Setting an import tolerance for 2,4‐D in soyabeans

Abstract

In accordance with Article 6 of Regulation (EC) No 396/2005, the applicant Dow AgroSciences submitted a request to the competent national authority in Greece to set an import tolerance for the active substance 2,4‐D in genetically modified (GM) soyabeans imported from Canada and the USA. The genetic modification confers tolerance to the herbicide 2,4‐D. The data submitted in support of the request provided sufficient evidence to conclude that residues of parent 2,4‐D and of the metabolite 2,4‐dichlorophenol (2,4‐DCP), which was found in the GM soybeans treated with 2,4‐D, are unlikely to present a risk for consumers. Sufficiently validated analytical methods are available to enforce the proposed maximum residue level (MRL) in soybeans.

Summary

In accordance with Article 6 of Regulation (EC) No 396/2005, the applicant Dow AgroScience submitted a request to the competent national authority in Greece (evaluating Member State (EMS)) to set an import tolerance for the active substance 2,4‐D in genetically modified (GM) soybeans expressing the aryloxyalkanoate dioxygenase‐12 (AAD‐12) protein, imported from Canada and USA. The genetic modification introduced confers tolerance to the herbicide 2,4‐D. The EMS drafted an evaluation report in accordance with Article 8 of Regulation (EC) No 396/2005 which was submitted to the European Commission and forwarded to the European Food Safety Authority (EFSA) on 5 March 2015.

EFSA bases its assessment on the revised evaluation report submitted by the EMS, the renewal assessment report (RAR) and its final addendum, the Commission revised review report on 2,4‐D, the conclusion on the peer review of the pesticide risk assessment of the active substance 2,4‐D and 2,4‐DB, as well as the European Union (EU) review of the existing maximum residue levels (MRLs) for 2,4‐D according to Article 12 of Regulation (EC) No 396/2005 and a previous EFSA opinion on GM maize.

The toxicological profile of the active substance 2,4‐D was re‐assessed within the framework of the EU pesticides renewal of the approval under Regulation (EC) No 1107/2009. An acceptable daily intake (ADI) of 0.02 mg/kg body weight (bw) per day and an acute reference dose (ARfD) of 0.3 mg/kg bw were finally established for 2,4‐D. The toxicological profile of the metabolite 2,4‐dichlorophenol (2,4‐DCP), which is a common metabolite to 2,4‐D and 2,4‐DB, was discussed during the pesticides peer review 186 Experts’ meeting in November 2018. The toxicological reference values derived for 2,4‐DB (and 2,4‐D) were concluded to be applicable also to 2,4‐DCP.

The metabolism of 2,4‐D in primary crops was previously investigated in conventional crops after foliar (cereals and root crops) and soil (fruit crops) applications. Based on the results of these studies, the residue definition for enforcement and risk assessment was established as sum of 2,4‐D, its salts, esters and conjugates, expressed as 2,4‐D.

A new metabolism study on soyabeans genetically modified to express the AAD‐12 protein was submitted in the framework of this MRL application. In seeds, no new metabolites were identified, but the largest component of the residue was represented by 2,4‐DCP and its conjugates, whereas parent 2,4‐D was found only in low amounts. The results of field residue trials confirmed the presence of 2,4‐DCP at levels up to 0.05 mg/kg in soyabean seeds. Based on the results of the metabolism study, EFSA proposes to include 2,4‐DCP in the residue definition for risk assessment for the GM soyabeans under assessment. Since 2,4‐DCP may be originated from other sources and cannot unequivocally be traced back to its origin, it should be excluded from the residue definition for enforcement. Therefore, it is proposed to apply the enforcement residue definition currently set in Regulation (EC) No 396/2005 also to GM soybeans under assessment.

A sufficient number of valid residue trials performed on GM soybeans expressing the AAD‐12 protein have been submitted in support to this application. In none of the trials, quantifiable residues of 2,4‐D (including salts, esters and conjugates) above the limit of quantification (LOQ) of 0.01 mg/kg have been found. Thus, the existing MRL for 2,4‐D in soybeans does not have to be raised. However, risk managers could consider the lowering of the existing MRL to the LOQ of 0.01 mg/kg, unless other uses of 2,4‐D require to maintain the current MRL at the level of 0.05 mg/kg (LOQ). Sufficiently validated enforcement analytical methods are available. In the framework of the review of existing MRLs under Article 12 of Regulation (EC) No 396/2005 a data gap for an independent laboratory validation (ILV) for the enforcement analytical method in high oil content matrices was identified. This data gap has been satisfactorily addressed in the framework of the EU pesticides peer review for the renewal of the approval of 2,4‐D.

Specific studies investigating the nature and magnitude of 2,4‐D residues in processed commodities are not required as significant residues are not expected in the raw agricultural commodities (RAC). As the uses of 2,4‐D are on imported crops, investigations of residues in rotational crops are not relevant. A change of the existing MRLs for 2,4‐D in products of animal origin resulting from the use of imported GM soyabean by‐product meal used as a feed item is not required.

The consumer risk assessment was performed with revision 2 of the EFSA Pesticide Residues Intake Model (PRIMo). For the chronic exposure, the existing uses at the EU level and the acceptable CXLs assessed in the framework of the MRL review were also taken into account. The acute risk assessment was performed for soyabeans only. The highest estimated long‐term consumer intake accounted for 34% of the ADI. The acute consumer exposure to 2,4‐D residues in GM soybean seeds was calculated as up to 0.02% of the ARfD.

EFSA concludes that the use of 2,4‐D according to the notified good agricultural practices assessed on GM soybeans expressing the AAD‐12 protein will not result in a consumer exposure exceeding the toxicological reference values for 2,4‐D. Hence, a risk for consumers is unlikely.

The information submitted was sufficient to propose the MRL summarised in the table below.

Codea	Commodity	Existing EU MRL (mg/kg)	Proposed EU MRL (mg/kg)	Comment/Justification
Enforcement residue definition: 2,4‐D (sum of 2,4‐D, its salts, its esters and its conjugates, expressed as 2,4‐D)
0401070	Soyabeans	0.05* (ft)	0.05* or 0.01* (further risk management consideration)	The submitted data are sufficient to perform a consumer risk assessment for the use of 2,4‐D in support the import tolerance request. The lowering of the existing MRL set at the LOQ of 0.05 mg/kg to the LOQ of 0.01 mg/kg as derived from the residue trials on 2,4‐D‐tolerant soybeans (AAD‐12 protein) and achievable with the validated enforcement analytical methods is an option to be discussed by risk managers. Risk for consumers unlikely MRL/Tolerance in the countries of origin (Canada and USA) is 0.02 mg/kg. The data gap identified by EFSA for additional information on the analytical method (ft) has been previously addressed

MRL: maximum residue level; LOQ: limit of quantification; AAD‐12: aryloxyalkanoate dioxygenase‐12.

*Indicates that the MRL is set at the limit of analytical quantification (LOQ).

^aCommodity code number according to Annex I of Regulation (EC) No 396/2005.

ft: The European Food Safety Authority identified some information on analytical methods as unavailable. When reviewing the MRL, the Commission will take into account the information referred to in the first sentence, if it is submitted by 17 December 2015, or, if that information is not submitted by that date, the lack of it.

The data gap identified in the framework of the MRL review (ILV for enforcement of residues in high oil content commodities) was satisfactorily addressed with the information submitted in the process of the EU pesticides renewal of the approval of the active substance 2,4‐D. Therefore, the footnote (ft) set in Commission Regulation (EU) No 1317/2013 can be deleted for soyabeans (code 0401070) and also for the whole subgroup of tree nuts (code 0120000), except chestnuts (code 0120040).

Background

Regulation (EC) No 396/20051 (hereinafter referred to as the MRL regulation) establishes the rules governing the setting of pesticide maximum residue levels (MRLs) in the European Union (EU). Article 6 of the Regulation lays down that commercially interested parties such as manufacturers or importers may submit an application requesting the setting of an import tolerance in accordance with the provisions of Article 7 of the MRL regulation.

Greece, the evaluating Member State (EMS), received an application from the company Dow AgroSciences2 to set an import tolerance for the active substance 2,4‐D on genetically modified (GM) soybeans expressing the aryloxyalkanoate dioxygenase‐12 (AAD‐12) protein imported from Canada and USA. The two events linked to the GM soybeans expressing AAD‐12: DAS‐68416‐4 and DAS‐44406‐6. The application was notified to the European Commission and the European Food Safety Authority (EFSA) and was subsequently evaluated by the EMS in accordance with Article 8 of the MRL Regulation.

According to the EMS, the notified uses of 2,4‐D on soybeans imported from Canada and USA does not require a change of the existing MRL set at the level of the limit of quantification (LOQ) of 0.05 mg/kg, but have an impact on the risk assessment, since a toxicologically relevant metabolite is expected to be present in higher concentrations than in the non‐GM soybeans.

EFSA proceeded with the assessment of the applications and the evaluation reports as required by Article 10 of the Regulation. EFSA identified data gaps which needed further clarification, and which were requested from the EMS. On 3 October 2018, the EMS submitted the requested information in a revised evaluation report (Greece, 2015), which replaced the previously submitted evaluation report.

In accordance with Article 10 of Regulation (EC) No 396/2005, EFSA shall, based on the evaluation report provided by the EMS, provide a reasoned opinion on the risks to the consumer associated with the application. A pesticides peer review experts’ meeting was held on 21–22 November 2018 to discuss the toxicological profile of the metabolite 2,4‐dichlorophenol (2,4‐DCP); (EFSA, 2018).

The evaluation reports submitted by the EMS (Greece, 2015) as revised in September 2018, the exposure calculations using the EFSA Pesticide Residues Intake Model (PRIMo), together with the report of the experts’ meeting on mammalian toxicology regarding 2,4‐D (EFSA, 2018), are considered as supporting documents to this reasoned opinion and, thus, are made publicly available.

The active substance and its use pattern

The details of the Good Agricultural Practices (GAPs) reported to be authorised for use on GM soybeans in Canada and USA are in Appendix A.

2,4‐D is the ISO common name for (2,4‐dichlorophenoxy)acetic acid (IUPAC). Different variants of 2,4‐D are used in commercial formulations, such as acid, salts and esters. The chemical structures of the active substance 2,4‐D and its main metabolites are reported in Appendix B.

For the two GM soybeans expressing the aryloxyalkanoate dioxygenase‐12 (AAD‐12) protein, DAS‐68416‐4 and DAS‐44406‐6, applications for authorisation for food and feed uses, import and processing were submitted in the EU in accordance with Regulation (EC) No 1829/20033. The two events express similar levels of AAD‐12 protein and equivalence tolerance to 2,4‐D applications. EFSA has carried out the scientific assessment and published the following Scientific Opinion:

• Application EFSA‐GMO‐NL‐2011‐91 for soybean DAS‐68416‐4, expressing the AAD‐12 and phosphinothricin acetyltransferase (PAT) proteins, which confer tolerance to aryloxyalkanoate herbicides (such as 2,4‐D) and glufosinate‐ammonium, respectively (EFSA GMO Panel, 2017a)

• Application EFSA‐GMO‐NL‐2012‐106 for soybean DAS‐44406‐6, expressing the AAD‐12, PAT and 2mEPSPS proteins, which confer tolerance to 2,4‐D, glufosinate‐ammonium and glyphosate, respectively (EFSA GMO Panel, 2017b)

In December 2017, a decision was taken to authorise products containing, consisting of, or produced from genetically modified soybean DAS‐68416‐44 and DAS‐44406‐65 pursuant to Regulation (EC) No 1829/20033.

Currently, the following two stack soybean applications, each one containing DAS‐68416‐4 and DAS‐44406‐6, are under the EFSA assessment:

• EFSA‐GMO‐NL‐2013‐115 for soybean DAS‐68416‐4 × MON‐89788‐1, expressing the AAD‐12, PAT and CP4 EPSPS proteins, which confer tolerance to 2,4‐D, glufosinate‐ammonium and glyphosate, respectively (Question No EFSA‐Q‐2013‐00281);

• EFSA‐GMO‐NL‐2016‐132 for soyabean DAS‐81419‐2 × DAS‐44406‐6, expression of the Cry1F, Cry1Ac, AAD‐12, PAT and 2mEPSPS proteins, which confer resistance against certain lepidopteran insects and tolerance to 2,4‐D, glufosinate‐ammonium and glyphosate, respectively (Question No EFSA‐Q‐2016‐00195).

According to Regulation (EU) No 2015/20336, the approval for the active substance 2,4‐D was renewed under Regulation (EC) No 1107/2009.7 The implementing regulation contains certain conditions and restrictions,8 and requested further confirmatory (eco)toxicological information to be submitted.9 The assessment is currently ongoing. The representative uses assessed during the renewal of the approval were on conventional wheat, barley, oats, rye and maize.

The EU MRLs for 2,4‐D are established in Annex II of Regulation (EC) No 396/2005. The review of the existing MRLs for 2,4 D according to Article 12 of Regulation (EC) No 396/2005 (MRL review) has been completed (EFSA, 2011). The proposed MRLs have been implemented in the MRL legislation by Commission Regulation (EU) No 1317/201310. For the MRLs not fully supported by data, including soybeans, footnotes were added in the regulation, which specifies which data have to be provided by 17 December 2015. After the MRL review EFSA has issued a reasoned opinion assessing the use of 2,4‐D on GM maize which did not required a change of the existing MRL (EFSA, 2017).

The MRL/tolerance set in the USA and Canada11 for 2,4‐D in soybean seeds is 0.02 mg/kg.

EFSA based its assessment on the evaluation report submitted by the EMS (Greece, 2015) as revised in September 2018, the renewal assessment report (RAR) and its addendum (Greece, 2013, 2014), the revised Commission renewal report on 2,4‐D (European Commission, 2017), the conclusion on the peer review of the pesticide risk assessment of the active substance 2,4‐D (EFSA, 2014, revised in 2017) and 2,4‐DB (EFSA, 2016), the review of the existing MRLs for 2,4‐D according to Article 12 of Regulation (EC) No 396/2005 (EFSA, 2011) and the reasoned opinion on the setting of an import tolerance for 2,4‐D in maize (EFSA, 2017). The assessment is performed in accordance with the legal provisions of the Uniform Principles for the Evaluation and the Authorisation of Plant Protection Products adopted by Commission Regulation (EU) No 546/201112 and the guidance documents relevant for the consumer risk assessment of pesticide residues applicable at time of submission of the application (European Commission, 1996, 1997a, b, c, d, e, f–g, 2000, 2010; OECD, 2011).

1. Method of analysis

1.1. Methods for enforcement of residues in food of plant origin

The EU pesticides peer review (renewal of the approval) concluded that analytical methods based on gas chromatography–mass spectrometry (GC–MS) and liquid chromatography with tandem mass spectrometry (LC–MS/MS) were available to enforce 2,4‐D residues (sum of 2,4‐D, its salts, esters and conjugates, expressed as 2,4‐D) in plants at the LOQ of 0.01 mg/kg. An independent laboratory validation (ILV) for the enforcement analytical method in high oil content commodities is available (EFSA, 2014). Further validation data on the hydrolysis step were identified as missing in the MRL review and it was noted that this data would be desirable. No new information has been submitted with the MRL application.

1.2. Methods for enforcement of residues in food of animal origin

The EU pesticides peer review (renewal of the approval) concluded that analytical methods based on LC–MS/MS were available to enforce 2,4‐D residues (sum of 2,4–D, its salts, esters and conjugates, expressed as 2,4–D) in muscle, fat, kidney, milk and eggs at the LOQ of 0.01 mg/kg (EFSA, 2014). Additional information was required on the hydrolysis step and extraction efficiency. This data gap is of no relevance for this application since a change of the existing MRLs in animal products was not requested.

2. Mammalian toxicology

2.1. Toxicological profile of the active substance

The toxicological profile of the active substance 2,4‐D was assessed in the framework of the renewal of the approval of the active substance under Regulation (EC) No 1107/2009 (EFSA, 2014). An acceptable daily intake (ADI) of 0.05 mg/kg body weight (bw) per day and an acute reference dose (ARfD) of 0.75 mg/kg bw were established (EFSA, 2014; European Commission, 2015). In 2016, EFSA recommended to amend the toxicological reference values for 2,4‐D in the light of the further consideration of studies common to the evaluation of the active substances 2,4‐D and 2,4‐DB, of which 2,4‐D is a major metabolite (EFSA, 2016; European Commission, 2017). The toxicological reference values formally approved in 2017 are compiled in Table 1.

Table 1.

Overview of the toxicological reference values

	Source	Year	Value	Study	Uncertainty factor
2,4‐D
ADI	European Commission	2017	0.02 mg/kg bw per day	1‐year dog study with 2,4‐DB	100
ARfD		2017	0.3 mg/kg bw	Developmental toxicity studies in rat and rabbit with 2,4‐D and 2,4‐DB	100

ADI: acceptable daily intake; ARfD: acute reference dose; bw: body weight,

2.2. Toxicological profile of metabolites

The toxicological profile of the metabolite 2,4‐DCP, which is a common metabolite to 2,4‐D and 2,4‐DB, was discussed during the pesticides peer review 186 Experts’ meeting in November 2018.

A number of toxicological studies are available from the open literature on 2,4‐DCP and information was reported in the assessment reports on 2,4‐D (Greece, 2013, 2014) and 2,4‐DB (Belgium, 2016). In the framework of the current application, a complete genotoxicity data package has been submitted (Greece, 2015).

The metabolite was found to be harmful upon acute oral administration according to Regulation (EC) No 1272/200813. Based on the genotoxicity data package covering in vitro gene mutation in bacterial and mammalian cells, chromosome aberration and in vivo micronucleus test resulting in negative results, the metabolite was considered devoid of genotoxic potential.

In the overall data package on the metabolite 2,4‐DCP, the critical lowest‐observed‐adverse‐effect level (LOAEL) was agreed at 25 mg/kg bw per day for transitory mammary gland effects (whitening, stiffening and swelling) from the two‐generation reproductive toxicity study in rats. The metabolite 2,4‐DCP taken together with its conjugated forms was considered a major metabolite of 2,4‐DB in rat urine after oral administration, although not retrieved in the rat metabolism after administration of 2,4‐D. Since the dog was considered the most sensitive species after administration of 2,4‐D and 2,4‐DB and as there are no studies available on the metabolite 2,4‐DCP in dogs, the experts agreed that the toxicological reference values derived for 2,4‐DB are applicable to 2,4‐DCP. Since the toxicological reference values of 2,4‐DB are also applicable to 2,4‐D, the ADI and ARfD reported in Table  1 for 2,4‐D are applicable to 2,4‐DCP. 2,4‐DCP was concluded to share the toxicological profile of 2,4‐DB and 2,4‐D.

3. Residues

3.1. Nature and magnitude of residues in plant

3.1.1. Primary crops

3.1.1.1. Nature of residues

Metabolism of 2,4‐D in primary crops was investigated in conventional crops in the framework of the MRL review under Article 12 of Regulation (EC) No 396/2005 and the EU peer review (renewal of the approval) (EFSA, 2011, 2014). An additional study on GM maize was assessed in a previous EFSA opinion (EFSA, 2017); furthermore, results of a study on GM cotton are reported in an assessment performed by JMPR (FAO, 2017).

In the framework of this MRL application, a metabolism study with 2,4‐D foliar applications to GM soyabean at approximately the notified rate (1N) was provided (Greece, 2015). An overview of the key parameters of the available metabolism studies is presented in Table 2.

Table 2.

Summary of available metabolism studies in plants

Crop group	Crops	Application	Sampling	Comments/Source
Conventional plants (peer reviewed studies)
Fruit	Apple	Soil, 2 × 2.13 kg/ha	56 DALA	[U‐phenyl‐14C]‐2,4‐D (EFSA, 2011)
Root	Potato	Foliar, 2 × 0.07 kg/ha	82 DALA	[U‐phenyl‐14C]‐2,4‐D (EFSA, 2011)
		Foliar, 0.14 + 0.28 kg/ha	29 DALA	[U‐phenyl‐14C]‐2,4‐D (EFSA, 2011)
Cereal/ Grass	Wheat	Foliar, 1 × 1.68 kg/ha	10 (forage), 49 (grain, straw) DAT	[U‐phenyl‐14C]‐2,4‐D (EFSA, 2011)
Genetically modified plants a (not peer reviewed studies)
Pulses/ oilseeds	Soyabean (AAD‐12)	Soil, 1 pre‐emergence + Foliar, 2 post‐emergence, Rate 1.12 kg/ha, up to BBCH 65	7 (forage) DAT2; 22 (hay), 41–62 (seed) DALA	[U‐phenyl‐14C]‐2,4‐D Event DAS‐68416‐4 (Greece, 2015)
	Cotton (AAD‐12)	Foliar, 1 pre‐emergence + 2 post‐emergence, Rate: 1.1 kg/ha, up to BBCH 65	56 DALA (seed, trash)	[U‐phenyl‐14C]‐2,4‐D (FAO, 2017)
Cereal/ Grass	Maize (AAD‐1)	Soil, 1 pre‐emergence + Foliar, 2 post‐emergence, Rate ca. 0.9‐1.1 kg/ha, up to BBCH 18	30 (immature plant); 57 (grain, cobs, fodder) DALA	[U‐phenyl‐14C]‐2,4‐D Event DAS‐40474‐7b (EFSA, 2017)
		Soil, 1 pre‐emergence + Foliar, 2 post‐emergence, Rate ca. 1.1‐1.2 kg/ha, up to BBCH 18		[U‐phenyl‐14C]‐2,4‐D Event DAS‐40278‐9 (EFSA, 2017)

DALA; day after last application; DAT days after treatment; DAT₂: days after 2nd treatment.

^aCrops which exhibit tolerance to application of 2,4‐D herbicide.

^bAccording to the EMS, in DAS‐40474‐7 more than a single copy of the AAD‐1 gene was inserted (EFSA, 2017).

In conventional crops, after soil applications to fruit crops, residues in apples were too low to allow further identification.

After foliar applications to root and cereal crops, the parent compound, including its conjugated forms, was the major component of residues. It accounted for 77% of total radioactive residues (TRR) in wheat forage, 72% TRR in wheat straw, 6% TRR in grain and 35% TRR in potato tuber. The metabolite 2,4‐DCP was found in small amounts, up to about 0.5 mg eq/kg (1% TRR) in maize straw (feed item) and in trace levels (< 0.01 mg eq/kg, 4% of TRR) in potato tubers. Based on the metabolism studies, the residue definition for enforcement and risk assessment was proposed as the sum of 2,4‐D, its salts, esters and conjugates, expressed as 2,4‐D (EFSA, 2011, 2014).

After foliar applications to maize genetically modified to express the AAD‐1 protein, a similar metabolic pattern was observed compared with conventional crops. In immature plant and mature fodder, 2,4‐D was the major component of residues (51–77% TRR) with 2,4‐DCP, its disaccharide and glucose conjugates representing a relevant part of residues as well (about 22–25% TRR). In maize grain, the parent compound was the only compound identified (7% TRR; 0.003 mg/kg) and 2,4‐DCP could not be detected. Considering that parent was the only compound found in the grain and that grain is the only commodity expected to be imported, EFSA concluded to apply the same residue definitions derived for conventional crops to the GM maize assessed (EFSA, 2017).

In the metabolism study in genetically modified soyabeans expressing the AAD‐12 protein, the majority of the radioactive residue in forage and hay was composed of 2,4‐D (up to 86% and 59% TRR, respectively) and free or conjugated 2,4‐DCP (up to 18% and 25% TRR, respectively). Total residue in mature soybean seeds was 0.37 mg eq/kg. 2,4‐DCP (free and conjugated) represented the major component of residues (5.6% TRR, 0.021 mg eq/kg), whereas parent compound was found in trace levels only (0.005 mg eq/kg, 1.4% TRR). The attempt to further characterise the large part of non‐extractable residues in seeds (approximately 70% TRR) resulted in highly polar and multi‐component fractions, none eluting in the region of 2,4‐D and 2,4‐DCP. According to the EMS, the non‐extractable compounds are attributed to natural plant constituents after incorporation of radiolabelled carbon (Greece, 2015). The results of field residue trials confirmed the presence of 2,4‐DCP in the seeds.

In addition, investigations on the metabolism in genetically modified cotton (AAD‐12 protein) were reported by JMPR (FAO, 2017). Total residue in mature cotton seeds was 1.18 mg eq/kg. The metabolite 2,4‐DCP and its conjugates were observed at higher concentrations than parent and represented the major component of the residues (22% TRR). This study confirmed that on GM cotton, similar to GM soyabeans, the genetical modification expressing the AAD‐12 protein was associated with increased metabolism of 2,4‐D to form 2,4‐DCP, which was then conjugated, mainly with glucose.

In conclusion, the metabolism of 2,4‐D in GM soybeans and cotton expressing the AAD‐12 protein showed to be qualitatively similar to the metabolism observed in conventional crops but the metabolic pattern was quantitatively different compared with conventional crops, showing higher amounts of 2,4‐DCP (free and conjugated).

Based on the results of the metabolism studies in GM soyabeans, EFSA proposes to include 2,4‐DCP in the residue definition for risk assessment for the GM soybeans under assessment.

As regards the enforcement residue definition, parent 2,4‐D (including salts, esters and conjugates) is not a good marker for GM soybeans. However, considering that the enforcement residue definition is applicable also to the conventional soybeans with parent compound expected to be the main residue and considering that 2,4‐DCP (free and conjugated) cannot unequivocally be related to the use of 2,4–D since it is a common metabolite generated also from other active substances belonging to the class of phenoxycarboxylic acids (e.g. 2,4‐DB), EFSA proposes no modification of the existing residue definition.

To summarise, the following residue definitions are proposed for genetically modified soybean seeds expressing the AAD‐12 protein conveying tolerance to 2,4‐D:

• Residue definition for enforcement: Sum of 2,4‐D, its salts, its esters and its conjugates, expressed as 2,4‐D. The residue definition for enforcement set in Regulation (EC) No 396/2005 is identical with the above mentioned residue definition.

• Residue definition for risk assessment: Sum of 2,4‐D, its salts, esters and conjugates, and 2,4‐DCP and its conjugates, expressed as 2,4‐D.

3.1.1.2. Magnitude of residues

In support of the MRL applications for soyabean, a total of 24 residue trials on GM soyabean expressing the AAD‐12 protein (event not specified) compliant with the notified critical GAP (see Appendix A, United States GAP) were submitted. Trials were conducted in Canada and the USA using a 2,4‐D dimethylamine salt. All supervised residue trials were performed using a non‐ionic surfactant in the spray mixture during a single season instead of at least two as required (European Commission, 1997b). Since the trials were located in different geographical regions, EFSA is of the opinion that the trials are sufficiently representative for the use and further trials performed in a different year were not requested. EFSA disregarded one trial since last application occurred at a very early growth stage (BBCH 35) compared to the notified GAP. All samples were analysed for 2,4‐D and 2,4‐DCP with an analytical method involving a hydrolysis step to cover also the conjugates of 2,4‐D and 2,4‐DCP. According to the EMS, the analytical methods used to analyse the residue trial samples have been sufficiently validated and were proven to be fit for purpose (Greece, 2015).

At harvest, 2,4‐D was not present in quantifiable concentrations (< LOQ) and 2,4‐DCP ranged from the LOQ (< 0.01 mg/kg) to 0.05 mg/kg. The results of the residue trials, the related risk assessment input values and the derived MRL are summarised in Table 3.

Table 3.

Overview of the available residues trials data

Commodity	Region/ Indoora	Residue levels observed in the supervised residue trials (mg/kg) b	Comments/Source	Calculated MRLc (mg/kg)	HR d (mg/kg)	STMR e (mg/kg)	CF f
Soyabeans	USA/CA	Mo = 23 × < 0.010 RA = 9 × < 0.024; 0.024; 0.026; 2 × 0.029; 0.032; 0.036; 0.039; 0.043; 0.045; 0.056; 0.058; 0.060; 0.069; 0.075	Residue trials compliant with the GAP on GM soyabean expressing the AAD‐12 protein. Samples harvest 51–104 days after last application. MRL/Tolerance set in the countries of origin is 0.02 mg/kg	0.01*	RA = 0.075	RA = 0.029	3.86

MRL: maximum residue level; GAP: Good Agricultural Practice; GM: genetically modified; Mo: according to the residue definition for enforcement; RA: according to the residue definition for risk assessment.

^aNEU: Outdoor trials conducted in northern Europe, SEU: Outdoor trials conducted in southern Europe, Indoor: indoor EU trials or Country code: if non‐EU trials.

^bResidues of 2,4‐DCP (free and conjugated) were adjusted for molecular weight (2,4‐D/2,4‐DCP = 221/163) by a factor of 1.36 to express them as 2,4‐D prior to be summed up.

^cThe asterisk indicates that the MRL is set at the limit of analytical quantification (LOQ).

^dHighest residue expressed on the basis of the residue definition for risk assessment.

^eSupervised trials median residue expressed on the basis of the residue definition for risk assessment.

^fMedian conversion factor to recalculate residues according to the residue definition for monitoring to the residue definition for risk assessment. Since in 9 trials residues were below the LOQ according to both residue definition for enforcement and risk assessment, the CF was not calculated.

Residues of 2,4‐D were found to be stable for up to 18 months in high water, high starch content and dry matrices and up to 12 months in high oil content matrices (EFSA, 2014). New frozen storage stability on 2,4‐D and 2,4‐DCP with samples stored at −20°C was submitted (Greece, 2015). In soyabean seeds, both parent and the metabolite 2,4‐DCP showed to be stable for a period of 28 months, which fully covered the storage of field trial samples (13.5 months).

EFSA concludes that a sufficient number of GAP‐compliant supervised residue trials is available to support the notified uses in GM soyabeans expressing the AAD‐12 protein. The data suggest that the lowering of the existing MRL set at the LOQ of 0.05 mg/kg to a lower LOQ of 0.01 mg/kg could be considered. The MRL/tolerance in the countries of origin set for 2,4‐D is 0.02 mg/kg.

3.1.1.3. Effect of industrial processing and/or household preparation

Standard hydrolysis studies addressing the nature of residues in processed products and studies on the magnitude of 2,4‐D residues in processed commodities are not required as the residue levels in raw agricultural commodities (RAC) did not exceed the trigger value of 0.1 mg/kg (European Commission, 1997d).

Two processing studies investigating the magnitude of residues in processed GM soyabeans expressing the AAD‐12 protein (event not specified) were assessed (Greece, 2015). The studies were conducted applying an exaggerate application rate (1.8N) compared to the rate defined in the critical GAP. Samples were analysed for 2,4‐D and 2,4‐DCP.

Parent 2,4‐D was not found in any of the unprocessed and the processed products (i.e. meal, hull, refined oil), except in soyabean aspirated grain fractions (AGF). 2,4‐DCP was found in quantifiable concentrations in unprocessed seeds and in processed soyabean meal, AGF and hulls, but not in refined oil. In meal and AGF, the concentrations of 2,4‐DCP were higher compared to unprocessed soybeans. The results of the submitted processing studies are reported in the Table 4.

Table 4.

Overview of the available processing studies

Crop (RAC), Processed product	Number of studies	Individual residues (mg/kg)		Transfer ratio for 2,4‐DCPa individual (mean)
		2,4‐D	2,4‐DCP
Soyabean, seed (RAC)	2	2 × ND	0.012; 0.109	–
Soyabean, meal	2	2 × ND	0.015; 0.155	1.29; 1.42 (1.35)
Soyabean, AGF	2	0.012; 0.016	0.074; 0.831	6.34; 7.62 (6.98)
Soyabean, hull	2	2 × ND	0.010; 0.046	0.042; 0.86 (0.64)
Soyabean, refined oil	2	2 × ND	2 × ND	–

RAC: raw agricultural commodity; 2,4‐DCP: 2,4‐dichlorophenol.

^aTransfer ratio was calculated as 2,4‐DCP residues in processed product/2,4‐DCP residues in raw commodity. When residues were not detectable (ND) in both raw and processed products, the ratio was not calculated.

Due to the lack of investigation on the nature of residues in processed commodities and the limited dataset, reliable processing factors (PF) could not be derived. However, the transfer ratio (TR) was used to perform the livestock dietary burden calculation related to soybean feed products (i.e. soybean meal).

3.1.2. Rotational crops

The residues of 2,4‐D and its metabolite 2,4‐DCP in rotational crops are not relevant for the assessment of import tolerances applications.

3.2. Nature and magnitude of residues in livestock

As soybean meal produced from imported GM soybeans may be fed to livestock, the potential transfer of residues into animal commodities was assessed (European Commission, 1996).

3.2.1. Dietary burden of livestock

The most recent livestock dietary burden was conducted in the framework of the MRL review taking into account the authorised EU uses of 2,4‐D (EFSA, 2011).14 In the framework of the current application, the dietary burden was updated including the expected contribution of residues in GM soybean meal, taking into account that residues of 2,4‐DCP in meal are likely to be higher than in soybean seeds. Thus, the median residue level expected in soybean seeds was multiplied by the TR of 1.35 derived in the processing studies (see Table 4). The dietary burden calculation was performed according to the methodology described in the EU guideline 7031/IV/95 rev.4 on livestock feeding studies (European Commission, 1996), which was applicable at the time of submission of the MRL application.

The input values for the dietary burden calculation are summarised in Table 5. The results of the calculations are presented in Table 6.

Table 5.

Input values for the livestock dietary burden calculation

Food commodity	Median dietary burden		Maximum dietary burden
	Input value (mg/kg)	Commenta	Input value (mg/kg)	Comment
Risk assessment residue definition: Sum of 2,4‐D, its salts, esters and conjugates expressed as 2,4‐D
Grass (fresh & silage)	12.60	STMR (EFSA, 2011)	26.00	HR (EFSA, 2011)
Maize silage	0.055	STMR (EFSA, 2011)	0.060	HR (EFSA, 2011)
Apple pomace	0.010	STMR (EFSA, 2011)	0.010	HR (EFSA, 2011)
Grass (hay)	50.40	STMR × PF (EFSA, 2011)	104.00	HR × PF (EFSA, 2011)
Cereal grain	0.05	STMR (EFSA, 2011)	0.05	STMR (EFSA, 2011)
Maize grain	0.05	STMR (EFSA, 2011)	0.05	STMR (EFSA, 2011)
Cereal bran	0.40	STMR × PF (EFSA, 2011)	0.40	STMR × PF (EFSA, 2011)
Cereal straw	0.05	STMR (EFSA, 2011)	1.88	HR (EFSA, 2011)
Risk assessment residue definition (GM soybean): Sum of 2,4‐D, its salts, esters and conjugates, and 2,4‐DCP and its conjugates, expressed as 2,4‐D
Soyabean meal	0.04	STMR × TR	0.04	STMR × TR

STMR: supervised trials median residue; HR: highest residue; PF: processing factor.

^aFor grass hay and cereal bran, in the absence of processing factors supported by data, default processing factors of 4 and 8, respectively, were included in the calculation to consider the potential concentration of residues in these commodities (EFSA, 2011). For soybean meal, to consider the observed concentration of 2,4‐DCP residues, instead of the default processing factor of 1.3, a mean transfer ratio (TR) of 1.35 derived for 2,4‐DCP only based on experimental data was used.

Table 6.

Results of the dietary burden calculation

	Maximum dietary burden (mg/kg bw per day)	Median dietary burden (mg/kg bw per day)	Highest contributing commodity	Max dietary burden (mg/kg DM)		Trigger exceed (Y/N)
				w/soyab	w/out
Dairy ruminants	4.727	2.291	Grass, fresh	130.00	130.00	Yes
Meat ruminants	5.571	2.700	Grass, fresh	130.00	130.00	Yes
Poultry	0.005	0.005	Wheat, bran	0.072	0.067	No
Pigs	0.784	0.382	Grass, silage	19.60	19.59	Yes

bw: body weight; DM: dry matter.

Comparing the results obtained with and without the contribution of expected residues in GM soybean meal, it becomes evident that the notified uses do not contribute to the current livestock dietary exposure of 2,4‐D in ruminants and pigs. The contribution of residue in soybean meal in the poultry diet was not significantly changing the livestock intake that was still not exceeding the trigger value of 0.1 mg/kg dry matter (DM). Thus, EFSA concludes that the import of soybean seeds treated in the countries of origin according to the notified uses will not trigger a revision of the existing MRLs.

4. Consumer risk assessment

The consumer risk assessment was performed with revision 2 of the EFSA PRIMo. This exposure assessment model contains the relevant European food consumption data for different subgroups of the EU population (EFSA, 2007).15 The estimated exposure was then compared with the toxicological reference values derived for 2,4‐D during the EU pesticides peer‐review renewal process (European Commission, 2017).

The most recent long‐term exposure assessment performed by EFSA (2017) for the residues of 2,4‐D was considering the existing uses at EU level and the acceptable CXLs (EFSA, 2011) and was updated to reflect the MRLs set by Regulation (EU) No 1317/2013 on certain products of animal origin. The calculation has been revised by including the STMR derived for GM soybeans from the trials submitted.

At the time of the MRL review, an acute consumer exposure assessment was not performed as an ARfD was not yet established (EFSA, 2011). During the renewal process of the active substance, an acute reference value was set for 2,4‐D (European Commission, 2017). Therefore, EFSA performed the acute exposure assessment for soyabeans, assuming the consumption of a large portion of the food item as reported in the national food surveys and that this item contained residues at the STMR level.

The input values used for the dietary exposure calculation are summarised in Table 7.

Table 7.

Input values for the consumer dietary exposure assessment

Commodity	Chronic exposure assessment		Acute exposure assessment
	Input (mg/kg)	Comment	Input (mg/kg)	Comment
Risk assessment residue definition (GM soybean): Sum of 2,4‐D, its salts, esters and conjugates, and 2,4‐DCP and its conjugates, expressed as 2,4‐D
Soyabeans	0.03	STMR	0.03	STMR
Risk assessment residue definition: Sum of 2,4‐D, its salts, esters and conjugates, expressed as 2,4‐D
Citrus fruits	0.31	STMR (EFSA, 2011)	Acute risk assessment undertaken only for soyabeans
Tree nuts	0.05	STMR (EFSA, 2011)
Pome fruits	0.01	STMR (EFSA, 2011)
Stone fruits	0.01	STMR (EFSA, 2011)
Berries and small fruits	0.05	STMR (EFSA, 2011)
Potatoes	0.05	STMR (EFSA, 2011)
Sweet corns	0.05	STMR (EFSA, 2011)
Asparagus	0.05	STMR (EFSA, 2011)
Barley and oats grain	0.05	STMR (EFSA, 2011)
Buckwheat grain	0.05	MRL (EFSA, 2011)
Maize, millet, sorghum grain	0.05	STMR (EFSA, 2011)
Rice grain	0.01	STMR (EFSA, 2011)
Wheat and rye grain	0.22	STMR (EFSA, 2011)
Sugar cane	0.01	STMR (EFSA, 2011)
Muscle, fata	0.125	STMR (FAO, 1998)
Liver, kidney, edible offala	2.75	STMR (FAO, 1998)
Poultry, products	0.05	MRL (LOQ)
Milks	0.01	MRL (LOQ)
Bird's eggs	0.01	MRL (LOQ)

STMR: supervised trials median residue;; MRL: maximum residue level; LOQ: limit of quantification.

^aProducts of swine, bovine, sheep, goat, equine, other farmed terrestrial animals.

The estimated acute exposure was then compared with the toxicological reference values derived for 2,4‐D (see Table 1). The results of the intake calculation using the EFSA PRIMo is a key supporting document and is made publicly available as a background document to this reasoned opinion.

Using the revised toxicological reference values, the highest chronic intake was calculated to be 34% of the ADI (IE adult diet). The contribution of residues in soybeans to the overall chronic exposure was low (maximum 0.1% of the ADI). The highest acute consumer exposure for soyabean was calculated to be 0.02% of the ARfD.

EFSA concludes that the residues of parent 2,4‐D and of the metabolite 2,4‐DCP, which was found in the GM soybeans expressing the AAD‐12 protein treated with 2,4‐D according to the notified good agricultural practices assessed, will not result in a consumer exposure exceeding the toxicological reference values for 2,4‐D. The data submitted in support of the request provided sufficient evidence to conclude that the risk for consumers is unlikely.

Conclusions and recommendations

The information submitted was sufficient to propose the MRL summarised in the table below:

Codea	Commodity	Existing EU MRL (mg/kg)	Proposed EU MRL (mg/kg)	Comment/Justification
Enforcement residue definition: 2,4‐D (sum of 2,4‐D, its salts, its esters and its conjugates, expressed as 2,4‐D)
0401070	Soyabeans	0.05* (ft)	0.05* or 0.01* (further risk management consideration)	The submitted data are sufficient to perform a consumer risk assessment for the use of 2,4‐D in support the import tolerance request. The lowering of the existing MRL set at the LOQ of 0.05 mg/kg to the LOQ of 0.01 mg/kg as derived from the residue trials on 2,4‐D‐tolerant soybeans (AAD‐12 protein) and achievable with the validated enforcement analytical methods is an option to be discussed by risk managers. Risk for consumers unlikely MRL/Tolerance in the countries of origin (Canada and USA) is 0.02 mg/kg The data gap identified by EFSA for additional information on the analytical method (ft) has been previously addressed

MRL: maximum residue level; LOQ: limit of quantification; AAD‐12: aryloxyalkanoate dioxygenase‐12.

*Indicates that the MRL is set at the limit of analytical quantification (LOQ).

^aCommodity code number according to Annex I of Regulation (EC) No 396/2005.

ft: The European Food Safety Authority identified some information on analytical methods as unavailable. When reviewing the MRL, the Commission will take into account the information referred to in the first sentence, if it is submitted by 17 December 2015, or, if that information is not submitted by that date, the lack of it.

Abbreviations

2,4‐DCP

2,4‐dichlorophenol

a.s.

active substance

AAD‐1

aryloxyalkanoate dioxygenase 1

AAD‐12

aryloxyalkanoate dioxygenase‐12

ADI

acceptable daily intake

ARfD

acute reference dose

BBCH

growth stages of mono‐ and dicotyledonous plants

bw

body weight

CF

conversion factor for enforcement to risk assessment residue definition

CIPAC

Collaborative International Pesticide Analytical Council

CXL

Codex maximum residue limit (Codex MRL)

DALA

days after last application

DAT

days after treatment

eq

equivalent

EMS

evaluating Member State

GAP

good agricultural practice

GC–MS

gas chromatography‐mass spectrometry

GM

genetically modified

HPLC

high‐performance liquid chromatography

HR

highest residue

ILV

independent laboratory validation

InChiKey

International Chemical Identifier Key

ISO

International Organisation for Standardisation

IUPAC

International Union of Pure and Applied Chemistry

LOAEL

lowest‐observed‐adverse‐effect level

LOQ

limit of quantification

MRL

maximum residue level

MS

Member State

MS/MS

tandem mass spectrometry detector

MW

molecular weight

NEU

northern Europe

OECD

Organisation for Economic Co‐operation and Development

PAT

phosphinothricin acetyltransferase

PF

processing factor

PHI

preharvest interval

PRIMo

(EFSA) Pesticide Residues Intake Model

RAC

raw agricultural commodity

RAR

renewal assessment report

SANCO

Directorate‐General for Health and Consumers

SEU

southern Europe

SMILES

simplified molecular‐input line‐entry system

SL

soluble concentrate

STMR

supervised trials median residue

TF

transfer ratio

TMDI

theoretical maximum daily intake

TRR

total radioactive residue

WHO

World Health Organization

Appendix A – Good Agricultural Practice (GAPs)

1.

Crop and/or situationa	MS or NEU/SEU or Country	F G or Ib	Pest or group of pests controlledc	Formulation		Application				Application rate per treatment			PHI (days)l	Remarksm
				Type d,e,f	Conc. a.s.i	Method kindf,g,h	Growth stage & seasonj	Number min–max k	Interval min–max	g/hL min–max	Water L/ha min–max	kg/ha min–max
GM Soyabean (expression of AAD‐12 protein)	CA	F	Annual and perennial weeds	SL	456 g/L	Spraying	Up to BBCH 65	2	12 days		50–200	0.82	n.a.
	US						Pre‐emergence (1)+ post‐emergence (2) up to BBCH 65	3	Post‐emergence: 12 days		50–94	1.12	n.a.	Critical GAP

NEU: northern Europe; SEU: southern Europe; MS: Member State; a.s.: active substance; AAD‐12: aryloxyalkanoate dioxygenase‐12; SL: soluble concentrate.

^aFor crops, EU or other classifications, e.g. Codex, should be used; where relevant, the usage situation should be described (e.g. fumigation of a structure).

^bOutdoor or field use (F), glasshouse application (G) or indoor application (I).

^ce.g. biting and sucking insects, soil‐borne insects, foliar fungi, weeds.

^de.g. wettable powder (WP), water‐soluble granule (WG).

^eGCPF Codes ‐ GIFAP Technical Monograph No 2, 1989.

^fAll abbreviations must be explained.

^gMethod, e.g. high volume spraying, low volume spraying, spreading, dusting, drench.

^hKind, e.g. overall, broadcast, aerial spraying, row, individual plant, between the plants. type of equipment used must be indicated.

ⁱg/kg or μg/L.

^jGrowth stage at last treatment (Meier U, 2001. Growth Stages of mono‐ and dicotyledonous plants. BBCH Monograph, 2nd Ed., Federal Biological Research Centre of Agriculture and Forestry, Braunschweig, Germany, 2001), including where relevant, information on season at time of application.

^kThe minimum and maximum number of application possible under practical conditions of use must be provided.

^lPHI: minimum preharvest interval.

^mRemarks may include: Extent of use/economic importance/restrictions.

Appendix B – Used compound codes

1.

Code/Trivial name	IUPAC name/SMILES notation/InChiKeya	Structural formulab
2,4‐D	(2,4‐dichlorophenoxy)acetic acid Clc1cc(Cl)ccc1OCC(=O)O OVSKIKFHRZPJSS‐UHFFFAOYSA‐N
2,4‐DCP	2,4‐dichlorophenol Clc1cc(Cl)c(O)cc1 HFZWRUODUSTPEG‐UHFFFAOYSA‐N
2,4‐DB	4‐(2,4‐dichlorophenoxy)butyric acid Clc1cc(Cl)ccc1OCCCC(=O)O YIVXMZJTEQBPQO‐UHFFFAOYSA‐N

IUPAC: International Union of Pure and Applied Chemistry; SMILES: simplified molecular‐input line‐entry system InChiKey: International Chemical Identifier Key.

^aACD/Name 2015 ACD/Labs 2015 Release (File version N20E41, Build 75170, 19 December 2014).

^bACD/ChemSketch 2015 ACD/Labs 2015 Release (File version C10H41, Build 75059, 17 December 2014).

Suggested citation: EFSA (European Food Safety Authority) , Anastassiadou M, Brancato A, Carrasco Cabrera L, Ferreira L, Greco L, Jarrah S, Kazocina A, Leuschner R, Magrans JO, Miron I, Pedersen R, Raczyk M, Reich H, Ruocco S, Sacchi A, Santos M, Stanek A, Tarazona J, Theobald A and Verani A, 2019. Reasoned Opinion on the setting an import tolerance for 2,4‐D in soyabeans. EFSA Journal 2019;17(4):5660, 20 pp. 10.2903/j.efsa.2019.5660