Document ID: EPA-HQ-OPP-2018-0245-0006
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2019-08-09T04:00Z

UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
                            WASHINGTON, D.C. 20460
                                       

                                                     OFFICE OF CHEMICAL SAFETY 
                                                       AND POLLUTION PREVENTION

MEMORANDUM

DATE:	May 3, 2019

SUBJECT: 	Lipochitooligosaccharide (LCO) MOR116: Summary of Hazard and Science Policy Council (HASPOC) Meeting on May 2, 2019:  Recommendations on the Need for the Following Subchronic Studies: 90-Day Oral Study (OCSPP 870.3000), 90-Day Dermal (OCSPP 870.3250), 90-Day Inhalation (OCSPP 870.3465), Prenatal Developmental Study (OCSPP 870.3700), and Genetic Toxicity Testing (OCSPP 870.5100, 870.5300, 870.5375).
 
PC Code:  006389

DP Barcode:  N/A
Decision No.:  N/A

Registration No.:  N/A
Petition No.:  N/A

Regulatory Action:  N/A
Risk Assessment Type: N/A

Case No.:  N/A
TXR No.: 0057865 

CAS No.: 163181-92-8
MRID No.:  N/A

40 CFR:  N/A

FROM:	Janet Camp, Executive Secretary
HASPOC
Health Effects Division (7509P)
				
THROUGH:	Hannah Pope-Varsalona, Ph.D., Co-Chair
Kelly Lowe, Co-Chair
HASPOC
Health Effects Division (7509P)

TO:	Elyse Osterweil, Ph.D., Toxicologist 
            Catherine Eiden, Senior Advisor
Risk Assessment Branch (RAB)
            Biopesticides and Pollution Prevention Division (7511P)

MEETING ATTENDEES:  
	
HASPOC Members:	Jonathan Chen, Evisabel Craig, Angela Gonzales, Kelly Lowe*, Elizabeth Mendez, Michael Metzger, Hannah Pope-Varsalona*, Elissa Reaves, Kristin Rickard, Brian VanDeusen
	* Co-chair

Presenter: 	Elyse Osterweil, Ph.D., Toxicologist

Other Attendees: 	Janet Camp**, Ruthanne Louden**
	** Secretary
 

 PURPOSE OF MEETING

The Biochemical Pesticide Branch (BPB) is evaluating a request for the registration of lipochitooligosaccharide (LCO) MOR116, a new active ingredient (a.i.). It is a synthetically derived member of the LCO chemical class and practically identical to the previously registered LCO SP104 (EPA File Symbol No 524-AUN). On October 2, 2017, HASPOC considered it acceptable to bridge data on LCOs and their metabolites (such as chitin, vaccenic acid, and GlcNAC) to LCO SP104. The toxicology database for LCO MOR116 is complete except for a 90-Day Oral Study (OCSPP 870.3000), 90-Day Dermal (OCSPP 870.3250), 90-Day Inhalation (OCSPP 870.3465), Prenatal Developmental Study (OCSPP 870.3700), and Genetic Toxicity Testing (OCSPP 870.5100, 870.5300, 870.5375), which are required in accordance with the current 40 CFR Part 158.2050 Toxicology Data Requirements.  The Hazard and Science Policy Council (HASPOC) met on May 2, 2019 to determine if the required studies are necessary to support the registration for LCO MOR116.

 SUMMARY OF USE PROFILE, EXPOSURE, AND HAZARD CONSIDERATIONS

 Use and Exposure Profile
A new active ingredient (a.i.), LCO MOR116, is intended for use as a plant growth regulator applied as a seed treatment for soybean seeds to facilitate root nodulation and colonization of bacteria on the root and promote mycorrhizal symbiosis. This a.i. is very functionally and structurally similar to other lipochitoosaccharides and the registered a.i. LCO SP104 (PC Code 006388), but differs by one N-acetyl-D-glucosamine (GlcNAC) group (see Figure 1). LCOs are signaling molecules involved in plant-microbe endosymbiotic interactions in an estimated 70-80% of land plants. LCOs and their metabolites (such as chitin, vaccenic acid, and GlcNAC) are ubiquitous in the environment in the roots of food crops and associated endosymbiotic bacteria and fungi, regularly consumed as part of a normal diet, easily digested, and not expected to bioaccumulate. Chitin backbone is composed of linked GlcNAC subunits, which are endogenously present in human tissues and breastmilk.

Figure 1. Structure of (a) LCO MOR116 and (b) LCO SP104

 LCO MOR116

   
   
   
   
   
   
   
   
   
   (b) LCO SP104

Pesticidal use of LCO MOR116 is unlikely to contribute significantly to overall human exposure. Dietary exposure is expected to be negligible as significant residues of the substance are not anticipated on treated commodities at the time of consumption when used as a seed treatment. Moreover, the proposed end-use product (EP) contains 2.66 X 10[-5] % LCO MOR116 and is composed of greater than 99% water. There is potential for occupational exposure; however, no toxicological endpoints were identified from the toxicology database for any routes of exposure and the application rates for the proposed use as a seed treatment are very low. No residential uses have been proposed. An aggregate risk assessment for dietary (food and drinking water) exposures was not conducted as no toxicological endpoints have been identified in the toxicity database.

According to the product labels, the proposed manufacturing-use product (MP) LCO MOR 116 (EPA File Symbol No. 524-ALU) contains 0.013% LCO and the EP LCO Liquid Additive (EPA File Symbol No. 524-ALG) contains 2.66 X 10[-][5] % LCO MOR116. The proposed use is as a plant growth regulator for use as a seed treatment on soybean seeds as a water-based slurry via mixing. The product is to be applied at a maximum application rate of 5.89 X 10[-][11] lb ai/lb seed. The proposed labels require the use of personal protective equipment (PPE; long-sleeved shirt and long pants, shoes and socks, and chemical resistant gloves) for mixers, loaders, applicators, and other handlers.

LCO MOR116 is characterized by the applicant as highly soluble in water and it is expected to degrade relatively rapidly once applied since it can function as a carbon source for microorganisms. Therefore, is not likely to result in significant residues on food or in water and is not likely to bioconcentrate. The physical and chemical properties of LCO MOR116 indicate it is readily biodegradable and will degrade rapidly in the environment through chitinases and soil microbes.

 Toxicity Profile

The Tier 1 acute mammalian toxicology studies were conducted in accordance with the biochemical pesticide data requirements (40 CFR§ 158.2050) and indicate that the proposed MP, LCO MOR116, is unlikely to present a human health hazard. Briefly, the acute Toxicity Categories for LCO MOR116 were determined to be: acute oral toxicity is Category IV (LD50 > 5000 mg/kg); dermal acute toxicity is Category IV (LD50 > 5000 mg/kg); inhalation toxicity is Category IV (LC50 > 5.14 mg/L); minimally irritating for acute eye irritation; minimally irritating for acute dermal irritation; and not a dermal sensitizer. Based on the information presented, LCO MOR116 is expected to have low acute toxicity (MRIDs 50464807 to 50464812).

No subchronic or chronic toxicity studies were submitted for LCO MOR116. In lieu of toxicity data on LCO MOR 116, studies from metabolites and constituents of LCO, such as chitin, GlcNAc, and OAG were used and are considered acceptable surrogates for LCO SP104 and LCO MOR116. Eight repeat dose studies are available for compounds related to LCOs that are adequate for use in risk assessment for LCO MOR116 and indicate a lack of adverse effects. A 90-day oral toxicity rat study performed on OAG indicates a POD value of 641 mg/kg/day, based on decreased weight of salivary glands. All other studies showed no adverse effects at or above the limit dose of 1000 mg/kg/day for structurally similar compounds chitin, GlcNAc, etc.

 LCO MOR116 Mode of Action (Taken from MRID 49819918)
   
LCOs comprise a class of molecules that act as Nod and Myc factors by promoting plant microbial symbiosis, soil microbial activity, and associated plant growth. When a plant senses the need for a nutrient such as nitrogen and has the capability of entering into Nod factor mediated symbiosis, it releases plant flavonoids that are recognized by rhizobia or other bacteria
capable of nodulation. The bacteria sense the flavonoids and produce LCO molecules that signal
the plant to initiate root nodulation, permitting the bacteria to colonize the plant roots and
provide atmospherically rich nitrogen to the plant. LCOs, acting as either Nod or Myc factors,
activate the DMI (stands for doesn't make infections) pathway by binding to transmembrane receptors that are comprised of extracellular lysine motifs and intracellular kinase domains (Gough and Cullimore, 2011). These receptors and the DMI pathway are not known to be present in humans or other animals and it therefore not considered a mammalian MOA. Furthermore, their presence in plants is not limited to leguminous species.

 History of Safe Use (Taken from MRID 49819919)
Nod and Myc factors are naturally occurring LCOs that have been involved in plant-microbe symbioses for millions of years. The majority of land plants rely on these signaling pathways to regulate effective microbe symbioses. They all belong to the same class of signaling molecules and share the common structural features of a chitin/chitooligomer backbone and fatty acid side chain. Food and feed crops, including corn and soybean, share in these signaling pathways with soil symbionts. LCO SP104 can act as a Nod and/or Myc factor, and act at concentrations similar to other LCOs, namely in the nano- to picomolar concentration range (Tanaka et al., 2015). Since LCOs acting as Nod and Myc factors evolved tens to hundreds of millions of years ago, and today are active signal transducers in the majority of land plants, there is a vast history of safe use/exposure to LCOs present in, or originating from, the rhizosphere.

   STUDY WAIVER REQUESTS

 Quantitative vs. Qualitative Risk Assessment

HASPOC recommends that a qualitative risk assessment is appropriate to support the proposed use of LCO MOR116. The qualitative determination is based on the following: (1) the low overall acute toxicity of LCO MOR116; (2) humans are already exposed to LCOs with structural similarity to LCO MOR116 as they are naturally occurring and have long been part of the normal diet (seafood and plants); and (3) dietary and non-dietary exposure from use as a seed treatment is expected to be negligible based on PPE requirements, very low application rates, and the composition of the proposed end-use product which contains more than 99% water.

 90-day Oral Toxicity (OCSPP 870.3100)
   
The 90-day oral toxicity study is required to register food uses for a pesticide. In MRID 50738202, data and information were cited from the open scientific literature to satisfy the data requirement. Eight studies (two 28-day, four 90-day, a 1-year, and a 2-year oral toxicity) are available for compounds related to LCOs that are adequate for use in risk assessments and indicate a lack of adverse effects. These structurally-similar compounds are chitooligomers (LCO degradation product), chitosan oligosaccharide (also an LCO degradation product), chitin-glucan, chitin, N-acetylglucosamine, and oligo-N-acetylglucosamine and are considered acceptable surrogates for LCO SP104 and LCO MOR116. Summaries of the eight studies are provided in Table 1 in the Appendix. Based on the results of the available toxicity studies, adverse effects of toxicological concern are not anticipated from exposure to LCO MOR116 as a biopesticide. Therefore, a qualitative assessment will be performed.

Prior data waivers for all subchronic toxicity requirements were granted by the Agency for use of
chitin as food additive, and chitosan and LCO SP104 as plant growth regulators. The rationales for the waivers were based on the low concentration of the active ingredient in pesticide products, the ingredient's low toxicity, and the natural ubiquity of the ingredient in the environment. In addition, the Agency issued a tolerance exemption for chitin and chitosan.

LCOs with structural similarity to LCO MOR116 are present in the roots of food crops, as they are signaling molecules that diffuse into the roots, in addition to being present in bacteria and fungi that are associated with the roots of food crops. Molecules identical to LCO breakdown products are also present in insects, crustaceans, fungi, bacteria, and humans, and are regularly consumed as part of a normal diet.

Based on a WOE approach, considering all the available LCO MOR116 hazard and other
data on similar compounds, the HASPOC recommends that a 90-day oral
study on LCO MOR116 is not required at this time. This approach included the following considerations: (1) several oral toxicity studies conducted on similar compounds to LCO MOR116 showed no adverse effects at the highest dose tested; (2) the acute toxicology studies for the MP LCO MOR116 demonstrate Toxicology Category IV, suggesting that the compound has low toxicity on an acute basis; (3) LCO MOR116 is naturally occurring and has long been part of the normal diet (seafood and plants); (4) prior data waivers for all subchronic toxicity requirements were granted by the Agency for use of chitin and chitosan as food additives and plant growth regulators, respectively; (5) mode of action is not relevant outside the plant kingdom; (6) there is an existing U.S. EPA tolerance exemption for chitin and chitosan; (7) naturally occuring-ubiquity in the environment; (8) due to low toxicity, a qualitative assessment will be performed; and 9) the low application rates of LCO MOR116.
   
 90-Day Dermal Toxicity
   
The 90-day dermal study is a conditional data requirement for pesticides with food use registrations. 

Based on a WOE approach, considering all the available LCO MOR116 hazard and other data on similar compounds, the HASPOC recommends that a 90-day dermal toxicity study on LCO MOR116 is not required at this time. This approach included the following considerations: (1) several oral toxicity studies conducted on similar compounds to LCO MOR116 showed no adverse effects at the highest dose tested; (2) the acute toxicology studies for the MP LCO MOR116 demonstrate Toxicology Category IV, suggesting that the compound has low toxicity on an acute basis; (3) LCO MOR116 is naturally occurring and has long been part of the normal diet (seafood and plants); (4) prior data waivers for all subchronic toxicity requirements were granted by the Agency for use of chitin and chitosan as food additives and plant growth regulators, respectively; (5) mode of action is not relevant outside the plant kingdom; (6) there is an existing U.S. EPA tolerance exemption for chitin and chitosan; (7) naturally occuring-ubiquity in the environment; (8) due to low toxicity, a qualitative assessment will be performed; and 9) the low application rates of LCO MOR116.

 90-Day Inhalation Toxicity

The 90-day inhalation study is a conditional data requirement for pesticides with food use registrations. 

Based on a WOE approach, considering all the available LCO MOR116 hazard and other data on similar compounds, the HASPOC recommends that a 90-day inhalation toxicity study on LCO MOR116 is not required at this time. This approach included the following considerations: (1) several oral toxicity studies conducted on similar compounds to LCO MOR116 showed no adverse effects at the highest dose tested; (2) the acute toxicology studies for the MP LCO MOR116 demonstrate Toxicology Category IV, suggesting that the compound has low toxicity on an acute basis; (3) LCO MOR116 is naturally occurring and has long been part of the normal diet (seafood and plants); (4) prior data waivers for all subchronic toxicity requirements were granted by the Agency for use of chitin and chitosan as food additives and plant growth regulators, respectively; (5) mode of action is not relevant outside the plant kingdom; (6) there is an existing U.S. EPA tolerance exemption for chitin and chitosan; (7) naturally occuring-ubiquity in the environment; (8) due to low toxicity, a qualitative assessment will be performed; and 9) the low application rates of LCO MOR116.

 Prenatal Developmental Toxicity
   
Prenatal developmental toxicity studies are required in the 2015 CFR Part 158 Toxicology Data Requirement because they provide important scientific information on the potential maternal and developmental toxicity effects from pesticide exposure.
   
 Evidence for prenatal development toxicity in the LCO MOR116 database: There are no repeated-dose toxicity studies for LCOs. The toxicity profile for LCO MOR116 does not indicate any specific organs that are the primary target organ.

Evidence for prenatal development toxicity in the toxicology database of similar chemicals:  Chemicals with structural similarity to LCO SP104 are present in the roots of food crops as they are signaling molecules which diffuse into the roots, in addition to being present in bacteria and fungi that are associated with the roots of food crops. Molecules identical to LCO breakdown products are also present in insects, crustaceans, fungi, bacteria and humans, and are regularly consumed as part of a normal diet. No toxicity information was provided on prenatal exposure to LCOs.  The use of LCO MOR116 as a plant growth regulator is not expected to increase exposure to women of childbearing age above background levels

Risk assessment considerations: A waiver is requested for the prenatal developmental toxicity data requirement for LCO MOR116 due to low acute oral, dermal, and inhalation toxicity (EPA Toxicity Category IV) and the 90-day oral toxicity of similarly-structured compounds having NOAELs greater than 2000 mg/kg in rats. LCOs are present in the roots of food crops, have long been part of the normal diet (e.g. seafood and plants). A qualitative assessment will be performed on this chemical.

Based on a WOE approach, considering all the available LCO MOR116 hazard and exposure data, the HASPOC recommends that a prenatal developmental study is not required at this time. This approach included the following considerations: (1) LCO MOR116 is naturally occurring and has long been part of the normal diet (seafood and plants); (2) the acute toxicology studies are classified as Toxicity Category IV, indicating low acute toxicity for LCO MOR116, and 90-day oral toxicity studies of similarly-structured compounds (chitin-glucan, chitin, N-acetylglucosamine, and oligo-N-acetylglucosamine) have found NOAELs greater than 2000 mg/kg in rats; (3) prior data waivers for all subchronic toxicity requirements were granted by the Agency for use of chitin and chitosan as food additives and plant growth regulators, respectively; (4) mode of action is not relevant outside the plant kingdom; (5) there is an existing U.S. EPA tolerance exemption for the related compounds, chitin and chitosan; (6) ubiquity in the environment; (7) due to the low toxicity for this chemical, a qualitative assessment will be performed; and (8) the low application rates of LCO MOR116.
 Genetic Toxicity Testing (OSCPP 870.5100, 5300, 5375)
   
Mutagenicity data are required to support food uses but are unavailable for LCO MOR116. The registrant cited three genotoxicity studies performed on structurally similar compounds to LCO (chitooligomers and N-acetylglucosamine). Fernandes et al. evaluated the genotoxicity of two low molecular weight chitooligosaccharides using fresh peripheral blood lymphocytes both in micronucleus and comet assays. No evidence of genotoxicity was observed in either assay (Fernandes et al., 2011). Qin et al. evaluated the genotoxicity of a chitooligomer preparation derived from shrimp chitosan (85% deacetylated) in Ames, mouse micronucleus, and mouse sperm abnormality assays. When tested up to 5 μg/plate using E. coli strains TA97, TA98, TA100 and TA102, no increase in mutation frequency was noted. Similarly, when tested in a mouse micronucleus assay, the chitooligomer preparation did not cause an increase in the number of micronuclei when tested up to 5,000 mg/kg (Qin et al., 2006). When tested in mice up to 5,000 mg/kg, there was no increase in sperm abnormalities, indicating a lack of mutagenic or genotoxic potential. Jamialahmadi et al. evaluated the potential genotoxicity of GlcNAc in an in vitro comet assay using human peripheral lymphocytes. Cells were exposed to 2.5, 5, 10, 20, or 40 mM GlcNAc and no genotoxic response was observed (Jamialahmadi et al., 2014).

Based on a WOE approach, considering all the available LCO MOR116 hazard and exposure data, the HASPOC recommends that genetic toxicity testing is not required at this time. This approach included the following considerations: 1) available literature studies demonstrated a lack of genotoxicity for LCOs; 2) structure-activity relationships demonstrating a lack of genotoxic potential; 3) prior genotoxicity data waivers granted by the Agency for use of (structurally similar) chitin and chitosan as food additives and plant growth regulators, respectively; 4) U.S. EPA has established a tolerance exemption for chitin and chitosan; 5) ubiquitous nature of LCOs in the environment; and 6) a History of Safe Use (HOSU) of LCOs.

   HASPOC CONCLUSIONS

Based on the summary information presented to HASPOC and a WOE approach, the HASPOC recommends that a qualitative risk assessment is appropriate for the proposed seed treatment use of LCO MOR116.  

Based on a WOE approach, considering all the available hazard and exposure data for LCO MOR116, HASPOC recommends that the studies for 90-day oral toxicity, 90-day dermal toxicity, 90-day inhalation toxicity, prenatal developmental toxicity, and genetic toxicity testing for LCO MOR116 are not required at this time.

 APPENDIX A

 Table 1. Oral Toxicity Data from the Open Literature for Structurally Similar Compounds (Taken from MRID 49819918).
Chemical
                                  Study Type
                                    NOAELs
                                  (mg/kg/day)
                                    LOAELs
                                  (mg/kg/day)
                               Effects at LOAEL
                                   Reference
Chitooligomers
28-day rat
3,000 (HDT)
-
No adverse effects at HDT
(Qin et al., 2006)
COS (chitosan oligosachharide)
28-day rat
2,000 (HDT)
-
No adverse effects at HDT
(Kim et al., 2001)
Chitin-glucan
90-day rat
6,600 M (HDT)
7,000 F (HDT)
-
No adverse effects at HDT
(Jonker et al., 2010)
Chitin
90-day rat
3,500 (HDT)
-
No adverse effects at HDT
(Niho et al., 1999)
GlcNAc
90-day rat
2,476 M (HDT)
2,834 F (HDT)
-
No adverse effects at HDT
(Lee et al., 2004)
OAG (oligo-N-acetylglucosamine)
90-day rat
641 M (HDT)
3,640 F (HDT)
5,000 ppm M
None F
M: decreased weight of submaxillary salivary gland
(Tago et al., 2007)
GlcNAc
1 yr rat
2,323 M (HDT)
2,545 F (HDT)
-
No adverse effects at HDT
(Takahashi et al., 2009)

GlcNAc
2 yr rat
2,323 M (HDT)
2,545 F (HDT)

-
No adverse effects at HDT
(not carcinogenic)
(Takahashi et al., 2009)
F = female; M = male; HDT = highest dose tested; LOAEL = lowest-observed-adverse-effect-level; NOAEL = no-observed-adverse-effect-level