled B. juncea seeds will be limited. Additionally, ingested proteins are typically degraded before entering the environment through manure or faeces of animals fed with GM B. juncea (Harmon & Swanson, 2020; Miner‐Williams et al., 2014; Mok & Urschel, 2020; Santos‐Hernández et al., 2018; van Bruchem et al., 1985). The data provided for the assessment of protein stability in the context of other applications expressing the same NEPs (see Section 3.4.1.2) also supports that the NEPs will be degraded. As compared to non‐GM CQ B. juncea, the GMO Panel considers that potential interactions of RF3 CQ B. juncea with non‐target organisms do not raise any additional environmental safety concern.

3.5.1.5. Interactions with biogeochemical cycles

Biogeochemical cycles encompass the microbiologically mediated movement, transformation and storage of carbon, nitrogen, and other compounds within the receiving environments. The GMO Panel evaluated the potential hazards of the NEPs and considered that the environmental exposure to spilled GM B. juncea material or occasional feral GM B. juncea plants arising from spilled RF3 CQ B. juncea seeds will be limited, whereas exposure to manure and faeces of animals fed with RF3 CQ B. juncea material is expected to be higher. However, ingested proteins are typically degraded before entering the environment through manure and faeces of animals fed with GM B. juncea (Harmon & Swanson, 2020; Miner‐Williams et al., 2014; Mok & Urschel, 2020; Santos‐Hernández et al., 2018; van Bruchem et al., 1985), and the data provided for the assessment of protein stability in the context of other applications expressing the same NEPs (see Section 3.4.1.2) supports that the NEPs will be also degraded. As compared to non‐GM CQ B. juncea, the GMO Panel considers that potential interactions of RF3 CQ B. juncea with biogeochemical cycles do not raise any environmental safety concern.

3.5.2. Post‐market environmental monitoring

The objectives of a post‐market environmental monitoring (PMEM) plan, according to Annex VII of Directive 2001/18/EC, are: (1) to confirm that any assumption regarding the occurrence and impact of potential adverse effects of the GMO, or its use, in the ERA are correct; and (2) to identify the occurrence of adverse effects of the GMO, or its use, on human health or the environment that were not anticipated in the ERA.

Monitoring is related to risk management, and thus a final adoption of the PMEM plan falls outside the mandate of EFSA. However, the GMO Panel gives its opinion on the scientific rationale of the PMEM plan provided by the applicant (EFSA GMO Panel, 2011b). As the ERA did not identify potential adverse environmental effects from RF3 CQ B. juncea, no case‐specific monitoring is required.

The PMEM plan proposed by the applicant for RF3 CQ B. juncea includes: (1) the description of a monitoring approach involving operators (federations involved in import and processing), reporting to the applicant, via a centralised system, any observed adverse effect(s) of GMOs on human health and the environment; (2) a coordinating system established by CropLife Europe for the collection of information recorded by the various operators; and (3) the review of relevant scientific publications retrieved from literature searches (Lecoq et al., 2007; Windels et al., 2008). The applicant proposes to submit a PMEM report on an annual basis for the duration of the authorisation period.

The GMO Panel considers that the scope of the PMEM plan provided by the applicant is consistent with the intended uses of RF3 CQ B. juncea. The GMO Panel agrees with the reporting intervals proposed by the applicant in its PMEM plan.

3.5.2.1. Conclusion of the environmental risk assessment and monitoring plan

The GMO Panel concludes that it is unlikely that RF3 CQ B. juncea would differ from conventional CQ B. juncea varieties in its ability to persist under European environmental conditions. Taking into account the scope of application EFSA‐GMO‐NL‐2019‐158, interactions of RF3 CQ B. juncea with the biotic and abiotic environment are not considered to be relevant issues. The analysis of HGT from RF3 CQ B. juncea to bacteria does not indicate a safety concern. Therefore, considering the introduced trait, the outcome of the agronomic and phenotypic analysis, and the routes and levels of exposure, the GMO Panel concludes that RF3 CQ B. juncea would not raise safety concerns in the event of release of processed GM RF3 CQ B. juncea or the accidental spillage of viable GM B. juncea seeds into the environment.

The scope of the PMEM plan provided by the applicant and the reporting intervals are in line with the intended uses of RF3 CQ B. juncea.

4. OVERALL CONCLUSIONS

The GMO Panel was asked to carry out a scientific assessment of RF3 CQ B. juncea for import, processing and food and feed uses in accordance with Regulation (EC) No 1829/2003. The RF3 event was originally developed in B. napus. and has previously been assessed by EFSA in application EFSA‐GMO‐RX‐MS8‐RF3 (EFSA GMO Panel, 2009). The molecular characterisation data establish that RF3 CQ B. juncea contains a single insert consisting of one copy of the barstar and PAT/bar expression cassettes. The quality of the sequencing methodology and datasets was assessed by the EFSA GMO Panel and complies with the requirements listed in the EFSA Technical Note. Bioinformatics analyses of the sequences encoding the NEPs and the sequences corresponding to ORFs predicted within the introgressed event do not raise safety concerns. The stability of the introgressed insert DNA and of the introduced trait has been confirmed over several generations. The methodology used to quantify the levels of the Barstar and PAT/bar protein is considered adequate. The protein characterisation data comparing the biochemical, structural and functional properties of the E. coli and RF3 CQ B. juncea Barstar and PAT/bar proteins indicate that these proteins are equivalent. Considering the selection of test materials, the field trial sites and the associated management practices, and the agronomic‐phenotypic characterisation as indicators of the overall field trial quality, the GMO Panel concludes that the field trials are appropriate to support the comparative analysis. None of the identified differences in the agronomic, phenotypic, or compositional characteristics tested between RF3 CQ B. juncea and its conventional counterpart needed further assessment, except for yield, crude fat, aspartic acid, and behenic acid (C22:0) which underwent additional evaluation and were found not to raise any environmental impact and nutritional or safety concerns. The GMO Panel does not identify safety concerns regarding the toxicity and allergenicity of the Barstar and PAT/bar proteins as expressed in RF3 CQ B. juncea and finds no evidence that the genetic modification would change the overall allergenicity of RF3 CQ B. juncea. In the context of this application, the consumption of food and feed from RF3 CQ B. juncea does not represent a nutritional concern in humans and animals. The GMO Panel concludes that RF3 CQ B. juncea is as safe as the conventional counterpart and non‐GM oilseed rape reference varieties tested, and no post‐market monitoring of food/feed is considered necessary. The GMO Panel concludes that there is a very low likelihood of environmental effects resulting from the accidental release of viable seeds from RF3 CQ B. juncea into the environment. The PMEM plan and reporting intervals are in line with the intended uses of RF3 CQ B. juncea. Based on the relevant publications identified through the literature searches, the GMO Panel does not identify any safety issues pertaining to the uses of RF3 CQ B. juncea. The GMO Panel concludes that RF3 CQ B. juncea is as safe as its conventional counterpart and the tested non‐GM CQ B. juncea reference varieties with respect to potential effects on human and animal health and the environment.

5. DOCUMENTATION AS PROVIDED TO EFSA

• Letter from the Competent Authority of The Netherlands received on 25 June 2019 concerning a request for authorization of the placing on the market of genetically modified Canola Quality Brassica juncea RF3, submitted in accordance with Regulation (EC) No. 1829/2003 by BASF Agricultural Solutions US LCC (EFSA‐GMO‐NL‐2019‐158; EFSA‐Q‐2019‐00412).

• The application was made valid on 14 September 2020.

• Additional information (1) was requested by EURL on  6 October 2020.

• Additional information (1) was received on 23 July 2021.

• Additional information (2) was requested on 23 December 2020.

• Additional information (2) was received on 11 March 2021 partial, and on 29 June 2021.

• Additional information (3) was requested on 16 February 2021.

• Additional information (3) was received on 16 April 2021 partial, and on 22 April 2021 complete.

• Additional information (4) was requested on 2 August 2021.

• Additional information (4) was received on 21 October 2021 partial, and on 21 December 2021 complete.

• Additional information (5) was requested by EURL on 13 August 2021.

• Additional information (5) was received on 20 July 2023.

• Additional information (6) was requested on 23 November 2022.

• Additional information (6) was received on 12 January 2023.

• Additional information (7) was requested on 21 July 2023.

• Additional information (7) was received on 29 February 2024, partial and on 5 March 2025.

• Additional information (8) was requested by EURL on 1 August 2023.

• Additional information (8) was received on 31 July 2025.

• Additional information (9) was requested on 4 April 2025.

• Additional information (9) was received on 10 April 2025.

• Additional information (10) was requested on 15 May 2025.

• Additional information (10) was received on 9 July 2025.

• Additional information (11) was requested on 7 August 2025.

• Additional information (11) was received on 20 October 2025.

• Additional information (12) was requested by EURL on 26 August 2025.

• Additional information (12) was received on 11 May 2026.

• Additional information (13) was requested on 20 November 2025.

• Additional information (13) was received on 19 January 2026.

• Additional information (14) was requested on 17 February 2026.

• Additional information (14) was received on 16 March 2026 partial, and on 2 April 2026 complete.

• Spontaneous information was received on 10 February 2023 and on 16 April 2021.

ABBREVIATIONS

bw

body weight

dw

dry weight

ELISA

enzyme‐linked immunosorbent assay

ERA

environmental risk assessment

fw

fresh weight

GM

genetically modified

GMO

genetically modified organisms

HGT

horizontal gene transfer

HR

homologous recombination

NEPs

newly expressed proteins

ORFs

open reading frames

PCR

polymerase chain reaction

PMEM

post‐market environmental monitoring plan

PMM

post‐market monitoring

REQUESTOR

Competent Authority of the Netherlands

QUESTION NUMBER

EFSA‐Q‐2019‐00412

COPYRIGHT FOR NON‐EFSA CONTENT

EFSA may include images or other content for which it does not hold copyright. In such cases, EFSA indicates the copyright holder and users should seek permission to reproduce the content from the original source.

PANEL MEMBERS

Josep Casacuberta, Francisco Barro, Albert Braeuning, Ruud de Maagd, Michelle M. Epstein, Thomas Frenzel, Jean‐Luc Gallois, Frits Koning, Antoine Messéan, F. Javier Moreno, Fabien Nogué, Giovanni Savoini, Alan H. Schulman, Christoph Tebbe and Eve Veromann.

Supporting information

Annex A: Cartagena protocol_Brassica juncea RF3

Annex B: Labelling proposal_Brassica juncea RF3

Annex C: PMEM Plan_Brasica juncea RF3

Annex D: Validation report_Brasica juncea RF3

Annex E: Validated method_Brasica juncea RF3

Annex F: CRM report_RF3

Annex G: DNA extraction

Annex H: Member State comments_RF3 Brassica Juncea

APPENDIX A. Additional studies

List of additional studies performed by or on behalf of the applicant with regard to the evaluation of the safety of RF3 CQ B. juncea for humans, animals or the environment.

Study identification	Title
M‐618733‐01‐1	RF3 Brassica juncea – Seed Cold Tolerance, 2017

APPENDIX B. List of relevant publications identified by the applicant through literature searches (March 2007–August 2025)

Reference
EFSA Panel on Genetically Modified Organisms; Mullins, Ewen; Bresson, Jean Louis; Dalmay, Tamas; Dewhurst, Ian Crawford; Epstein, Michelle M.; Firbank, Leslie George; Guerche, Philippe; Hejatko, Jan; Moreno, Francisco Javier; Naegeli, Hanspeter; Nogue, Fabien; Rostoks, Nils; Sanchez Serrano, Jose Juan; Savoini, Giovanni; Veromann, Eve; Veronesi, Fabio; Ardizzone, Michele; Camargo, Ana M.; Fernandez, Antonio; Goumperis, Tilemachos; Lenzi, Paolo; Lewandowska, Aleksandra; Raffaello, Tommaso; Streissl, Franz (2023). Assessment of genetically modified oilseed rape MS8, RF3 and MS8 × RF3 for renewal authorisation under Regulation (EC) No 1829/2003 (application EFSA‐GMO ‐RX ‐024). EFSA Journal (2023), 21(4), e07934. CODEN: EJFOA6; ISSN: 1831‐4732
Mackie, S. J. W. (2021). Comparison of RF3 B. juncea to RF3 B. napus. Food and Nutrition Sciences (2021), Volume 12, Number 11, pp. 1041‐1061, 40 refs. ISSN: 2157‐944X; 2157‐9458. https://doi.org/10.4236/fns.2021.1211077. Published by: Scientific Research Publishing, Irvine

APPENDIX C. Animal dietary exposure

TABLE C.1.

Animal body weight, feed total daily intake, feed materials inclusion rate and daily dietary intake.

Animal		Feed	Oilseed rape full fat seed		Oilseed rape meal
Species	BW	TDI	IR	DDI	IR	DDI
Dairy cow	650	25	na	na	10	3.85
Beef cattle	500	12	na	na	20	4.80
Dairy sheep	80	2.8	na	na	20	7.00
Dairy goat	60	3.4	8	4.53	4.2	2.38
Rabbit	2	0.15	na	na	20	15.00
Fattening pig	100	3	na	na	20	6.00
Lactating sow	200	6	na	na	20	6.00
Piglet	20	1	na	na	20	10.00
Broiler	2	0.158	na	na	18	14.22
Laying hens	1.9	0.13	na	na	10	6.84
Turkey	7	0.5	na	na	20	14.29
Salmon	5	0.03	na	na	na	na
Carp	1	0.02	na	na	13	2.60
Cat	4	0.06	5	0.75	5	0.75
Dog	25	0.36	5	0.72	30	4.32
Horse	450	9	na	na	8	1.60

Abbreviations: BW, body weight (kg); DDI, daily dietary intake of feed material (g^DM/kg^BW); IR, inclusion rate percentage of feed material; na, not applicable (EFSA GMO Panel, 2023b); TDI, total daily intake of feed (kg^DM/animal).

TABLE C.2.

Newly expressed protein levels in feed materials.

NEP (μg/g dw)	Oilseed rape full fat seed	Oilseed rape meal a
PAT/bar	2.43	3.89
Barstar	0.063	0.10

^a Conversion factor: ×1.6.

TABLE C.3.

Dietary exposure based on the consumption of oilseed rape full fat seed.

Animal species	Daily dietary exposure to PAT/bar		Daily dietary exposure to Barstar
Category	μg/kg bw	mg/kg bw	μg/kg bw	mg/kg bw
Dairy Goat	11.02	0.011	0.29	0.0003
Cat	1.82	0.002	0.05	0.00005
Dog	1.75	0.002	0.05	0.00005

TABLE C.4.

Dietary exposure based on the consumption of oilseed rape meal.

Animal species	Daily dietary exposure to PAT/bar		Daily dietary exposure to Barstar
Category	μg/kg bw per day	mg/kg bw per day	μg/kg bw per day	mg/kg bw per day
Dairy cow	14.96	0.01	0.38	0.0004
Beef cattle	18.67	0.02	0.48	0.0005
Dairy sheep	27.23	0.03	0.70	0.0007
Dairy goat	9.26	0.01	0.24	0.0002
Rabbit	58.35	0.06	1.50	0.0015
Fattening pig	23.34	0.02	0.60	0.0006
Lactating sow	23.34	0.02	0.60	0.0006
Piglet	38.90	0.04	1.00	0.0010
Broiler	55.32	0.06	1.42	0.0014
Laying hens	26.62	0.03	0.68	0.0007
Turkey	55.57	0.06	1.43	0.0014
Carp	10.11	0.01	0.26	0.0003
Cat	2.92	0.00	0.08	0.0001
Dog	16.80	0.02	0.43	0.0004
Horse	6.22	0.01	0.16	0.0002

EFSA GMO Panel (EFSA Panel on Genetically Modified Organisms) , Casacuberta, J. , Barro, F. , Braeuning, A. , de Maagd, R. , Epstein, M. M. , Frenzel, T. , Gallois, J.‐L. , Koning, F. , Messéan, A. , Moreno, F. J. , Nogué, F. , Savoini, G. , Schulman, A. H. , Tebbe, C. , Veromann, E. , Ardizzone, M. , De Sanctis, G. , Fernandez Dumont, A. , … Raffaello, T. (2026). Assessment of genetically modified RF3 Canola Quality (CQ) Brassica juncea (application EFSA‐GMO‐NL‐2019‐158). EFSA Journal, 24(8), e10178. 10.2903/j.efsa.2026.10178