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> **UNITED STATES ENVIRONMENTAL PROTECTION AGENCY**

**WASHINGTON, D.C. 20460**

> **OFF ICE OF PREVENTION,**

**OFFICE OF PESTICIDES AND TOXIC SUBSTANCES**

**MEMORANDUM**

**DATE:** April 7, 2008

**SUBJECT**: Non-Target Organism and Endangered Species Assessment in
Support of the Registration Review of Chitin
(Poly-N-Acetyl-D-glucosamine) and Chitosan (Poly-D-Glucosamine).
Re-review of Existing Non-target Organism Studies

**Registration Review Case No.**: 6063

**Chemical Class:** Biochemical

**PC Codes:** 128991 (Chitin) & 128930 (Chitosan)

**CAS Numbers:** 1398-61-4 (Chitin) & 9012-76-4 (Chitosan)

> **Tolerance Exemptions:** 40 CFR 180.1089 (Chitin) & 40 CFR 180.1072
> (Chitosan)
>
> **MRID Numbers:** None

**FROM:** Russell S. Jones, Ph.D., Senior Biologist /s/ 04/07/2008

> Biochemical Pesticides Branch
>
> Biopesticides & Pollution Prevention Division (7511P)

**TO:** Chris Pfeifer, Regulatory Action Leader

> Biochemical Pesticides Branch
>
> Biopesticides & Pollution Prevention Division (7511P)

**BACKGROUND**

The Food Quality Protection Act of 1996 mandated the continuous review
of existing pesticides. All pesticides distributed or sold in the United
States must generally be registered by EPA, based on scientific data
showing that they will not cause unreasonable risks to human health,
workers, or the environment when used as directed on product labeling.
The Initial Docket for the Registration Review of Chitin and Chitosan
was established for September 2007 and the Final Workplan is scheduled
for January 2008. In support of the Registration Review, BPPD has
re-evaluated the existing non-target organism studies that have been
submitted in support of the registration of products containing Chitin,
Chitosan, and Chitosan Hydrolysate (a hydrolyzed form of Chitosan).
These data are re-reviewed in this document to support the Endangered
Species Risk Assessment for the Registration Review of Chitin and
Chitosan. No new studies were submitted.

**EXECUTIVE SUMMARY**

Chitin and its closely related active ingredients, Chitosan and Chitosan
Hydrolysate, are not expected to cause any adverse effects in any
non-target organisms, including threatened and endangered species. A
**No Effects (NE)** determination has been made for Chitin and Chitosan
(including Chitosan Hydrolysate). Chitin is ubiquitous in nature and is
found in many terrestrial and aquatic species (see Effects on Non-Target
Organisms and Endangered Species below). Chitosan is a partially
deacylated version of Chitin. Chitosan Hydrolysate is an acid hydrolyzed
version of Chitosan and is comprised of smaller, more water-soluble
chains of Chitosan. Chitin and its derivatives are functionally
identical and have a non-toxic mode of action (see Description of the
Active Ingredients below). There is no direct activity of the active
ingredients against the target pest.

**RECOMMENDATIONS AND CONCLUSIONS**

Based on the existing data, adverse effects on non-target organisms are
highly unlikely and there will be **NO EFFECTS (NE)** on threatened or
endangered species resulting from application of products containing
Chitin and Chitosan (including Chitosan Hydrolysate) when products are
used in accordance with approved labeling.

**NON-TARGET ORGANISMS AND ENDANGERED SPECIES ASSESSMENT**

[Description of the Active Ingredients]{.underline}

Chitin and Chitosan (which includes Chitosan Hydrolysate) are both
comprised of a naturally-occurring chain of glucose molecules linked via
*B*-1,4-linkages, that is structurally related to cellulose. Both
substances are slightly different forms of the same molecule (see Figure
1 below).

Chitin (Poly- N-Acetyl-D-Glucosamine) is commonly found in nature and is
usually derived from crustacean shells, particularly from crabs and
shrimp. Historically, it has been used as a food additive and a
fertilizer. As a pesticide active ingredient, it acts by stimulating the
growth of certain microorganisms in soil, which release substances that
kill pathenogenic nematodes and their eggs. The compound is also reputed
to play a role as a plant growth regulator by bolstering plant defenses
against disease (Systemic Acquired Response, or SAR inducer) (Kramer and
Muthukrishnan, 1998). Chitin does not have any direct toxicity to pests
or non-target organisms.

Chitosan (Poly-D-Glucosamine) is a synthetic compound prepared through
the partial or complete deacetylation of Chitin. Thus, if deacetylation
is incomplete, a Chitosan chain will contain molecules of Chitin
randomly distributed along its length. Chitosan also differs from Chitin
in that it contains an amine group, whereas Chitin contains an amide
group. It is

somewhat more water-soluble than Chitin. Chitosan has biomedical
applications. as a haemostatic agent that is hypoallergenic and, similar
to Chitin, it is known to possess anti-microbial activity. These
properties also allow for its use as an active ingredient in
anti-microbial pesticides. As a pesticidal active ingredient, Chitosan
is best known as a plant growth enhancer that boosts the ability of
plants to defend against fungal infections (also an SAR inducer).
Similar to Chitin, Chitosan has no direct toxicity to pests or
non-target organisms.

Chitosan Hydrolysate is prepared via the acid hydrolysis of Chitosan and
is comprised of smaller chains of Chitosan (also an SAR inducer).
Biologically, there is little functional difference between Chitin,
Chitosan, and Chitosan Hydrolysate.

**Figure 1. The structures of Chitin and Chitosan**.

![](media/image2.png){width="2.7319444444444443in"
height="1.7173611111111111in"} **Chitin**

![](media/image3.png){width="2.954861111111111in"
height="1.7694444444444444in"}

**The actual chemical structure of Chitosan is subject to substantial
variations based on the degree of deacetylation of Chitin; the structure
above depicts a completely deacetylated chain of Chitosan molecules.**

[Current USEPA-registered Products]{.underline}

There is one USEPA-registered product containing Chitin as its active
ingredient: Clandosan 618 \[EPA Reg. No. 54137-1 (formerly EPA Reg. No.
58200-9)\]. There are two USEPA-registered products containing Chitosan
as their active ingredient: ELEXA-4 (EPA Reg. No. 81045-2) and
ChitoSante (EPA Reg. No. 81446-1). There are two products containing
Chitosan Hydrolysate as its active ingredient that are currently in
registration review: Chitosan

Hydrolysate (EPA File Symbol No. 73512-A) and Keyplex 350 (EPA File
Symbol No. 73512-L).

Clandosan 618 is registered for non-food use on ornamentals, lawn, and
turf grasses. Elexa-4 is registered for use on field crops, ornamentals,
turf, home gardens, and nurseries. Chitosante is registered for use
non-food use as an antimicrobial pesticide on textiles and surfaces.
Chitosan Hydrolysate (registration pending) is intended for
manufacturing use only. Keyplex 350 (registration pending) in intended
for use food use on commercial crops, ornamentals, and turf.

The amounts of active ingredient in each product are listed in Table 1
below:

Table 1. Farnesol and Nerolidol Content in USEPA Registered End-Use
Products

+------------------------+-----------+----------------+----------------+
| **Product**            | **P       |                |                |
|                        | ercentage |                |                |
| **(EPA Reg. No.)**     | Active    |                |                |
|                        | In        |                |                |
|                        | gredients |                |                |
|                        | ^1^**     |                |                |
+------------------------+-----------+----------------+----------------+
|                        | *         | **Chitosan**   | **Chitosan     |
|                        | *Chitin** |                | Hydrolysate**  |
+------------------------+-----------+----------------+----------------+
| Clandosan 618          | 25        | \-             |                |
| (54137-1)              |           |                |                |
+------------------------+-----------+----------------+----------------+
| Elexa-4 (81045-2)      | \-        | 4              |                |
+------------------------+-----------+----------------+----------------+
| ChitoSante (81446-1)   | \-        | 6              |                |
| ^2^                    |           |                |                |
+------------------------+-----------+----------------+----------------+
| Chitosan Hydrolysate   | \-        |                | 8.33           |
| (73512-A)^3^           |           |                |                |
+------------------------+-----------+----------------+----------------+
| Keyplex 350 (73512-L)  | \-        | \-             | 0.8            |
| ^4^                    |           |                |                |
+------------------------+-----------+----------------+----------------+

^1^ All active ingredients are closely-related forms of
poly-D-glucosamine.

^2^ An antimicrobial product having no outdoor exposure

^3^ Manufacturing-use only.

^4^ Also contains 1.5% Salicylic acid as an active ingredient

[Effects on Non-Target Organisms and Endangered Species]{.underline}

Chitin and Chitosan (which includes Chitosan Hydrolysate) are both
comprised of a naturally-occurring chain of glucose molecules linked via
*B*-1,4-linkages, that is structurally related to cellulose. Both
substances are slightly different forms of the same molecule (see Figure
1 above).

Chitin is one of the most common polymers found in nature. It is a major
component of the exoskeletons of aquatic and terrestrial arthropods
(insects, crustaceans), the radula of molluscs, and the beaks of
cephalpods (squid, octopus), and the cell walls of fungi (Campbell,
1996). In addition, many species possess chitinases (Bishop et al.,
2000; Kramer and Muthukrishnan, 1998), enzymes that metabolize chitin,
particularly those that consume chitin-containing organisms (MacDonald,
2006; Matsumiya et al. 2006). Chitinases are also found in humans
(Escott and Adams, 1995; Paolettia et al. 2007).

**Table 2. Summary of Non-Target Organism Data/Information for Chitin
(C), Chitosan (CS), and Chitosan Hydrolysate (CSH) End-Use Products
(EP)**

+----------------------+-----------+----------+----------------+------+
| **[Study Type/OPPTS  | **[L      | *        | **[Toxicity    | **   |
| Guid                 | D~50~/LC~ | *[Active | Category]      | [MRI |
| eline]{.underline}** | 50~]{.und | I        | {.underline}** | D]{. |
|                      | erline}** | ngredien |                | unde |
|                      |           | t]{.unde |                | rlin |
|                      |           | rline}** |                | e}** |
+======================+===========+==========+================+======+
| Acute Oral Toxicity  | \>5000 mg | CSH      | Practically    | 4493 |
| (rat) OPPTS 870.1100 | EP/kg ^1^ |          | non-toxic for  | 1205 |
|                      |           |          | EP ^1^         |      |
|                      | (200 mg   |          |                |      |
|                      | a.i./kg)  |          |                |      |
+----------------------+-----------+----------+----------------+------+
|                      | \>5000 mg | CSH      | Practically    | 4679 |
|                      | EP/kg ^2^ |          | non-toxic for  | 0003 |
|                      |           |          | EP ^2^         |      |
|                      | (40 mg    |          |                |      |
|                      | a.i./kg)  |          |                |      |
+----------------------+-----------+----------+----------------+------+
| Freshwater Fish      | See       | C and CS | Practically    | \-   |
| LC50/OPPTS 850.1075  | N         |          | non-toxic ^3^  |      |
| and                  | on-target |          |                |      |
|                      | Aquatic   |          |                |      |
| Freshwater           | Organism  |          |                |      |
| Invertebrate/OPPTS   | summary   |          |                |      |
| 850.1010             | below     |          |                |      |
+----------------------+-----------+----------+----------------+------+
| Avian Acute          | See       | C and CS | Practically    | \-   |
| Oral/OPPTS 850.2100  | N         |          | non-toxic ^3^  |      |
|                      | on-target |          |                |      |
| and                  | Bird      |          |                |      |
|                      | summary   |          |                |      |
| Avian Dietary/OPPTS  | below     |          |                |      |
| 850.2200             |           |          |                |      |
+----------------------+-----------+----------+----------------+------+
| Non-target           | See       | C        | Practically    | \-   |
| Plants/OPPTS         | N         |          | non-toxic ^3^  |      |
| 850.4000             | on-target |          |                |      |
|                      | Plants    |          |                |      |
| and                  | and       |          |                |      |
|                      | Insects   |          |                |      |
| Non-target           | summary   |          |                |      |
| Insects/OPPTS        | below     |          |                |      |
| 850/3020; 850.3030;  |           |          |                |      |
| and/or 850.3040      |           |          |                |      |
+----------------------+-----------+----------+----------------+------+

^1^ Elexa-4 (EPA Reg. No. 81045-2) contains 4.0% CSH.

^2^ Keyplex 350 (EPA File Symbol No. 73512-L) contains 0.8% CSH.

^3^ Based on the non-toxic mode of action of the active ingredients, the
presence of chitin as a naturally occurring component of many non-target
organisms, and the presence of chitinases (enzymes that metabolize
chitin) in many non-target organisms that consume chitin-containing
organisms in their normal diets.

[Non-Target Mammals]{.underline}

An end-use product containing 4% Chitosan hydrolysate has been shown to
be practically non-toxic to rats on acute oral basis (see MRID
44931205). No adverse effects are expected to mammalian wildlife when
chitin and chitosan-containing products are applied in accordance with
approved labeling.

[Non-Target Aquatic Organisms]{.underline}

Chitin is ubiquitous in nature and is a major component of the
exoskeletons of aquatic arthropods (insects, crustaceans), the radula of
molluscs, and the beaks of cephalpods (squid, octopus), the cell walls
of fungi (Campbell, 1996), and the scales of fish (Uawonggul et al.
2002). Chitin is a normal component in the diets of fish (e.g. in the
exoskeletons of aquatic arthropods and in the scales of prey fish) and
fish possess chitinases that metabolize these dietary chitins (Matsumiya
et al. 2006). No mortalities have been observed in fish fed with diets
supplemented with chitin and chitosan. No adverse effects are expected
to non-target aquatic organisms when chitin and chitosan-containing
products are applied in accordance with approved labeling.

[Non-Target Birds]{.underline}:

Chitin is present in the exoskeletons of arthropods and, therefore, is a
regular component of the diets of insectivorous birds. Chitin and
chitosan have been shown to be relatively indigestible by some birds
(Akaki and Duke, 1999; and Razdan and Pettersson, 1994). However, many
birds (starlings, raptors, and many seabirds) possess chitinases that
aid in the digestion of chitin, which serves as a source of protein
(MacDonald, 2006). No mortalities have ever been observed in birds fed
with diets supplemented with chitin and chitosan. No adverse effects are
expected to birds when chitin and chitosan-containing products are
applied in accordance with approved labeling.

[Non-target Plants and Insects]{.underline}

Chitin is ubiquitous in nature and is a major component of the
exoskeletons of aquatic arthropods (insects, crustaceans). Chitin,
Chitosan, and Chitosan Hydrolysate are intended for use as plant defense
\"boosters\" to protect plants from fungal pathogens via he induction of
Systemic Acquired Response (SAR). SAR is a mechanism that stimulates the
internal defense mechanisms of plants to resist pathogen infection. In
plants, chitin stimulates the production of chitinases which are used to
degrade the chitin-containing cell walls of pathogenic fungi (Kramer and
Muthukrishnan, 1998). There is no direct activity of Chitin or Chitosan
(including Chitosan Hydrolysate) against the pathogen. No adverse
effects to non-target insects and plants are anticipated.

**REFERENCES**

Bishop, J. A. Dean, and T. Mitchell-Olds. 2000. PNAS 97(10): 5322-5327.

Campbell, N. A. 1996. *Biology* (4th edition) Benjamin Cummings, New
York. [ISBN
0-8053-1957-3](http://en.wikipedia.org/wiki/Special:BookSources/0805319573)

Escott GM, Adams DJ (1995). Infection and Immunity 63(12): 4770-4773. 

Kaspari, M. and A. Joern. 1993. OIKOS 68: 410-430.

Kramer, K. and S. Muthukrishnan. 1998. Insect Biochemistry and Biology
27(11): 887-900.

MacDonald, D. 2006. Protein Requirements of Birds.
<http://www.drmacs.com/ProteinRequirementsofBirds.pdf>

Matsumiya, M., Y. Arikane, A. Haga, S. Muthukrishnan, and K. Kramer.
2006. Bioscience, Biotechnology, and Biochemistry 70(4): 971-979.

Paolettia MG, L. Norbertob, R. Daminia, S. Musumeci . 2007. Annals of
Nutrition and Metabolism 51(3): 244-251.

Razdan, A. and D. Pettersson. 1994. British Journal of Nutrition. 1994
72: 277-288.

Uawonggul, N., C. Ruksakulpiwat, and S. Chanthai. 2002. Poster Abstract.
28th Congress on Science and Technology of Thailand, Queen Sirikit
National Convention Center, Bangkok, Thailand, Oct. 24-26.

cc: R. S. Jones, BPPD Subject File/IHAD

R. S. Jones, FT, OPY: 04/08/2008