1 

### Study Report

Title: Visualization of Aggregation State of Test Substance Suspended in Commercial

Aqueous Solution Using Cryo-Electron Microscopy


### Report Date: October 30, 2014



### File Reference: { }



### Pri ncipal Investigator(s): {

}



### Test ing Facility: {

}


### Sponsor: { }



### EPA Reference: TS -ZF02NT2 

### St udy Report
Visualization of Aggregation State of Test Substance Suspended in Commercial Aqueous Solution Using Cryo-Electron Microscopy
1) Introduction: The study used transmission electron microscopy of rapidly cryogenically frozen sample solutions (i.e., emerging cryo-TEM techniques) to generate micrographs of the Test Substance suspended in typical commercial Solutions, to permit visual characterization of the degree of agglomeration of the Test Substance in that phase. .



### 2) Purpose & Scope: This study provides characterization data on the aggregation state of the Sponsor’s Test Substance when in normal liquid suspensions (Test Solution). This summary is to be used to inform chemical identification processes, { }. 

### 3) A dministrative Organization: This report was prepared by principal investigator, { ,} based upon cryo-TEM ima ging carried out by { }. 

### 4) Test Sub stance: The study was conducted on the TS -ZF02NT new chemical substance (Test Substance), after processing as described in TS -ZF02NT, forming stable aqueous suspensions of the Test Substance (Test Solutions) (i.e., the form in which the Test Substance is typically distributed). Test Solution sample, { }
, sent for analysis were taken from larger typical full-sized batches. A full QC characterization report is provided in Appendix 1

.



### 5) Principle and Study Design: Samples of the Test Solution were vitrified using cryogenic techniques to create a fixed “snapshot” of the Test Substance as dispersed in a Test Solution in a manner that can be examined by transmission electron microscopy (TEM). Random fields are examined at a number of magnifications and digital electron micrographs are collected of particles that illustrate the aggregation state of the Test Substance within the Test Solution. See Appendix 3

. 

### 6) Sample Handling and Preparation: {

}3

en route to the Testing Facility. { } The sample w as prepared under the direction of { ,} from a full lot provided by the Sponsor’s { } . Final packaging and transfer to the UPS carrier was by { } Shipping and Receiving. The sample was shipped intact, in a secure containe r to the Testing Facility. 

### 7) Study Results: Micrograph images of the Test Substance as dispersed in the Test Solution sample are provided in the Testing Facility’s report, attached hereto as Appendix 3 . {

}.



### Appendices:

1. { }

2. { .}107/29/14

Section 1: Project Summary and Observations
The purpose of this project was to perform transmission electron microscopy (TEM) cryo imaging analysis of one sample. This work is conducted under price quotation

Details of the experiments, methods used and the results obtained are provided in sections 2-4 below. Here we provide a brief summary of the main conclusions. Only a limited set of images was selected to be shown in this report; the client can examine the entire set of images provided as described in section 4.
The first figure shows thumbnails summarizing the results for each sample at nominal magnifications of 110,000x, 52,000x and 21,000x, followed by one image for each sample showing annotated areas of interest. These are followed by a set of images presented at the following magnifications: 6,500x, 110,000x, 52,000x and 21,000x. The lower magnification images provide a more general overview of the variety of structures present and the higher magnification images provi de more details of the structures.
Note that for this project the sample was imaged using a high electron dose to increase contrast, and areas where particles were embedded in a thicker layer vitreous ice were specifically targeted to provide an idea of how the particles might be distributed in bulk solution.
Brief impressions of samples are below.

We would be happy to discuss these results in more detail with the client.

207/29/14

Section 2: Sample shipping, receiving, and handling
Package received in good condition at room temperature. Date/Time of sample arrival:2014-07-16 10:25:00 Carrier/Tracking Number:UPS The package was labeled as and contain 1 vials labeled as follows:

pro cedures were used for sample preparation and imaging.
Section 3: EM grid preparation
Samples were imaged undiluted.
Each sample was preserved in vitrified ice supported by Each sample was prepared by applying a 3μL drop of sample suspension to a cleaned grid, blotting away with filter paper, and immediately proceeding with vitrification in liquid ethane. Grids were stored under liquid Nitrogen until transferred to the electron microscope for imaging.
Section 4: EM Imaging
Electron microscopy was performed using an FEI Tecnai T12 electron microscope, operating at 120keV equipped with an FEI Eagle 4k x 4k CCD camera. Vitreous ice grids were transferred into the electron microscope using a cryostage that maintains the grids at a temperature below -170 °C.
Images of each grid were acquired at multiple scales to assess the overall distribution of the specimen. After identifying potentially suitable target areas for imaging at lower magnifications, high magnification images were acquired at nominal magnifications of 110,000x (0.10 nm/pixel), 52,000x (0.21 nm/pixel) and 21,000x (0.50 nm/pixel). The images were acquired at a nominal underfocus of -2 μ m (110,000x), -3 μ m (52,000x) and -4 μ m (21,000x) and electron doses of ~10-40 e/Å 2

. The experiments performed were as follows:

N o te that the images are reduced in size in this written report and some images for the samples can be found only in the full set of images. The full set of images at full resolution is provided on a web-based portal. Documentation describing how to access and use the web server is also available at:

The images can be viewed using a standard web browser connected to the secure client web site:

307/29/14

We encourage the client to browse through the set of images in order to get an overall feel for the distribution and morphology of the particles in the samples.

407/29/14

507/29/14

607/29/14

707/29/14

807/29/14

907/29/14

1007/29/14

1107/29/14

1207/29/14

13