SRC TR

- 13

- 060

HPV3 TEST RULE DATA ADEQUACY R EVIEW ( REVISED FINAL ): TOXICITY TO ALGAE , CHRONIC TOXICITY T O DAPHNIA , AND

CHROMOSOMAL ABERRATI ON S

Propanoic acid, 2

- methyl

- , 3

- (benzoyloxy)

- 2,2,4

- trimethylpentyl ester

, CASRN 22527

- 63

- 5

Prepared by:

Amy Nguyen, Jennifer Rayner, Teresa Manyin, and William L. Richards

Defense and Environmental Solutions Division

SRC, Inc. 7502 Round Pond Road North Syracuse, NY 13212

Contract No. EP

- W

- 09

- 027 Task FG007. 4 .009 .006

Submitted to:

OCSPP/OPPT/RAD (7403M) U.S. Environmental Protection Agency 1200 Pennsylvania Avenue Washington, D.C. 20460

Bryan Lobar, Project Officer David Brooks, Work Assignment Manager Amy Benson, Alter nate Work Assignment Manager

January 7, 2014Final HPV 3 Chemical Data Adequacy Review (January 2014) CAS RN 22527

- 63

- 5

i TABLE OF CONTENTS

A. SOURCE MATERIAL USED FOR ASSESSMENT

................................ .............................. 1
B. EVALUATING DATA ADEQUACY

................................ ................................ ..................... 1

C

. SUMMARY OF DATA ADEQUACY FOR TESTING REQUIRED BY TEST RULE

........ 2

D. DATA FOR SIDS ENDPOINTS

................................ ................................ .............................. 7

D

- 1. Toxicity to Aquatic Plants

................................ ................................ ................................ . 7

D

- 2. Chronic Toxicity to Aquatic Invertebrates

................................ ................................ ....... 14

D

- 3. Genetic Toxicity

– Chromosomal Aberrations

................................ ................................ 23
E. DATA ADEQUACY SUMMARY TABLE

................................ ................................ ........... 28 ADDENDUM: Test Guideline Source Material to Support Reliability Ratings Assigned to Submitted Studies That Are Considered Inadequate to Satisfy Test Rule Requirements

.......... A

- 1Final HPV 3 Chemical Data Adequacy Review (January 2014) CAS RN 22527

- 63

- 5

1 A. SOURCE MATERIAL USED FOR ASSESSMENT

This document evaluates toxicity tests for p ropanoic acid, 2

- methyl

- , 3

- (benzoyloxy)

- 2,2,4

- trimethylpentyl ester (CASRN 22527

- 63

- 5 ) submitted by Eastman Chemical Company ( on June 20, 2013) to EPA in response to the final test rule, “Testing of Certain High Production Volume Chemicals; Third Group of Chemicals” ( 76 FR 65385

, October 21, 2011 ; referred to herein as HPV3; available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

2009

- 0112

- 0086 as of January 7, 2014 ).

The HPV3 test rule requires the following toxicological testing for CASRN 22527

- 63

- 5: C1 (consisting of acute toxicity to fish, acute toxicity to Daphnia , and toxicity to algae testing if log K ow < 4.2 or chronic toxicity to Daphnia and toxicity to algae testing if log K ow ≥ 4.2) , D (acute mammalian toxicity test ), E1 ( ba cterial reverse mutation test ), E2 (chromosomal aberration or micronucleus test) , and F1 (combined repeated dose toxicity study with the reproduction/developmental toxicity screening test) . Since the log K ow of CASRN 22527

- 63

- 5 has been experimentally det ermined to be ≥ 5.4 ( based on measured log K ow values of 5.4, 5.5, and 5.8 for the three main components of the test item) 1 , which is > 4.2, the C 1 aquatic toxicity testing requirement is considered to include chronic toxicity to Daphnia and toxicity to al gae testing.

The June 20, 2013 submission from Eastman Chemical Company (“Eastman”) , available online
at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2009

- 0112

- 0145 as of January 7, 2014

, contained the following toxicological test reports:

 “ Benzoflex 1046: Algal Growth Inhibition Test ”

, submitted for the C1 testing requirement

 “ Benzoflex 1046: D aphnia magna Reproduction Test ”, submitted for the C1 testing requirement

 “ Benzoflex 1046: Chromosome Aberration Test in Human Lymphocytes In Vitro ”

, submitted for the E2 testing requirement

These newly submitted data are evaluated for adequacy herein. No other toxicity data are evaluated in this report.

B. EVALUATING DATA ADEQUACY

Data available for SIDS toxicity endpoints are evaluated for adequacy in Section D. Each study was summari zed according to a standard format and assigned a Klimisch reliability code based on its scientific merit and conduct, using professional judgment. To determine whether data were adequate to satisfy SIDS endpoints, study details were compared to current s tandard test guidelines to establish whether the study met current testing requirements. If the data available for the endpoint were determined to be inadequate, a basis for that conclusion is supplied. A table summarizing data availability/adequacy for each SIDS endpoint is included in Section E of this document.

1 Data for log K ow and other physicochemical properties of Benzoflex 1046 (a “bulk reaction product” containing CASRN 22527

- 63

- 5 ) were submitted to EPA on June 20, 2013 (available online at http://www.regulations.gov/#!documentDet ail;D=EPA

- HQ

- OPPT

- 2009

- 0112

- 0145 as of January 7, 2014 ).Final HPV 3 Chemical Data Adequacy Review (January 2014) CAS RN 22527

- 63

- 5

2 C

. SUMMARY OF DATA ADEQUACY FOR TESTING REQUIRED BY TEST RULE

Justification for Use of Test Substance: The test rule classifies propanoic acid, 2

- methyl

- , 3

- (benzoyloxy)

- 2,2,4

- trimethylpentyl ester (CASRN 22527

- 63

- 5) as a Class 1 Chemical Substance for which testing must be conducted with material having a purity of 99% or greater. However, in a letter to EPA dated February 20, 2012 (available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

2009

- 0112

- 0107 as of January 7, 2014 ), Eastman proposed the use of a bulk reaction product of the following composition : 60

- 65% propanoic acid, 2

- methyl

- , 3

- (benzoyloxy)

- 2,2,4

- trimethylpentyl ester (CASRN 22527

- 63

- 5), 20

- 25% 1,3

- pentanediol, 2,2,4

- trimethyl

- 1,3

- dibenzoate (CASRN 68052

- 23

- 3), and 10

- 15% 2,2,4

- trimethyl

- 1,3

- pentanediol diisobutyrate (CASRN 6846

- 50

- 0).

Eas tman’s rationale for the use of this mixture (the “bulk reaction product”) was based on the fact that “The manufacture of benzoate ester for commercial use has been as a bulk reaction and there is no readily available process to synthesize the chemical sub stance by direct reaction to achieve the purity required for Class 1 substances. Eastman believes that use of the bulk reaction product which is formulated into finished products for commercial sale will more accurately represent exposure and use of the b enzoate ester and avoid a costly and technically uncertain purification procedure.” Given that the major constituent (60

- 65%) of the bulk reaction product is the test rule substance, propanoic acid, 2

- methyl

- , 3

- (benzoyloxy)

- 2,2,4

- trimethylpentyl ester ( C ASRN 22527

- 63

- 5 ), and the remaining two major components (CASRNs 68052

- 23

- 3 and 6846

- 50

- 0) can be considered close analogs of CASRN 22527

- 63

- 5, EPA approved Eastman’s plan to conduct aquatic and mammalian toxicity tests on this mixture via email from P. Ca mpanella (EPA) to G. Shrum (Eastman) on April 19, 2012. This mixture, referred to as “Benzoflex 1046” in the submitted study reports, can be considered a supporting substance for CASRN 22527

- 63

- 5; as such, the toxicity of Benzoflex 1046 is expected to be representative of the toxicity of CASRN 22527

- 63

- 5.

Evaluation of Toxicological Data Submitted on June 20, 2013: Data submitted for the c hromosomal aberration test for Benzoflex 1046, supplied on June 20, 2013 by Eastman and reviewed herein, are adequate to satisfy the E2 testing requirement promulgated in the final HPV3 test rule.

Data submitted for toxicity to algae and c hronic toxicity to Daphnia for Benzoflex 1046, supplied on June 20, 2013 by Eastman

, are not considered adequate to satisfy the C 1 testing requirement for CASRN 22527

- 63

- 5 promulgated in the final HPV3 test rule.

The toxicity to algae study is considered inadequate because the test was conducted at concentrations lower than the water solubility limit of the test substance and the test failed to identify a discrete EC 50 for algal growth. Given that inhibition of algal growth rate , biomass, and yield at the highest test concentration was less than 50% in the definitive test, the study only determined that the EL 50 (based on WAF load ing rates) was greater than 100 mg/L and that the EC 50 (based on mean measured concentrations) was greater than 0.19 mg/L. The highest testFinal HPV 3 Chemical Data Adequacy Review (January 2014) CAS RN 22527

- 63

- 5

3 concentration (mean measured concentration of 0.19 mg/L) was considerably less than the reported water solubility l imit of the test substance (1.01 mg/L) 1

. Additional deficiencies of the toxicity to algae study included the following: (1) The solubility, stability, and volatility of the test substance in the growth medium were not determined prior to the initiation of the algae test, as recommended by ASTM and OPPTS guidelines. (2) Analytical measurements demonstrated a disparity (greater tha n 3

- fold) between the test substance concentrations of the WAFs prepared for the range

- finding and definitive tests. Specifically, the initial (time = 0) measured test substance concentration of the 100 mg test item/L loading rate WAF sample was 0.380 mg/ L in the definitive test, but was 1.23 mg/L in the range

- finding test. The author did not provide an explanation for the difference between the measured concentrations of the WAFs used in the range

- finding and definitive tests. The discrepancy in measure d concentrations at test initiation raises concerns about the consistency and reproducibility of the methods used to prepare the test solutions for the two tests and/or the analytical methods used to determine test substance concentrations. The study repo rt did not provide a detailed description of the procedures for storage and handling of WAF preparations. Missing information included, for example, whether the WAF was stored prior to use and, if so, for how long and under what conditions. (3) During the 9 6

- hour exposure period, measured test substance concentrations declined by approximately 98% during the definitive study and 96% in the range

- finding study. In the definitive test, measured concentrations of the 100 mg Benzoflex 1046/L loading rate WAF sa mples were 0.380 and 0.00638 mg/L at 0 and 96 hours, respectively. The reason for the decline in test item concentrations during the test was unclear. Although the study author stated that the decline in test item concentration was due to the unstable na ture of the test item, the report did not provide data to substantiate the author’s claim of instability of the test substance in the algae growth medium. (4) Growth rate in control cultures was not constant during the exposure. After 72 hours, a marked decl ine in growth rate was observed in controls. The average specific growth rate in control cultures from 72 to 96 hours (0.022 cells/mL/hour) was less than one third of the growth rate observed during the first 72 hours (0.068 cells/mL/hour). No explanatio n was provided by the study author for this decrease.

The chronic toxicity to Daphnia study i s considered inadequate because the test failed to identify a discrete EC 50 or accurate LOEC and NOEC values for chronic effects on survival, reproduction, and growth of daphnids. Although adverse effects were observed on reproduction and growth, inhibition of each parameter was less than 50% at the highest concentration tested and, therefore, the study did not determine a discrete EC 50 . Furthermore, accurate NOEC and LOEC values could not be identified because mean measured concentrations ( 0.14, 0.13, 0.18, 0.12, and 0.21 mg/L, respectively, for nominal loading rates of 1, 3.2, 10, 32, and 100 mg/L) did not consistently increase with increasing loading rate concentrations (see #4 below)

. The large

1 An experimentally measured water solubility limit for Benzoflex 1046 of 1.01 mg/L at 20 °C was reported in a submission from Eastman on June 20, 2013 (available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2009

- 0112

- 0145 as of January 7, 2014 ).Final HPV 3 Chemical Data Adequacy Review (January 2014) CAS RN 22527

- 63

- 5

4 variations in measured test substance concentrations prevent the interpretation of the biological data to characterize the concentra tion

- response relationship.

Additional deficiencies of the chronic toxicity to Daphnia study included the following: (1) The test item was detected in control samples collected on Days 0, 2, 7, 12, 16, and 21, suggesting unreliable analytical techniques for measuring concentrations or inadequate quality control. (2) Measured test substance concentrations among “fresh” WAF test solutions (sampled at the time of solution renewal) of the same nominal loading rate were highly variable at different measurement time points. For example, the measured concentrations of “fresh” solutions of WAFs at a nominal loading rate of 100 mg/L we re 0.927, 0.0522, 0.489, 0.467, and 0.0573 mg/L on Days 0, 5, 9, 14, and 19, respectively. During the study, measured concentrations of “fresh” test solutions varied by a factor of at least 15 (highest measured “fresh” value divided by lowest measured “fr esh” value) for each treatment level. For loading rates of 1, 3.2, 10, 32, and 100 mg/L, individual measured concentrations of “fresh” test solutions varied by as much as 140, 180, 134, 144, and 133% from the mean measured “fresh” concentration for that l oading rate during the 21

- day exposure period. This variability raises concerns about the consistency and reproducibility of the methods used to prepare the test solutions during the test and/or the analytical methods used to determine test substance conc entrations. No explanations for the variability among measured concentrations of “fresh” test solutions were provided in the study report. The study report did not provide a detailed description of the procedures for storage and handling of WAF preparati ons. Missing information included, for example, whether the WAF was stored prior to use and, if so, for how long and under what conditions. The inconsistencies among test item concentrations for individual WAF loading rates leads to uncertainty in the re ported endpoint values. (3) Substantial declines in measured test substance concentrations were observed between test solution renewals. Measured concentrations of “old” test solutions (sampled prior to solution renewal) were up to 99% less than the respect ive measured “fresh” concentrations. No explanation for these declines was provided in the study report and no analysis was conducted to determine the cause. It is unknown whether the decline in test substance concentration could be due to degradation of the test substance because no data were provided to describe stability of the test substance in the test media. (4) The combination of highly variable test substance concentrations at test solution renewal and steep declines in test substance concentration be tween renewals resulted in large variations in measured test substance concentrations at each WAF loading rate over the 21

- day exposure period. When measurements from “old” and “fresh” test solutions were combined, measured concentrations varied by a fact or of at least 144 (highest measured value divided by lowest measured value) for a given treatment level. For loading rates of 1, 3.2, 10, 32, and 100 mg/L, individual measured concentrations (“old” and “fresh” combined) varied by as much as 272, 311, 349 , 374, and 350% from the mean measured concentration for that loading rate during the 21

- day exposure period. The ranges for the measured concentrations (“fresh” and “old” combined) were < LOQ

- 0.521, < LOQ

0.529, 0.00517

- 0.814, < LOQ

- 0.546, and < LOQ

- 0.92 7 mg/L, respectively, for nominal loading rates of 1, 3.2, 10, 32, and 100 mg/L

. The mean measured concentrations ( 0.14, 0.13, 0.18, 0.12, and 0.21 mg/L, respectively, for nominal loading rates of 1, 3.2, 10, 32,Final HPV 3 Chemical Data Adequacy Review (January 2014) CAS RN 22527

- 63

- 5

5 and 100 mg/L) did not consistently increas e with increasing loading rate concentrations, which precluded the identification of accurate NOEC and LOEC values. (5) The solubility and stability of the test substance in the dilution water (culture medium) were not determined prior to the initiation of the test, as recommended by ASTM E1193

- 97. (6) The study did not include data on observations of first

- and second

- generation daphnids for abnormal development or aberrant behavior as recommended by section 12.10.5 of ASTM E1193

- 97. (7) The 100 mg/L loading rate WAF was filtered through a glass wool plug on day 9 to remove microscopic particles of the test item in the water column. The use of glass wool for removal of undissolved test material is not recommended. Instead, the use of centrifugation or a separatory f unnel is suggested.

Therefore, t he algae and daphnid studies should be repeated to assure that CASRN 22527

- 63

- 5 is tested at concentrations up to its solubility limit in a test medium appropriate for algae and Daphnia , respectively. It is recommended that the study design for further toxicity testing of algae and Daphnia include the conduct of preliminary testing to determine what procedures are necessary to assure that the test substance reaches maximum possible solubility in the int ended test medium (i.e., dilution water, culture media) at each concentration that is to be tested in the main toxicity study. Procedures for preparation of WAF solutions should also be refined to minimize variability among measured test substance concent rations of fresh test solutions at renewal. In addition, s tability trials should be conducted to determine if the test substance significantly de grades during the intended testing period (algae) or renewal cycle ( Daphnia ). It is recommended that t est concentration measurements be conducted more frequently (every 24 hours) to better characterize exposure concentration s during the test period. The study reports should contain detailed procedures for storage and handling conditions of prepared WAFs p rior to use, as well as detailed procedures for collection , storage, and analysis of test solution samples

. Evaluation of Previously Submitted Toxicological Data: In a previous submission (dated February 26, 2013; available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2009

- 0112

- 0138 January 7, 2014 ), Eastman supplied data from acute mammalian toxicity and gene mutation test s . These data were reviewed in a separate report (SRC Technical Report #13

- 050) and were determined to be adequate to satisfy the D and E1 testing requirements promulgated in the HPV3 te st rule.

Remaining H

PV3 Test Rule Toxicity Testing Requirements: In an email dated December 18, 2013 (from L.W. Velsor [Eastman] to J. Schaeffer [EPA]), Eastman supplied d ata from a combined repeated dose toxicity study with the reproduction/developmental toxicity screening t est in response to the F1 testing requirement

. These data are currently under review.

Summary of Data Adequacy for Toxicological Endpoints : Data submitted by Eastman for the chronic toxicity to Daphnia and toxicity to algae endpoints are not considered adequate; these tests need to be repeated. Adequate data have been submitted by Eastman for acute mammalian toxicity, gene mutation, and chromosomal aberrationsFinal HPV 3 Chemical Data Adequacy Review (January 2014) CAS RN 22527

- 63

- 5

6 endpoints. Data submitted by Eastman for the repeated

- dose, reproductive, and developmental toxicity endpoints are currently under review

.Final HPV 3 Chemical Data Adequacy Review (January 2014) CAS RN 22527

- 63

- 5

7 D. DATA FOR SIDS ENDPOINTS

D

- 1 . Toxicity to Aquatic Plants

Summary of endpoint: Not A dequate

Study 1: Algal g rowth inhibition toxicity test in Pseudokirchneriella subcapitata ( Vryenhoef , 2013)

Title: Benzo flex 1046 : Algal growth inhibition te st

This study is: Adequate Not Adequate

This study is not considered adequate because the test was conducted at concentrations lower than the water solubility limit of the test substance and the test failed to identify a discret e EC 50 for algal growth. Given that inhibition of algal growth rate , biomass, and yield at the highest test concentration was less than 50% in the definitive test, the study only determined that the EL 50 (based on WAF loading rates) was greater than 100 m g/L and that the EC 50 (based on mean measured concentrations) was greater than 0.19 mg/L. The highest test concentration (mean measured concentration of 0.19 mg/L) was considerably less than the reported water solubility limit of the test substance (1.01 mg/L) 1

.

TEST SUBSTAN CE

Identity: Benzoflex 1046; supporting substance for propanoic acid, 2

- methyl

- , 3

- (benzoyloxy)

- 2,2,4

- trimethylpentyl ester (CASRN 22527

- 63

- 5)

Remarks: Clear colorless liquid; Batch No. V046212201; Composition: 68.8% p ropanoic acid, 2

- methyl

- , 3

- (benzoyloxy)

- 2,2,4

- trimethylpentyl ester (CASRN 22527

- 63

- 5), 19.5% 1,3

- pentanediol, 2,2,4

- trimethyl

- 1,3

- dibenzoate (CASRN 68052

- 23

- 3), 7% 2,2,4

- trimethyl

- 1,3

- pentanediol diisobutyrate (CASRN 6846

- 50

- 0), 4.2% miscellaneous esters, 0.5% volatiles, and 0.02 % moisture. 2

METHOD

Method/guideline followed: OPPTS 850.5 4 00 (“Algal Toxicity, Tiers I and II”) and ASTM E1218

- 04 (“Standard Guide for Conducting Static Toxicity Tests with Microalgae”)

1 An experimentally measured water solubility limit for Benzoflex 1046 of 1.01 mg/L at 20 °C was reported in a submission from Eastman on June 20, 2013 (available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- O PPT

- 2009

- 0112

- 0145 as of January 7, 2014 ).

2 The certificate of analysis provided in the study report describes the c omposition as follows: 68.8% “diester”

, 19.5% “dibenzoate”, 7% “TXIB”, 4.2% miscellaneous esters, 0.5% volatiles, and 0.02% moisture. In a n email dated July 3, 2013 from G. Shrum (Eastman) to J. Schaeffer (EPA), Eastman indicated that “ diester” was synonymous with p ropanoic acid, 2

- methyl

- , 3

- (benzoyloxy)

- 2,2,4

- trimethylpentyl ester (CASRN 22527

- 63

- 5), “dibenzoate” was synonymous with 1,3

- pe ntanediol, 2,2,4

- trimethyl

- 1,3

- dibenzoate (CASRN 68052

- 23

- 3), and “TXIB” was synonymous with 2,2,4

- trimethyl

- 1,3

- pentanediol diisobu t yrate (CASRN 6846

- 50

- 0).Final HPV 3 Chemical Data Adequacy Review (January 2014) CAS RN 22527

- 63

- 5

8 Test type: Toxicity to p lants ( algae )

– static

GLP compliant ?: Yes

Year study was performed: 2013

Test species: Green algae ( Pseudokirchneriella subcapitata ) strain CCAP 278/4; obtained from the Culture Collection of Algae and Protozoa, SAMS Research Services Ltd, Scottish Marine Institute , located in Oban, Argyll, Scotland

Analytical monitoring: Yes

Exposure duration: 96 hours

Concentrations tested: The test item, Benzoflex 1046, a multi

- component mixture, was prepared as a water accommodated fraction ( WAF ) due to its low aqueous solubility

. Range

- finding test: Loading rate (WAFs) : 0, 10, and 100 mg/L . Measured concentrations for the 100 mg/L WAF loading rate at 0 and 96 hours : 1.23 and 0.0454 mg/L, respectively. The mean measured concentration (calculated by the EPA reviewer) was 0.64 mg/L. Definitive test: Loading rate ( WAFs ): 0 and 100 mg/L

. Measured concentrations for the 100 mg/L WAF loading rate at 0 and 96 hours : 0.380 and 0.00638 mg/L , respectively

. The mean measured concentration (calculated by the EPA reviewer) was 0.19 mg/L.

Test method/conditions remarks: “The culture medium was prepared using reverse osmosis purified deionized water and the pH [was] adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl. The water hardness (calculated) of the culture mediu m was 0.15 mmol/L (= 15 mg/L as CaC O 3 ).” [Vryenhoef 2013, p. 31]

In pre

- study tests, the test item was stirred in deionized reverse osmosis water for 23, 47, or 95 hours to examine the effect of increased stirring time on the concentration of dissolved test item

. In these tests, stirring the test item at a loading rate of 100 mg/L for longer than 23 hours (47 or 95 hours) did not substantially increase the amount of dissolved test substance, as indicated by total organic carbon (TOC) measurements. T OC values for the aqueous phase were 1.20, 1.22, and 1.28 mg carbon/L (corrected for control) for stirring periods of 23, 47, and 95 hours, respectively.

Range

- Finding Test Procedure : In the range

- finding test, Pseudokirchneriella subcapitata cells were exposed to a series of nominal loading rates o f 10 and 100 mg/L. Negative control cells were not exposed to the test item.

“The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with po lyurethane foam bungs to reduce evaporation. Two replicate flasks were prepared for each control and test concentration.” [Vryenhoef 2013, p. 12]Final HPV 3 Chemical Data Adequacy Review (January 2014) CAS RN 22527

- 63

- 5

9 “Amoun ts of test item (20 and 200 mg) were each separately added to the surface of 2 liters of culture mediu m to give the 10 and 100 mg/L loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtur es [were] allowed to stand for 1 hour... The aqueous phase or WAF was removed by mid

- depth siphoning (the first 75

- 100 mL discarded) to give the 10 and 100 mg/L loading rate WAFs. Microscopic inspection of the WAFs show ed no micro

- dispersions or undissolved test item to be present.” [Vryenhoef 2013, p. 12]

An aliquot of each of the WAFs was inoculated with algal suspension , resulting in an initial cell density of approximately 1 x 10 4 cells/mL.

“The flasks were... incubated

... at 24 ± 2 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380

- 730 nm) and constantly shaken at approximately 150 rpm for 96 hours.” [Vryenhoef 2013, p. 12]

Cell densities w ere determined at 0 and 96 hours using a Coulter ® Multisize Particle Counter.

“Samples of each loading rate WAF were taken for chemical analysis at 0 and 96 hours in order to determine the stability of the test item over the test duration

. All samples we re stored at approximately

- 20 °C prior to analysis.” [Vryenhoef 2013, p. 12]

Definitive Test Procedure : Based on the results of the range

- finding test, a loading rate of 100 mg/L was selected for the definitive (limit) test

. Negative control cells were not exposed to the test item.

“ An amount of test item (200 mg) was added to the surface of 2 liters of culture medium to give the 100 mg/L loading rate. After the addition of the test item, the culture medium was stirred by

m agnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture [was] allowed to stand for 1 hour... The aqueous phase or WAF was removed by mid

- depth siphon ing (the first 75

- 100 mL discarded) t o give the 100 mg/L loading rate WAF.” [Vryenhoef 2013, pp. 12

- 13]

“At the start of mixing the 100 mg/L loading rate WAF was observed to have formed a clear colorless media column with oily globules of test item floati ng on the surface and settled on the base of the mixing vessel. After stirring, and following a 1

- hour standing period, the WAF was observed to have formed a clear colorless media column with an oily slick of test item floating on the surface and a few oi ly globules dispersed throughout. Microscopic examination of the WAF showed there to be no micro

- dispersions or globules of test item present.” [Vryenhoef 2013, p. 19]

Exposure flasks were 250 mL glass conical flasks , each containing 100 mL of test solution . Six replicates were tested for both the control and 100 mg/L WAF loading rate.Final HPV 3 Chemical Data Adequacy Review (January 2014) CAS RN 22527

- 63

- 5

10 “Pre

- culture conditions gave an algal suspension in log phase growth characterized by a cell density of 3.30 x 10 5 cells per mL. Inoculation of 1 liter of test me dium with 30.3 mL of this algal suspension gave an initial nominal cell density of 1 x 10 4 cells per mL...” [Vryenhoef 2013, p. 13]

“The flasks were plugged with polyurethane foam bungs and incubated

... at 24 ± 1 °C under continuous illumination (intensity a pproximately 7000 lux) provided by warm white lighting (380

- 730 nm) and constantly shaken at approximately 150 rpm for 96 hours.” [Vryenhoef 2013, p. 13]

“Samples were taken at 0, 24, 48, 72 and 96 hours and the cell densities determined using a Coulter ® Multisizer Particle Counter.” [Vryenhoef 2013, p. 13]

The pH was measured at the start and end of the study and t emperatures within the incubator were recorded hourly

.

“ Samples were taken from the control and the 100 mg/L loading rate WAF test group (replicates R 1

– R 6 pooled) at 0 and 96 hours for quantitative analysis. All samples were stored at approximately

- 20 °C prior to analysis. Duplicate samples were taken at each occasi on and stored at approximately

- 20 °C for further analysis if nec essary. ” [Vryenhoef 2013, p. 1 4 ]

Test c oncentrations were analyzed using high performance liquid chromatograph y (HPLC) with UV detection ( wavelength of 230 nm). T he l imit of q uantitation [LOQ] was 0.0036 mg/L.

“ A Student's t

- test incorporating Bartlett ’ s test for homogeneity of variance (Sokal and Rohlf 1981) was carried out on the growth rate, yield and biomass integral data after 96 hours for the control and the 100 mg/L loading rate to determine any statistically significant differences between the te st and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999

- 2001). ” [Vryenhoef 2013, p. 16]

“ The results of the test were considered valid if the following criteria were met:

 The cell concentration of the control cultures must increase by a factor of at least 16 after 72 hours (ASTM Guidelines) and by a factor of at least 100 after 96 hours (OPPTS Guidelines)

. 1

 The coefficient of variation in replicate control cultures after 72 hours must not exceed 7% for average specific growth rate and 20% for yield (ASTM Guidelines ).

 The coefficient of variation of the cell density values in replicate control cultures must not exceed 20% after 72 and 96 hours (OPPTS Guidelines). 2 ” [ Vryenhoef 2013, p. 16 ]

1 OPPTS 850.5400 (guideline cited by test report) does not specify a minimum increase in cell concentration of control cultures. The requirement for an increase by a factor of at least 100x after 96 hours appears to have been derived from OCSPP 850.4500, a n EPA harmonized test guideline for algal toxicity.

2 OPPTS 850.5400 does not specify a requirement for coefficient of variation of cell density values. OCSPP 850.4500 specifies that the coefficient of variation in control cultures should be less than 15% for yield and growth rate at test termination (96 hours)

.Final HPV 3 Chemical Data Adequacy Review (January 2014) CAS RN 22527

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11 RESULTS

Endpoint value(s): The following results were based on WAF loading rates : 96

- hour E L 50 (growth rate, yield, and biomass) > 100 mg/L 96

- hour NOEL 1 (growth rate, yield, and biomass) = 100 mg/L 96

- hour LOEL 2 (growth rate, yield, and biomass) > 100 mg/L

The following values are based on mean measured concentrations (definitive test) that correspond with nominal loading rates: 96

- hour E C 50 (growth rate, yield, and biomass) > 0.19 mg/L 9 6

- hour NOEC (growth rate, yield, and biomass) = 0.19 mg/L 96

- hour LOEC (growth rate, yield, and biomass) > 0.19 mg/L

Results remarks: Range

- Finding Test Results: “Chemical analysis of the 100 mg/L loading rate WAF at 0 hours showed a measured test concentration of 1.2 mg/L was obtained. A significant decline in measured test concentration [96%] was observed at 96 hours to 0.045 mg/L. This decline was considered [by the study author] to be due to the unstable nature of the test item.” [Vryenhoef 2013, p. 17]

The percent i nhibition of “yield/biomass integral” was 22% and 28% at loading rates of 10 and 100 mg/L, respectively. No inhibition of growth rate was observed at the nominal loading rates of 10 and 100 mg/L.

Definitive Test Results: “At the start of the test all c ontrol and 100 mg/L loading rate WAF test cultures were observed to be clear colorless solutions. After the 96

- hour test period all control and test cultures were observed to be dark green dispersions.” [Vryenhoef 2013, p. 19]

“Analysis of the 100 mg/L l oading rate WAF test preparation at 0 hours

... showed [that] a measured test concentration of 0.38 mg/L was obtained. A significant decline in measured test concentration [98%] was observed at 96 hours to 0.0064 mg/L. This decline in measured concentratio n was considered [by the study author] to be due to the unstable nature of the test item.” [Vryenhoef 2013, p. 20]

“[T] he growth rate, yield and biomass integral of Pseudokirchneriella subcapitata (CCAP 278/4) were not affected by the presence of the test item over the 96

- Hour exposure period.” [Vryenhoef 2013, p. 18]

The percent inhibition values for growth rate, yield, and biomass integral were 1%, 0%, and 2%, respectively, after 96 hours at a nominal loading rate of 100 mg/L. No statistically signific ant differences (p ≥ 0.05) between the control and 100 mg/L loading rate WAF test group were

1 NOE L = No Observed Effect Loading R ate
2 LOEL = Lowest Observed Eff ect Loading R ateFinal HPV 3 Chemical Data Adequacy Review (January 2014) CAS RN 22527

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12 observed and

, therefore

, the No Observed Effect Loading Rate (NOEL) for growth rate, yield, and biomass was 100 mg/L based on the WAF loading rate

. “All test and control cultures were inspected microscopically at 96 hours. There were no abnormalities detected in any of the control or test cultures.” [Vryenhoef 2013, p. 19]

“Temperature was maintained at 24 ± 1 °C throughout the test.” [Vryenhoef 201 3, p. 19] The pH ranged from 7.1 to 7.8.

The study author considered the test to be valid based on the following :

 The cell density in control cultures increased by a factor of 122 after 72 hours and a factor of 206 after 96 hours

.

 The coefficient of variation for average specific growth rate and yield in controls after 72 hours was 1% and 4%, respectively

.

 The coefficient of variation for cell density values in controls was 4% after 72 hours and 6% after 96 hours

.

CONCLUSIONS

The 96

- hour NOEL was 100 mg/L based on WAF loading rate s ; the 96

- hour NOEC was 0.19 mg/L based on mean measured concentrations . The 96

- hour E L 50 values for growth rate, yield, and biomass were > 100 mg/L based on WAF loading rate s ; the 96

- hour EC 50 values for growth rate, yield, and biomass were > 0.19 mg/L based on mean measured concentrations

.

STUDY RELIABILITY

[3 ] Not reliable

. This study is considered unreliable because the test was conducted at concentrations lower than the water solubilit y limit of the test substance and the test failed to identify a discrete E C 50 for algal growth

. Given that inhibition of algal growth rate , biomass, and yield at the highest test concentration was less than 50% in the definitive test, the study only determined that the EL 50 (based on WAF loading rates) was greater than 100 mg/L and that the EC 50 (based on mean measured concentrations) was greater than 0.19 mg/L. The highest test concentration ( mean measured concentration of 0.19 mg/L) wa s considerabl y less than the reported water solubility limit of the test substance (1.01 mg/L) 1

. Additional deficiencies include the following: (1) The solubility, stability, and volatility of the test substance in the growth medium were not determined prior to the initiation of the algae test, as recommended by ASTM and OPPTS guidelines. (2) Analytical measurements demonstrated a disparit y (greater tha n 3

- fold) between the test substance concentrations of the WAFs prepared for the range

- finding and definitive test s

. Specifically, the initial (time = 0) measured test substance concentration of the 100 mg test item/L loading rate WAF sample was 0.380 mg/ L in the definitive test, but was 1.23

1 An experimentally measured water solubility limit for Benzoflex 1046 of 1.01 mg/L at 20 °C was reported in a submission from Eastman on June 20, 2013 (available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2009

- 0112

- 0145 as of January 7, 2014 ).Final HPV 3 Chemical Data Adequacy Review (January 2014) CAS RN 22527

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13 mg/L in the range

- finding test. The author did not provide an explanation for the difference between the measured concentrations of the WAFs used in the range

- finding and definitive tests. T he discrepancy in measured concentrations at test initiation raises concerns about the consistency and reproducibility of the methods used to prepare the test solutions for the two tests and/or the analytical methods used to determine test substance concentrations. The stu dy report did not provide a detailed description of the procedures for storage and handling of WAF preparation s . Missing information included, for example, whether the WAF was stored prior to use and, if so, for how long and under what conditions. (3) Durin g the 96

- hour exposure period, measured t est substance concentrations declined by approximately 98% during the definitive study and 96% in the range

- finding study

. In the definitive test, measured concentrations of the 100 mg Benzoflex 1046 /L loading rate WAF sample s were 0.380 and 0.00638 mg/L at 0 and 96 hours, respectively. The reason f or the decline in test item concentrations during the test was unclear. Although t he study author stated that the decline in test item concentration was due to the unstable nature of the test item

, the report did not provide data to substantiate the author’s claim of in stability of the test substance in the algae growth medium

. (4) Growth rate in control cultures was not constant during the exposure. After 72 h ours, a marked decline in growth rate was observed in controls

. The average specific growth rate in control cultures from 72 to 96 hours (0.022 cells/ mL/hour) was less than one third of the growth rate observed during the first 72 hours (0.068 cells/mL/hour). No explanation was provided by the study author for this decrease.

REFERENCE

Vryenhoef, H. 2013. Benzoflex 1046: Algal growth inhibition test

. Harlan Laboratories Ltd.

, Shardlow, Derbyshire

, UK
, Project No. 41202705 . Sponsor: Eastman Chemical Company

, Kingsport, TN

. Study report available online at htt p://www.regulations.gov/#!documentDetail; D=EPA

- HQ

- OPPT

- 2009

- 0112

- 0145 as of January 7, 2014

. Text in quotes is taken directly from Vryenhoef (2013).Final HPV 3 Chemical Data Adequacy Review (January 2014) CAS RN 22527

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14 D

- 2

. Chronic Toxicity to Aquatic Invertebrates
Summary of endpoint: Not a dequate

Study 1: Chronic toxicity study in Daphnia magna ( Harris, 2013 )

Title: Benzoflex 1046: Daphnia magna reproduction t est

This study is: Adequate Not Adequate

This study is not considered adequate because the test failed to identify a discrete EC 50 or accurate LOEC and NOEC values for chronic effects on survival, reproduction, and growth of daphnids. Although adverse effects were observed on reproduction and growth, inhibition of each parameter was less than 50% at the highest concentration tested and, therefore, the study did not determine a discrete EC 50 . Furthermore, accurate NOEC and LOEC values could not be identified because mean measured concentrations ( 0.14, 0.13, 0.18, 0.12, and 0.21 mg/L, respectively, for nominal loading rates of 1, 3.2, 10, 32, and 100 mg/L) did not consistently increase with increasing loading rate concentrations

. The large variations in measured test substance concentrations prevent the interpretation of the biological data to characterize the concentration

- response r elationship.

TEST SUBSTANCE

Identity: Benzoflex 1046; supporting substance for propanoic acid, 2

- methyl

- , 3

- (benzoyloxy)

- 2,2,4

- trimethylpentyl ester (CASRN 22527

- 63

- 5)

Remarks: C lear colorless liquid; Batch No. V046212201; Composition: 68.8% p ropanoic acid, 2

- methyl

- , 3

- (benzoyloxy)

- 2,2,4

- trimethylpentyl ester (CASRN 22527

- 63

- 5), 19.5% 1,3

- pentanediol, 2,2,4

- trimethyl

- 1,3

- dibenzoate (CASRN 68052

- 23

- 3), 7% 2,2,4

- trimethyl

- 1,3

- pentanediol diisobutyrate (CASRN 6846

- 50

- 0), 4.2% miscellaneous esters , 0.5% volatiles, and 0.02% moisture. 1

METHOD

Method/guideline followed: Method was designed to be compatible with A STM Standard E1193

- 97 (“ Standard Guide for Conducting Daphnia magna Life

- Cycle Toxicity Tests ”)

Test type: Chronic t oxicity to Daphnia

– Semi

- Static

GLP compliant?: Yes

1 The certificate of analysis provided in the study report describes the c omposition as follows: 68.8% “diester”

, 19.5% “dibenzoate”, 7% “TXIB”, 4.2% miscellaneous esters, 0.5% volatiles, and 0.02% moisture. In an email dated July 3, 2013 from G. Shrum (Ea stman) to J. Schaeffer (EPA), Eastman indicated that “ diester” was synonymous with p ropanoic acid, 2

- methyl

- , 3

- (benzoyloxy)

- 2,2,4

- trimethylpentyl ester (CASRN 22527

- 63

- 5), “dibenzoate” was synonymous with 1,3

- pentanediol, 2,2,4

- trimethyl

- 1,3

- dibenzoate (C ASRN 68052

- 23

- 3), and “TXIB” was synonymous with 2,2,4

- trimethyl

- 1,3

- pentanediol diisobu t yrate (CASRN 6846

- 50

- 0).Final HPV 3 Chemical Data Adequacy Review (January 2014) CAS RN 22527

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15 Year study was performed: 2013

Test species: Daphnia magna , less than 24 hours old ; obtained from laboratory cultures maintained in

- house at Harlan Laboratories, Ltd., in Shardlow, Derbyshire, UK

Analytical monitoring: Yes

Exposure duration: 21 d ays

Concentrations tested: The test item, Benzoflex 1046, a multi

- component mixture, was prepared as a water accommodated fraction ( WAF ) due to its low aqueous solubility. Loading rates (WAFs) : 0 , 1.0, 3.2, 10, 32, and 100 mg/ L Mean measured concentrations (calculated by the EPA reviewer ) : 0.14 , 0.13, 0.18, 0.1 2 , and 0.21 mg/L 1

Test method/conditions remarks: Deionized reverse osmosis water was used to prepare the
test medium (Elendt M7 medium). The pH of the prepared medium was 8.0 ± 0.2.

In pre

- study tests, the test item was stirred in deionized reverse osmosis water for 23, 47, or 95 hours to examine the effect of increased stir ring time on the concentration of dissolved test item. In these tests, stirring the test item at a loading rate of 100 mg/L for longer than 23 hours (47 or 95 hours) did not substantially increase the amount of dissolved test substance, as indicated by total organic carbon (TOC) measurements. TOC values for the aqueous phase were 1.20, 1.22, and 1.28 mg carbon/L (corrected for control) for stirring periods of 23, 47, and 95 hours, respectively

. “Amounts of test item (20, 64, 200, 640 and 2000 mg) were each separately added to the surface of 20 lit ers of Elendt M7 to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rates, respectively. After the addition of the test item, the Elendt M7 was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures [were] allowed to stand for 1 hour... The aqueous phase or WAF was removed b y mid

- depth siphoning (the first approximate 75

- 100 mL discarded) to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAFs.” [Harris 2013, pp. 12

- 13]

“At the start of the mixing period the 1.0, 3.2, 10, 32, and 100 mg/L loading rates were observed to be clear colorless water columns with an oily slick of test item on the surface and oily globules of test item dispersed throughout. After 23 hours stirri ng and a 1

- hour standing period the 1.0, 3.2, 10, 32, and 100 mg/L loading rates were observed to be clear colorless water columns with oily globules of test item on the surface and on the base of the vessel. Microscopic inspection of the WAFs showed no m icro

- dispersions or undissolved test item to be present [with the exception of the 100 mg/L loading rate WAF on day 9]

. After siphoning and for the duration of the test, the 1.0, 3.2, 10, 32, and 100 mg/L loading rates were observed to be clear, colorless solutions.” [Harris 2013, p. 17]

1 For the purposes of calculating mean measured concentrations, a value of 0.0036 (limit of quantitation for analytical measurements) was substituted for measured concentrations reported as < LOQ.Final HPV 3 Chemical Data Adequacy Review (January 2014) CAS RN 22527

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16 “On day 9, microscopic examination of the 100 mg/L loading rate WAF showed oily globules of test item to be present in the water column and therefore it was considered justifiable to remove the WAF by filtering through a glass wool plug (2

- 4 cm in length). Microscopic examination after filtering showed the glass wool plug had removed all test item from the water column.” [Harris 2013, p. 17] “This difference on day 9 may have been due to the media being stirred slightly faster, causing more test item to be drawn into the water column.” [Harris 2013, p. 1 3 ]

Daphnia magna (10 replicates per test concentration, each containing a single daphni d) were exposed to WAFs of the test item at nominal loading rates of 0, 1.0, 3.2, 10, 32

, and 100 mg/L for a period of 21 days. The WAF test solution s were renewed 3 times per week (on days 0, 2, 5, 8, 9, 12, 14, 16, and 19)

.

Test vessels were 150 mL glass flasks containing 100 mL of test medium and covered with a plastic lid to reduce evaporation. The daphnids were fed algae and Tetramin ® flake food suspensions daily.

“ The flasks were maintained at approximately 20 °C with a photoperiod of 16 hours light (681 to 965 lux) and 8 hours darkness with 20 minute dawn and dusk transition periods

... Each vessel was randomly assigned to a position in the laboratory. The test vess els were not aerated. The diluent water only was aerated prior to use.” [Harris 2013, p. 13]

The size and general condition of the parental daphnids were evaluated daily. The length of each surviving parent al daphnid was determined at the end of the test. The number of daphnids with eggs or young in the brood pouch and the number of discarded unhatched eggs were recorded daily. The number of live and dead daphnids of the adult ( parental

, P 1 ) and filial (F 1 ) generations were also recorded daily.

“Young daphnids were considered to be dead if no sign of movement was apparent during microscopic examination. Adult Daphnia which were unable to swim for approximately 15 seconds after gentle agitation (i.e., immobile) were considered to be dead.” [Harris 2013, p. 14]

“ Temperature of the test preparations and light intensity were recorded daily throughout the test. Dissolved oxygen concentrations, pH and temperature were recorded before and after each test med ia renewal

... Measurements were made on one replicate for each test concentration. The water hardness, alkalinity and conductivity of the control and the highest surviving test concentration in the old media were measured once per week. The room temperatu re and the temperature in one control vessel were recorded continuously throughout the test.” [Harris 2013, p. 14]

“Water samples were taken from the control and each surviving test group (replicates pooled) for quantitative analysis. Samples of the fresh test preparations were taken on D ays 0, 5, 9, 14 and 19 and of the expired test preparations on D ays 2, 7, 12, 16 and 21. Samples were stored at approximately

- 20 °C prior to analysis. Duplicate samples were taken and stored at

a ppro ximately

- 20 °C for further analysis if necessary.” [Harris 2013, p. 14]Final HPV 3 Chemical Data Adequacy Review (January 2014) CAS RN 22527

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17 “The test item concentration in the test samples was determined by high performance liquid chromatography with UV detection (HPLC/UV) [at a wavelength of 230 nm] using an external standard. The test item gave a chromatographic profile consisting of two peaks.” [Harris 2013, p. 44]

“[T]he limit of quantitation [LOQ] was determined to be 0.0036 mg/L. ” [Harris 2013, p. 48]

“ For the estimation of the “ Lowest Observed Effect Loading Rate ” (LOEL) and the “ No Observed Effect Loading Rate ” (NOEL)

, the numbers of live young produced per adult over the duration of the test for the control, and 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAF test groups were compared using one way analysis of variance incorporating the Williams test for differences between treatment means when several dose levels are compared with a zero dose control

... Results from the control and 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAF test
groups [for] Daphnia length data, determined for the surviving daphnids on termination of the test, were compared using one way analysis of variance incorporating Bartlett ’ s test for homogeneity of variance

... and Dunnett ’ s multiple comparison procedure for comparing several treatments with a control

... All statistical analyses were performed using the SAS computer software package (SAS 1999

- 2001). ” [Harris 2013, p. 15]

The test w as considered valid if the following criteria were met:

 Control mortality was ≤ 30%

.

 Dissolved oxygen content was > 3 mg O 2 /L

.

 Control group pH did not deviate by > 1.5 units

.

 The mean number of live young produced per surviving adult daphnid in the control group was ≥ 60 after 21 days

.

 The coefficient of variation for the control group was ≤ 25%

.

 The first offspring in the control group we re born before a maximum of nine days

.

RESULTS

Endpoint value(s):

The following values are based on WAF loading rates : 21

- day E L 50 (mortality/immobility) > 100 mg/L 21

- day E L 50 (reproduction) > 100 mg/L 21

- day E L 50 (growth) > 100 mg/L

21

- day NOE L (mortality/immobility ) = 100 mg/L 21

- day NOEL ( reproduction) = 10 mg/L 21

- day NOEL (growth) = 1.0 mg/L

21

- day LOEL (mortality/immobility) > 100 mg /L 21

- day L OEL ( reproduction) = 32 mg/L 21

- day LOEL (growth) = 3.2 mg /L

MATC (growth) = 1 .79 mg/L ( loading rate )Final HPV 3 Chemical Data Adequacy Review (January 2014) CAS RN 22527

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18 The following values are based on mean measured concentrations that correspond with nominal loading rates: 21

- day E C 50 (mortality/immobility) > 0.21 mg/L 21

- day E C 50 (reproduction) > 0.21 mg/L 21

- day E C 50 (growth) > 0.21 mg/L

21

- day NOE C (mortality/immobility ) = 0.21 mg/L 21

- day NOEC ( reproduction) = 0.18 mg/L * 21

- day NOEC (growth) = 0.14 mg/L *

21

- day LOEC (mortality/immobility) > 0.21 mg/L 21

- day LOEC ( reproduction) = 0.1 2 mg/L * 21

- day LOEC (growth) = 0.13 mg/L *

MATC (growth) = 0.13 mg/ L *

*Based on mean measured concentrations that correspond with the reported LOEL and NOEL values, the LOEC values for reproduction and growth are less than the NOEC values. Given that L OEC values, by definition, must be greater than NOEC values , the LOEC

, NO EC , and MATC values identified above are not considered accurate

. T he large fluctuations in measured test substance concentrations for each nominal loading rate preclude the accurate estimation of LOEC

, NOEC , and MATC values

.

Results remarks: Measured concentrations are summarized in Table 1 . Based on combined “old” and “ fresh ” samples for the 0 (dilution control), 1, 3.2, 10, 32, and 100 mg/L groups, mean measured concentrations were < LOQ, 0.14 (14% of nominal), 0.13 (4% of nominal), 0.18 ( 2 % of nominal), 0.1 2 (0. 4 % of nominal), and 0.21 (0.21% of nominal) mg/L , respectively. The test substance was detected in several control samples, but an explanation was not provided for these findings.

Table 1 . Measured Concentrations of Test Substa n ce Time point (day) Nominal concentration (mg/L)

0 1 3.2 10 32 100

0 ( fresh ) 0.00414/

< LO Q a 0.521 0.529 0.814 0.546 0.927

2 (old) 0.00372/

< LO Q a 0.301 0.330 0.0163 0.0102 0.0441
5 (fresh) < LO Q 0.14 0.0475 0.509 0.0168 0.0522

7 (old) 0.00441/

< LO Q a < LO Q < LO Q 0.00517 < LO Q < LO Q
9 (fresh) < LO Q < LO Q < LO Q 0.0870 0.061 0.489

12 (old) 0.0115/

< LO Q a < LO Q < LO Q 0.0209 0.00371 0.00486
14 (fresh) < LO Q 0.088 8 0.121 0.281 0.151 0.467Final HPV 3 Chemical Data Adequacy Review (January 2014) CAS RN 22527

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19 Table 1 . Measured Concentrations of Test Substa n ce Time point (day) Nominal concentration (mg/L)

0 1 3.2 10 32 100

16 (old) 0.0192/

< LO Q a < LO Q < LO Q 0.018 0.0123 0.00943
19 (fresh) < LO Q 0.331 0.242 0.0513 0.342 0.0573

21 (old) 0.0180/

< LO Q a < LO Q < LO Q 0. 0 114 0.00588 < LO Q

Mean measured concentration ( m g/L) b 0.14 0.13 0.18 0.1 2 0.21

a Duplicate sample stored at

- 20 ° C prior to analysis

b Mean of combined fresh and old samples ; f or the purposes of calculating mean measured concentrations, a value of 0.0036 (limit of quantitation for analytical measurements) was substituted for measured concentrations reported as < LOQ. LOQ = Limit of Quantitation ( 0.0036 mg/L)

“Anal ysis of the test preparations showed variable measured concentrations on a day

- to

- day basis. This variation is common in the analysis of WAFs of low solubility, complex reaction mixtures. The results of the analysis did however show a decline in measured concentrations between the fresh and old test media samples on each preparation/exposure period.” [Harris 2013, p. 19]

“No significant mortalities occurred at the 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAF test concentrations throughout the test. There was one [adult] daphnid observed to be immobilized in the 32 mg/L loading rate WAF group, however, since no more than 10% mortality occurred, this is considered not to have affected the outcome of the test.” [Harris 2013, p. 18]

“ After 21 days the l ength of each surviving adult [of the parental generation] was determined

... The results showed that there were no statistically significant differences (p ≥ 0.05 ) between the control and the 1.0 mg/L loading rate WAF test group in terms of length of the daphnids after 21 days exposure to the test item

... However, there was a significant difference [in daphnid length at the end of exposure] between the control and 3.2, 10, 32 and 100 mg/L loadin g rate WAF test concentrations.” [Harris 2013, p. 18]

Mean dap hnid lengths at day 21 in the control

, 1.0

, 3.2, 10, 32, and 100 mg/L WAF loading rate groups were 4.4 ± 0.13

, 4.4 ± 0.16, 4.2 ± 0.13, 4.1 ± 0.20, 4.0 ± 0.12, and 3.8 ± 0.20 mm, respectively. Daphnid lengths, expressed as percentages of control values, were 100%, 95%, 93%, 91%, and 86% at WAF loading rates of 1, 3.2, 10, 32, and 100 mg/L, respectively.

“After 21 days there were no statistically significant differences between the control and the 1.0, 3.2 and 10 mg/L loading rate WAF test groups in terms of the number of live young produced per adult. The 32 and 100 mg/L loading rate WAF test groups showed a statistically significant difference from the control and the remaining test groups after 21 days in terms of producing fewer numbers of live young per adult.” [Harris 2013, p. 18]

The mean numbers of live young produced per adult by day 21 in the control

, 1.0

, 3.2, 10, 32, and 100 mg/L WAF loading rate groups were 120.2 ± 17.3, 133.9 ± 10.8

, 134.2 ± 16.6

, 124.3 ±Final HPV 3 Chemical Data Adequacy Review (January 2014) CAS RN 22527

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20 17.7

, 106.0 ± 18.8 , and 99.9 ± 12.2, respectively. L ive young per adult , expressed as percentages of control values, were 111%, 11 2 %, 103%, 88%, and 83% at WAF loading rates of 1, 3.2, 10, 32, and 100 mg/L, respectively.

“ [A]n assessment made at each media renewal showed the “ filial ” daphnids produced by all the test groups were in the same general condition as the young produced by the controls over the duration of the test... There were no unhatched eggs or dead young in a ll control and treatment groups surviving to maturation.” [Harris 2013, p. 1 9 ]

Water temperatures measured daily in one replicate test chamber per concentration ranged from 19 to 21 °C. Dissolved oxygen concentrations were maintained at 7.7 to 9.1 mg O 2 /L throughout the test period (remained ≥ 85 % of saturation ) . Measurements of pH in the test chambers ranged from 7 .3 to 8. 3 throughout the test period. Light intensity ranged from 681 to 965 lux throughout the test period. Ranges of w ater hardness, con ductivity , and alkalinity measurements in the control and 100 mg/L nominal loading rate treatments during the study were 216

- 260 mg CaCO 3 /L, 680

- 833 μ S/cm, and 39.6

- 51.6 mg CaCO 3 /L, respectively.

The results of the test were considered valid based on the following:

 Control mortality was 0%

.

 Dissolved oxygen content was > 7.7 mg O 2 /L

.

 The maximum deviation in pH in the control group was 1.0 units

.

 The mean number of live young produced per surviving adult daphnid in the control
group was 120 after 21 days

.

 The coefficient of variation for the control group was 14.4%

.

 The first offspring in the control group were recorded on day 8

.

CONCLUSIONS

Daphnia magna exposed to the test item (Benzoflex 1046 mixture) as a WAF at nominal loading rates of 1.0 to 100 mg/L for 21 days showed no treatment

- related reductions in survival /immobility . Treatment

- related reductions in growth (length) of the surviving daphnids of the parental generation were seen at loading rates ≥ 3 .2 mg/L. Treatment

- related inhibition of reproduction

, as measured by a reduction in the number of live young per daphnid, was seen at loading rates ≥ 32 mg/L. The NOEL and LOEL for this study, based on the most sensitive endpoint tested (growth), were 1

. 0 and 3.2 mg/L , respectively . The MATC was calculated to be 1.79 mg/L based on WAF loading rate s . The 21

- day EL 50 value s for adult mortality/immobility

, reproduction , and growth were > 100 mg/L based on WAF loading rate s

. T he corresponding 21

- day EC 50 values for adult mortality/immobility, reproduction, and growth

, based on mean measured concentrations, were > 0.21 mg/L. The large fluctuations in measured test substance concentrations for each nominal loading rate precluded the accurate estimation of LOEC, NOEC, and MATC values.

STUDY RELIABILITY

[3] Not reliable. This study is considered unreliable because the test failed to identify a discrete E C 50 or accurate LOEC and NOEC values for chronic effects on survival, reproduction, andFinal HPV 3 Chemical Data Adequacy Review (January 2014) CAS RN 22527

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21 growth of daphnids. Although adverse effects were observed on reproduction and growth, inhibition of each parameter was less than 50% at the highest concentration tested and

, therefore, the study did not determine a discrete EC 50

. Furthermore, accurate NOEC and LO EC values could not be identified because mean measured concentrations ( 0.14, 0.13, 0.18, 0.12, and 0.21 mg/L, respectively, for nominal loading rates of 1, 3.2, 10, 32, and 100 mg/L) did not consistently increase with increasing loading rate concentration s (see #4 below)

. The large variations in measured test substance concentrations prevent the interpretation of the biological data to characterize the concentration

- response relationship.

Additional deficiencies included the following: (1) The test item was detected in control samples collected on Days 0, 2, 7, 12, 16, and 21

, suggesting unreliable analytical techniques for measuring concentrations or inadequate quality control

. (2) Measured test substance concentrations among “fresh” WAF test solutions (sampled at the time of solution renewal) of the same nominal loading rate were highly variable at different measurement time points (see Table 1 above)

. For example, the measured concentrations of “fresh” solutions of WAFs at a nominal loading rate of 100 mg/L were 0 .927, 0.0522, 0.489, 0.467, and 0.0573 mg/L on Days 0, 5, 9, 14, and 19, respectively. During the study, measured concentrations of “fresh” test solutions varied by a factor of at least 15 (highest measured “fresh” value divided by lowest measured “fresh” value) for each treatment level. For loading rates of 1, 3.2, 10, 32, and 100 mg/L, individual measured concentrations of “fresh” test solutions varied by as much as 140, 180, 134, 144, and 133% from the mean measured “fresh” conc entration for that loading rate during the 21

- day exposure period. Th is variability raises concerns about the consistency and reproducibility of the methods used to prepare the test solutions during the test and/or the analytical methods used to determine test substance concentrations. No explanations for the variability among measured concentrations of “fresh” test solutions were provided in the study report. The study report did not provide a detailed des cription of the procedures for storage and handling of WAF preparations. Missing information included, for example, whether the WAF was stored prior to use and, if so, for how long and under what conditions. The inconsistencies among test item concentrat ions for individual WAF loading rates leads to uncertainty in the reported endpoint values. (3) Substantial declines in measured test substance concentrations were observed between test solution renewals. M easured concentrations of “old” test solutions (sam pled prior to solution renewal) were up to 99% less than the respective measured “fresh” concentrations. No explanation for these declines was provided in the study report and no analysis was conducted to determine the cause. It is unknown whether the decline in test substance concentration could be due to degradation of the test substance because n o data were provided to describe stability of the test substance in the test media. (4) The combination of highly variable test substance concentrations at test solution renewal and steep declines in test substance concentration between renewals resulted in large variations in measured test substance concentrations at each WAF loading rate over the 21

- day exposure period. When measurements from “old” and “fresh” test solutions were combined, measured concentrations varied by a factor of at least 144 (highest measured value divided by lowest measured value) for a given treatment level. For loading rates of 1, 3.2, 10, 32, and 100 mg/L, individual measured concentr ations (“old” and “fresh”Final HPV 3 Chemical Data Adequacy Review (January 2014) CAS RN 22527

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22 combined) varied by as much as 272, 311, 349, 374, and 350% from the mean measured concentration for that loading rate during the 21

- day exposure period. The ranges for the measured concentrations (“fresh” and “old” combined) wer e < LOQ

- 0.521, < LO Q

0.529, 0.00517

- 0.814, < LOQ

- 0.546, and < LOQ

- 0.927 mg/L, respectively, for nominal loading rates of 1, 3.2, 10, 32, and 100 mg/L

. The mean measured concentrations ( 0.14, 0.13, 0.18, 0.1 2 , and 0.21 mg/L, respectively, for nominal loading rates of 1, 3.2, 10, 32, and 100 mg/L ) did not consistently increase with increasing loading rate concentrations

, which precluded the identification of accurate NOEC and LOEC values. (5) The solubility and stability of the test substance in the dilution water (culture medium ) were not determined prior to the initiation of the test, as recommended by ASTM E1193

- 97. (6) The study did not include data on observations of first

- and second

- generati on daphnids for abnormal development or aberrant behavior as recommended by section 12.10.5 of ASTM E1193

- 97. (7) The 100 mg/L loading rate WAF was filtered through a glass wool plug on day 9 to remove microscopic particles of the test item in the water colum n. The use of glass wool for removal of undissolved test material is not recommended. Instead, the use of centrifugation or a separatory funnel is suggested.

REFERENCE

Harris, S . 20 13

. Benzoflex 1046: Daphnia magna reproduction test

. Harlan Laboratories Ltd.

, Shardlow, Derbyshire, UK

, Project No. 41202706 . Sponsor: Eastman Chemical Company

, Kingsport, TN

. Study report available online at http://www .regulations.gov/#!documentDetail; D=EPA

- HQ

- OPPT

- 2009

- 0112

- 0145 as of January 7, 2014

. Text in quotes is taken directly from Harris (2013).Final HPV 3 Chemical Data Adequacy Review (January 2014) CAS RN 22527

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23 D

- 3 . Genetic Toxicity

– Chromosomal Aberrations

Summary of endpoint: Adequate

Study 1: Chromosome aberration assay in human lymphocytes ( Bowles , 2013 )

Title: Benzoflex 1046: Chromosome aberration test in human lymphocytes in vitro

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Benzoflex 1046; supporting substance for propanoic acid, 2

- methyl

- , 3

- (benzoyloxy)

- 2,2,4

- trimethylpentyl ester (CASRN 22527

- 63

- 5)

Remarks: C lear colorless liquid; Batch No. V046212201; Composition: 68.8% p ropanoic acid, 2

- methyl

- , 3

- (benzoyloxy)

- 2,2,4

- trimethylpe ntyl ester (CASRN 22527

- 63

- 5), 19.5% 1,3

- pentanediol, 2,2,4

- trimethyl

- 1,3

- dibenzoate (CASRN 68052

- 23

- 3), 7% 2,2,4

- trimethyl

- 1,3

- pentanediol diisobutyrate (CASRN 6846

- 50

- 0), 4.2% miscellaneous esters, 0.5% volatiles, and 0.02% moisture. 1

METHOD

Method/guideline followed: Compatible with OECD TG 47 3 (“ In Vitro Mammalian Chromosome Aberration Test”) ; Method B10 of Commission Regulation (EC) No. 440/2008

Test type: In vitro m ammalian chromosome aberration t est

GLP compliant?: Yes ; with the following exception: “ No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within two hours of it being applied to the test system; it is assumed that the formulati on was stable for this duration. ” [Bowles 2013, p. 3 ]

Year study performed: 2012

Test species: Human peripheral blood lymphocytes

Metabolic activation: With (rat S9 microsomal fraction) and without

1 The certificate of analysis provided in the study report d escribes the c omposition as follows: 68.8% “diester”

, 19.5% “dibenzoate”, 7% “TXIB”, 4.2% miscellaneous esters, 0.5% volatiles, and 0.02% moisture. In an email dated July 3, 2013 from G. Shrum (Eastman) to J. Schaeffer (EPA), Eastman indicated that “ diest er” was synonymous with p ropanoic acid, 2

- methyl

- , 3

- (benzoyloxy)

- 2,2,4

- trimethylpentyl ester (CASRN 22527

- 63

- 5), “dibenzoate” was synonymous with 1,3

- pentanediol, 2,2,4

- trimethyl

- 1,3

- dibenzoate (CASRN 68052

- 23

- 3), and “TXIB” was synonymous with 2,2,4

- trim ethyl

- 1,3

- pentanediol diisobu t yrate (CASRN 6846

- 50

- 0).Final HPV 3 Chemical Data Adequacy Review (January 2014) CAS RN 22527

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24 Concentrations tested: Experiment 1: 4

- hour exposure followed by 20

- hour incubation without test item : 0 ( vehicle control) * , 5 * , 10 * , 20 * , 40, 60, and 80 μg Benzoflex 1046/mL (without S9) 0 (vehicle control) *

, 10

, 20 , 40 *

, 8 0 *

, 12 0 * , and 160 μg Benzoflex 1046/mL ( with S9) Experiment 2: Without S9, 24

- hour continuous exposure: 0 (vehicle control) * , 5, 10, 20 * , 40 * , 60 * , and 80 μg Benzoflex 1046/mL With S9, 4

- hour exposure followed by 20

- hour incubation without test item 0 (vehicle control) * , 5, 10, 20 * , 40 * , 80 * , and 120 μg Benzoflex 1046/mL *Concentrations selected for metaphase analysis

Statistical methods used: “ The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. ” [Bowles 2013, p. 15]

Vehicle: Dimethyl sul f

oxide

Test method/conditions remarks: Human lymphocytes were collected from a blood sample isolated from the peripheral circulation of a screened volunteer

. A pproximately 48 hours after mitogenic stimulation with phytohemagglutinin, duplicate cultures were treated with the test item

or appropriate control in the presence or absence of S9 metabolic activation at 37°C

.

“The test item was... dissolved in dimethyl sulphoxide and serial dilutions [were] prepared. The molecular weight of the test item was unknown and

, therefor e , designated to be 500 (because it was a mixture of multiple chemicals). The maximum dose level [in the preliminary toxicity test] was 5000 μg/ mL , which was calculated to be equivalent to the 10 mM maximum recommended dose level .” [Bowles 2013, p. 9]

“The test item was formulated within two hours of it being applied to the test system.” [Bowles 2013, p. 10]

“There was no significant change in pH when the test item was dosed into dimethyl sulphoxide and the osmolality did not increase by more than 50 mO sm. ” [Bowles 2013, p. 9]

The S9 homogenate was produced from homogenized liver samples collected from ma le rats that had received three daily oral doses of a phenobarbitone (80 mg/kg

- bw /day ) and β

naphthoflavone (100 mg/kg

- bw /day ) mixture prior to S9

- mix preparation on the fourth day. S9 batches were routinely verified for activation of known mutagens in the Ames assay.

Preliminary Toxicity Test: “A preliminary toxicity test was performed on cell cultures using a 4

- hour exposure time with and without metabolic activation followed by a 20

- hour recovery period, and a continuous exposure of 24 hours without metabolic activation. The dose range of test item used was 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 μg/mL. Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so thatFinal HPV 3 Chemical Data Adequacy Review (January 2014) CAS RN 22527

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25 test item precipitate observati ons could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.” [Bowles 2013, p. 1 2 ]

Chromosome Aberrations Assay : Experiment 1 : Lymphocytes were exposed to the test item or control for 4 hours with out or with S9 (2%) , followed by a 20

- hour incubation period

. C oncentrations without S9 activation selected for metaphase analysis were 0 (vehicle control), 5, 10, and 20 μg Benzoflex 1046/mL. C oncentrations with S9 activation selected for metaphase analysis were 0 (vehicle control), 40, 80, and 120 μg Benzoflex 1046/mL.

Experiment 2 : Lymphocytes were exposed to the test item or control continuously for 24 hours without S9, or exposed to the test item for 4 hours with S9 (1%) followed by a 20

- hour incubation period. C oncentrations without S9 activation selected for metaphase analysis were 0 (vehicle control), 20, 40, and 60 μg Benzoflex 1046/mL. C oncentrations with S9 activation selected for metaphase an a lysis were 0 (ve hicle control), 20, 40, and 80 μg Benzoflex 1046/mL.

Lymphocytes were arrested in mitosis with demecolcine ( Colcemid

, 0.1 μ g /mL) two hours before scheduled harvest. The c ells were treated with 0.075M hypotonic KCl, fixed with methanol/glacial acetic acid (3:1 v/v), mounted on microscope slides, and stained w ith 5% Giemsa stain . Slides were randomly coded using a computerized number generator.

“The slides were checked microscopically to determine the quality of the metaphases and also the toxicity a nd extent of precipitation, if any, of the test item. These observations were used to select the dose levels for mitotic index evaluation.” [Bowles 2013, p. 14]

“A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.” [Bowles 2013, p. 14]

“Where possible the first 100 consecutive well

- sp read metaphases from each culture were counted, where there were approximately 30 to 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44

- 48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing. Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.” [Bowles 2013, p. 14]

“ ...[C] ell s with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported.” [Bowles 2013, p. 14]

“A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose

- relationship. For modest increases in aberration frequ ency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.” [Bowles 2013, p. 30]Final HPV 3 Chemical Data Adequacy Review (January 2014) CAS RN 22527

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26 Positive Control Substances: Experiment 1: Without S9: Mitomycin C at 0.4 μg/mL; With S9: Cyclophosphamide at 5 μg/mL Experiment 2: Without S9: Mitomycin C at 0.2 μg/mL; With S9: Cyclophosphamide at 5 μg/mL

RESULTS

Genotoxicity result: Negative

Cytotoxic concentration : 4

- and 24

- h ou r exposures without S9: 78.13 μg/mL; 4

- h ou r exposures with S9: 156.25 μg/mL (based on ≥ 50% inhibition of mitotic index in the preliminary toxicity test)

Results remarks: Positive and negative c ontrol responses were appropriate.

Preliminary Toxicity Test Results: P recipitate was observed at ≥ 156.25 μg/mL in cultures in the 4

- hour and 24

- hour exposure
groups without S9, and at ≥ 312.5 μg/mL in the 4

- hour exposure group with S9

. Hemolysis was observed in the 4

- hour exposure group at ≥ 312.5 μg/mL with S9 ( but not in the 4

- hour exposure
group without S9 ) and in the 24

- hour continuous exposure group without S9 at ≥ 78.13 μg/mL

. “Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 78.13 μ g/m L in the absence of S9 and 156.25 μ g/m L in the presence of S9 , respectively

. There were no scorable metaphases present at dose levels above these stated in any exposure group... [ T ] he test item demonstrated steep toxicity curves in all three exposure groups which made achieving optimum toxicity very difficult.” [Bowles 2013, p. 16]

Chromosomal Aberrations Assay : The test item did not induce any statistically significant increases in the frequency of cells with chromosome aberrations or in the number of polyploid cells in either Experiment 1 or 2.

Experiment 1: Scorable metaphases were present at concentrations up to 40 μg/mL without S9 and up to 120 μg/mL with S9

. However, “[t]he toxicity observed at 40 μg /ml in the absence of S 9 [i.e., a 77% inhibition of the mitotic index compared with the control] was considered to be excessive and precluded the dose from chromosome analysis. ” [Bowles 2013, p. 1 7 ] No precipitate or hemolysis was observed in the blood cultures at the end of the exposure period either in the presence or absence of S9

.

Mitotic ind ices w ere inh ibited both with and without S9. Mitotic indices (% of control) were 99, 93, 62, and 23%, respectively, at 5, 10, 20, and 40 μg/mL for the 4

- hour exposure without S9. Mitotic indices (% of control) were 102, 100, 98, and 54%, respectively, at 20, 40, 80, and 120 μg/mL for the 4

- hour exposure with S9.

Experiment 2: Scorable metaphases were present at the maximum test item concentration of 8 0
μ g/mL without S9 and at 80 μ g/mL with S9. However, “[t]he toxicity observed at 80 μg /ml in the absence of S 9 [i.e., a 77% inhibition of the mitotic index compared with the control] wasFinal HPV 3 Chemical Data Adequacy Review (January 2014) CAS RN 22527

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27 considered to be excessive and precluded the dose from chromosome analysis. ” [Bowles 2013, p. 1 8 ] No precipitate or hemolysis of the test item was observed at the end of exposure in the absence of S9 ; however, hemolysis was observed at 120 μg /m L in the presence of S9.

Mitotic indices were inhibited both with and without S9. Mitotic indices (% of control) were 102, 87, 64, and 23%, respectively, at 20, 40, 60, and 80 μg/mL for the 24

- hour continuous exposure without S9. Mitotic indices (% of control) were 136, 100, 87, and 42%, respectively, at 10, 20, 40, and 80 μg/mL for the 4

- hour exposure with S9.

CONCLUSIONS

The test item , Benzoflex 1046, d id not induce chromosomal aberrations in human peripheral blood lymphocytes in vi tro

.

STUDY RELIABILITY

[ 2 ] Reliable with restrictions. Missing study details included sex of blood donor, the cell density seeded at the start of the experiment , justification for choice of vehicle, and stability of the test item in the vehicle

.

REFERENCE

Bowles

, A . 201 3 . Benzoflex 1046: Chromosome aberration test in human lymphocytes in vitro

. Harlan Laboratories Ltd., Shardlow, Derbyshire, UK, Project No. 4120191 8 . Sponsor: Eastman Chemical Company

, Kingsport, TN

. Study report available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2009

- 0112

- 0145 as of January 7, 2014

. Text in quotes is taken directly from Bowles (2013).Final HPV 3 Chemical Data Adequacy Review (January 2014) CAS RN 22527

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28 E. DATA ADEQUACY SUMMARY TABLE

Endpoint Study Information Available Acceptable Data Gap

Aquatic Effects Acute Toxicity to Fish No

– – 1 Acute Toxicity to Aquatic Invertebrates No

– – 1 Chronic Toxicity to Aquatic Invertebrates Yes No Yes

Toxicity to Aquatic Plants Yes No Yes Health Effects

Acute Toxicity Yes Yes No Repeated

- Dose Toxicity Yes

– 2

– 2 Gene Mutation Yes Yes No Chromosomal Aberrations Yes Yes No Reproductive Toxicity Yes

– 2

– 2 Developmental Toxicity Yes

– 2

– 2

1 Acute toxicity to fish and acute toxicity to aquatic invertebrate testing are not required because the measured log K ow of CASRN 22527

- 63

- 5 is < 4.2.
2 Data submitted by Eastman on December 18 , 201 3 are currently under review

.Addendum to HPV 3 Chemical Data Adequacy Review (Final ) CASRN 22527

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A

- 1 ADDENDUM: Test Guideline Source Material to Support Reliability Ratings Assigned to Submitted Studies That Are Considered Inadequate to Satisfy Test Rule Requirements

This addendum provides additional information regarding deficiencies of each of the submitted studies that is considered inadequate to satisfy a test rule requirement. For each deviation from relevant test guidelines (i.e., standard test guidelines purportedly followed by each study according to the study repo rt), quotes of relevant passages from the guidelines are supplied, followed by a brief summary of the quoted material and, where appropriate, an interpretation by a qualified scientist. Emphasis has been added to quotes (in the form of italics) by the rev iewer.

Study 1: Toxicity to Aquatic Plants (Vryenhoef 2013) Relevant Guidelines: The study was conducted according to OPPTS 850.5400 and ASTM E1218

- 04. The study report cites OECD Series on Testing and Assessment Number 23 (Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures) as supporting the use of Water Accommodated Fractions (WAFs), given that the test item is a complex mixture that includes components with poor water solubility.

In the ASTM E1218

- 04 guideline, p. 2 , Section 3 “Terminology”, Sub

- section 3.1, “Definitions”, item 3.1.1 indicates that the words “must” and “should” have very specific meanings. Sub

- item 3.1.1.2 states: “ must

— is used to express an absolute requirement, that is, to state that the test ough t to be designed to satisfy the specified condition, unless the purpose of the test requires a different design. ‘ Must ’ is only used in connection with factors that directly relate to the acceptability of the test (see 13.1).” Sub

- item 3.1.1.3 states: “ should

— is used to state that the specified condition is recommended and ought to be met if possible. Although violation of one

‘ should ’ is rarely a serious matter, violation of several will often render the results questionable.”

[A] Deviation 1: This study failed to identify a discrete EC 50 or IC 50 because inhibition of algal growth rate, biomass, and yield at the highest test concentration was less than 50% in the definitive test. The study only determined that the EL 50 (based on WAF loading rates) w as greater than 100 mg/L and that the EC 50 (based on mean measured concentrations) was greater than 0.19 mg/L, which is considerably less than the reported water solubility limit of the test substance (1.01 mg/L at 20 °C). OPPTS 850.5400, p. 2, Section ( d) “Test Procedures”, Sub

- section (3) “Definitive test”, item (i) states: “The purpose of the definitive test is to determine the concentration response
curves, the EC50’s for algal growth for each species tested, with a minimum amount of testing beyond th e range

- finding test.” OPPTS 850.5400, p. 2, Section (d) “Test procedures”, Sub

- section (2) “Range

- finding test”, item (ii) states: “Algae are exposed to a widely spaced (e.g., log interval) chemical concentration series [in the range

- finding test]. The lowest value in the series, exclusive of controls, should be at the chemical’s detection limit. The upper value, for water soluble compounds, should be the saturation concentration . A minimum of three replicates is required: Nominal concentrations of the chemical are acceptable if it is determined that definitive testing is not required.”; and item (iv) states: “Definitive testing may not beAddendum to HPV 3 Chemical Data Adequacy Review (Final ) CASRN 22527

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A

- 2 necessary if the highest chemical concen tration tested ( water saturation concentration or 1000 mg/L) results in less than a 50 percent reduction in growth.” 1 OPPTS 850.5400, p. 2, Section (d) “Test procedures”, Sub

- section (1) “Summary of the test”, item (ii) states: “At the end of 96 h, and at the end of 24, 48, and 72 h if possible, enumerate the algal cells in all containers to determine inhibition or stimulation of growth and the pattern of growth in test containers compared to controls. Use data to define the concentration

- response curve, a nd calculate the EC50 value at these times.” OPPTS 850.5400, p. 8, Section (f) “Reporting” states: “... In addition to the reporting requirements as specified under EPA Good Laboratory Practice Standards, 40 CFR Part 792, subpart J, the following specific in formation is to be reported :”; and item (6) states: “The 96

- h EC50 values , and when sufficient data have been generated, the 24

- , 48

- , and 72

- h EC50s and 95 percent confidence limits, the methods used to derive these values, the data used to define the shape of the concentration

- response curve and the goodness

- of

- fit determination.” ASTM E1218

- 04, p. 6, Section 9 “Test Material”, Sub

- section 9.3 “Test Concentration(s)”, item 9.3.2.1 states: “... To ensure that solubility has been achieved in the toxicity t est, it may be appropriate to test up to approximately twice the solubility limit in the medium being used. However, in this case toxicity should be expressed as the solubility limit (that is, no effect at the solubility limit, or 20% reduction in cell nu mbers at the solubility limit). Analytical verification of the solubility under the test conditions can be critical for test materials of limited aqueous solubility.” ASTM E1218

- 04, p. 6, Section 9 “Test Material”, Sub

- section 9.3 “Test Concentration(s)”, item 9.3.3 states: “In some (usually regulatory) situations, it is only
necessary to determine whether a specific concentration of test material affects growth of the test species or whether the IC50 is above or below a specific concentration. For example, the specific concentration might be the concentration occurring in a surface water, the concentration resulting from the direct application of the material to a body of water, or the solubility limit of the material in water . When there is only interest in a specific concentration, it is often only necessary to test that concentration (see 11.1.1.2), and it is not necessary to actually determine the IC50.”

Summary of Quoted Material: A primary objective of a toxicity to algae test is to determine a discrete EC 50 or IC 50 value (EL 50 or IL 50 value if expressed as loading rate). The highest concentration tested should be equivalent to the water solubility limit. Interpretation: If a discrete EC 50 or IC 50 value is not determined because insufficient (< 50%) inhibition of growth rate, biomass, and yield occurs at the highest concentration tested and this concentration is below the aqueous solubility limit, the test is not considered acceptable because it fails to provide useful information for regulation of the test substance.

[B] Deviation 2: There was no evidence that the solubility, stability, and volatility of the test substance in the growth medium were dete rmined prior to the initiation of the algae test; the

1 Although the maximum concentration tested in the range

- finding test was approximately equivalent to the water solubility limit at initiation of the test (measured concentration of 1.2 mg/L at 0 hours ) and inhibition of growth rate , biomass and yield was less than 50%, the range

- finding test used only 2 replicates, rather than the 3 replicates required. Therefore, a definitive test i s required.Addendum to HPV 3 Chemical Data Adequacy Review (Final ) CASRN 22527

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A

- 3 study report did not provide measurements of these physical

- chemical properties in the growth medium. OPPTS 850.5400, p. 2, Section (d) “Test procedures”, Sub

- section (2) “Range

- finding test”, item (i ) states: “... Water solubility of the test chemical (as well as other physical chemical characteristics, e.g. volatility) should be determined before definitive testing.” ASTM E1218

- 04, p. 4, Section 9 “Test Material”, Sub

- section 9.1 “General” states: “... Before a test is begun, the following should be known about the test material:”; and item 9.1.2 states: “ Solubility, stability, and volatility in the growth medium.” ASTM E1218

- 04, p. 6, Section 9 “Test Material”, Sub

- section 9.3 “Test Concentration(s)” , item 9.3.2.1 states: “... To ensure that solubility has been achieved in the toxicity test, it may be appropriate to test up to approximately twice the solubility limit in the medium being used. However, in this case toxicity should be expressed as the so lubility limit (that is, no effect at the solubility limit, or 20% reduction in cell numbers at the solubility limit). Analytical verification of the solubility under the test conditions can be critical for test materials of limited aqueous solubility.” A STM E1218

- 04, p. 10, Section 15 “Report”, Sub

- section 15.1 states: “ Include the following information on the record of the results of an acceptable algal test, either directly or by reference to available documents:”; and item 15.1.2 states: “Source of the test material, its lot number, composition (identities and concentrations of major ingredients and major impurities), known chemical and physical properties , and the identity and concentration(s) of any solvent used, ...” OECD 23, p. 15, Section 1 “Preliminary assessment of stability of test substance” states: “Data providing an indication of the stability of the test substance under the test conditions should therefore be obtained before commencing testing.” OECD 23, p. 20, Section 3. 1 “Poorly water

- soluble substances”, Sub

- section 3.1.1 “Preliminary experiment” states: “A preliminary experiment should be considered for all studies with poorly water

- soluble substances to determine the maximum dissolved concentration that can be achieve d under test conditions, which is defined as the saturation concentration, and the preparation conditions that are required to achieve it. The results of the preliminary experiment will form a basis of and justification for the media preparation procedure s adopted for the toxicity tests and a reference point (i.e. water solubility under test conditions) against which the test results can be evaluated. In view of its importance the preliminary experiment should be fully reported.”

Summary of Quoted Materi al: The solubility, stability, and volatility of the test substance in the growth medium should be determined prior to initiation of a test of toxicity to algae. The solubility of the test substance in the growth medium should be provided in the study re port so the adequacy of test concentrations can be assessed. Other known physical

- chemical properties, such as the stability and volatility of the test substance in the growth medium, should also be reported. Interpretation: It is important to know the solubility of the test substance in the dilution water so that the appropriateness of the media preparation procedures can be assessed. Data describing test substance solubility, stability, and volatility in the growth medium that are generated prior to t he study are useful in optimizing the study design; without such data, an optimal design is difficult to achieve.Addendum to HPV 3 Chemical Data Adequacy Review (Final ) CASRN 22527

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A

- 4 [C] Deviation 3: Analytical measurements demonstrated a disparity (greater than 3

- fold) between the test substance concentrations of the WAFs prepared for the range

- finding and definitive tests. Specifically, the initial (time = 0) measured test substance concentration of the 100 mg test item/L loading rate WAF sample was 0.380 mg/L in the definitive test, but was 1.23 mg/L in the range

- finding test. The discrepancy in measured concentrations at test initiation raises concerns about the consistency and reproducibility of the methods used to prepare the test solutions for the two tests and/or the analytical methods used to determine test substance concentrations. The author did not provide an explanation for this disparity. In addition, the study report did not provide a detailed description of the procedures for storage and handling of WAF preparations, including whether the WAF was stored prior to use and, if so, for how long and under what conditions. OPPTS 850.5400, p. 5, Section (d) “Test procedures”, Sub

- section (4 ) “Analytical measurements”, item (i) “Chemical”, sub

- item (A) states: “... The concentration of test chemical in the test containers is to be determined at the beginning and end of the definitive test by standard analytical methods which have been validated prior to the test.” OPPTS 850.5400, p. 8, Section (f) “Reporting” states: “... In addition to the reporting requirements as specified under EPA Good Laboratory Practice Standards, 40 CFR Part 792, subpart J, the following specific information is to be repor ted :”; and item (7) states: “Methods and data records of all chemical analyses and test substance concentrations, including method validations and reagent blanks.” ASTM E1218

- 04, p. 9, Section 12 “Analytical Methodology”, Sub

- section 12.1 states: “If samples of stock solution or test solutions cannot be analyzed immediately, they should be handled and stored appropriately to minimize loss of test material by such things as microbial degradation, hydrolysis, oxidation, photolysis, reduction, sorptio n, and volatilization.”; and Sub

- section 12.2 states: “Chemical and physical data should be obtained using appropriate ASTM standards whenever possible. For those measurements for which ASTM standards do not exist or are not sensitive enough, methods shou ld be obtained from other reliable sources.” ASTM E1218

- 04, p. 10

- 11, Section 15 “Report”, Sub

- section 15.1 states: “ Include the following information on the record of the results of an acceptable algal test, either directly or by reference to available d ocuments:”; and item 15.1.7 states: “Methods used for, and results (with standard deviations or confidence limits) of, chemical analysis of concentration(s) of test material, impurities, and reaction and degradation products, including validation studies

a nd reagent blanks.”; and item 15.1.12 states: “Anything unusual about the test, any deviation from these procedures, and other relevant information.”; and Sub

- section 15.2 states: “Published reports should contain enough information to clearly identify the procedures used and the quality of the results.”

Summary of Quoted Material: Any unusual findings (e.g., large disparities between initial test concentrations in range

- finding and definitive tests) are to be addressed in the study report. In addition, samples should be handled and stored appropriately to minimize test material losses. Analytical measurement of test substance concentrations should be conducted using appropriate ASTM standards or comparable reliable methods. The analytical method used f or measurement of test substance concentrations is to be fully described in the study report, and documentation of the validity of the sampling, storage, and analytical methods should be sufficient to demonstrate that the methods conformed to acceptable st andards. Interpretation: If highly

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- 5 substance concentrations result when identical procedures are used for preparation, handling, storage, and/or analysis of test solutions, the reliability of the reported endpoint values for toxicity to alg ae is questionable.

[D] Deviation 4: Measured test substance concentrations declined substantially during the 96

- hour exposure period; decreases of approximately 98% and 96% were observed during the definitive and range

- finding tests, respectively. The study author stated that the declines in test substance concentration were due to the unstable nature of the tes t substance, but the report did not provide data to substantiate the author’s claim of instability of the test substance in the algae growth medium. OPPTS 850.5400, p. 2, Section (d) “Test procedures”, Sub

- section (2)(i) “Range

- finding test” states: “... Wa ter solubility of the test chemical (as well as other physical chemical characteristics , e.g. volatility) should be determined before definitive testing.” ASTM E1218

- 04, p. 4, Section 9 “Test Material”, Sub

- section 9.1 “General” states: “... Before a test is begun, the following should be known about the test material:”; and item 9.1.2 states: “ Solubility, stability, and volatility in the growth medium.” ASTM E1218

- 04, pp. 10

- 11, Section 15 “Report”, Sub

- section 15.1 states: “ Include the following information on the record of the results of an acceptable algal test, either directly or by reference to available documents:”; and item 15.1.2 states: “Source of the test material, its lot number, composition (identities and concentrations of major ingredients and major impurities), known chemical and physical properties , and the identity and concentration(s) of any solvent used, ...”; and item 15.1.12 states: “Anything unusual about the test, any deviation from these procedures, and other rel evant information.” OECD 23, p. 15, Section 1 “Preliminary assessment of stability of test substance” states:

“ Modifications to media preparation and exposure systems may be required where exposure concentrations of a test substance are likely to decline s ignificantly over the test period. In OECD guidelines for aquatic toxicity testing a decline in concentration of 20% is considered sufficient to warrant consideration of measures to reduce the decline. Data providing an indication of the stability of the test substance under the test conditions should therefore be obt ained before commencing testing... ” OECD 23, pp. 16

- 17, Section 1.2 “Preliminary stability study” states: “The possibility of losses during sampling, sample treatment and analysis must be considered. Where samples are stored before analysis, losses during sample storage should also be assessed.” OECD 23, p. 37, Section 3.11 “Multi

- component substances”, Sub

- section entitled “Partially soluble” states: “... The method used to prepare the WAF should therefore be fully described in the test report and evidence of its compositional stability over time provided... Chemical specific analysis is required to demonstrate attainment of equilibrium in WAF preparation and stability during the test. M ethods capable of identifying gross changes in the composition of WAFs with time are required...”

Summary of Quoted Material: The stability of the test substance in the growth medium should be determined before study initiation. Data on the compositional stability of the WAF over time should be provided. Unusual findings (e.g., large declines in test substance concentrations d uring the test) are to be addressed in the study report. The possibility that test substance was lost from solution during sampling, handling of samples, and analysis of samples must beAddendum to HPV 3 Chemical Data Adequacy Review (Final ) CASRN 22527

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- 6 considered. Interpretation: If test substance concentrations decli ne by 20% or more during the test, modification of test methods may be required to reduce this decline.

[E] Deviation 5: Growth rate in control cultures was not constant during the exposure. After 72 hours, a marked decline in the average specific growt h rate was observed in control cultures. The average specific growth rate in control cultures from 72 to 96 hours was less than one third of the growth rate observed during the first 72 hours. No explanation was provided by the study author for this decr ease in growth rate. ASTM E1218

- 04, p. 10, Section 14 “Calculation”, Sub

- section 14.1.2 states: “ The average specific growth rate is the growth rate calculated over the entire test period. In addition, the specific growth rate during the course of the tes t (days 0

- 1, 1

- 2, 2

- 3, etc.), also called the section

- by

- section growth rate, should be calculated to assess effects of the test material during the exposure period, such as an increased lag phase. Substantial differences between the section

- by

- section gr owth rates and the average specific growth rates indicate deviation from theoretical exponential growth and that close examination of the data is warranted. In this instance, the recommended approach is to compare specific growth rates from exposed cultur es during time period of maximum inhibition to controls during the same time period.” ASTM E1218

- 04, pp. 10

- 11, Section 15 “Report”, Sub

- section 15.1 states: “Include the following information on the record of the results of an acceptable algal test, eithe r directly or by reference to available documents:”; and item 15.1.12 states: “Anything unusual about the test, any deviation from these procedures, and other relevant information.”

Summary of Quoted Material: Substantial differences in section

- by

- section growth rates indicate deviation from theoretical exponential growth; such data require close examination. ASTM E1218

- 04 describes calculations that can be performed to assess deviations from theoretical expon ential growth. Special considerations are recommended for the interpretation of growth data from tests in which growth rate was not constant. The study report is to address any unusual findings, such as declining growth rates in controls.

REFERENCE

Vry enhoef, H. 2013. Benzoflex 1046: Algal growth inhibition test. Harlan Laboratories Ltd.

, Shardlow, Derbyshire, UK, Project No. 41202705. Sponsor: Eastman Chemical Company, Kingsport, TN. Study report available online at http://www.regulations.gov/#!documentDetail; D=EPA

- HQ

- OPPT

- 2009

- 0112

- 0145 as of January 7, 2014.Addendum to HPV 3 Chemical Data Adequacy Review (Final ) CASRN 22527

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- 7 Study 2: Chronic Toxicity to Daphnia (Harris 2013) Relevant Guidelines: The study method was designed to be compatible with ASTM Standard E1193

- 97. The study report cites OECD Series on Testing and Assessment Number 23 (Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures) as supporting the use of Water Accommodated Fractions (WAFs), given that the test item is a complex mixture that includes components with poor water solubility.

In the ASTM E1193

- 97 guideline, p. 217, Section 3 “Terminology”, defines the words “must” and “should” as having very specific meanings. Sub

- section 3.2 states: “ must

—used to express an absolute requirement, that is, to state that the test ought to be designed to satisfy the specified condition, unless the purpose of the test requires a different design. ‘Must’ is used only in connection with factors that directly relate to the acceptability of the test (see 14.1).” Sub

section 3.3 states: “ should

— used to state that the specified condition is recommended and ought to be met if possible. Although violation of one ‘ should’ is rarely a serious matter, violation of several will often render the results questionable.”

[A] Deviation 1: This study failed to identify a discrete EC 50 or accurate LOEC and NOEC values for chronic effects on survival, reproduction, and growt h of daphnids. Although adverse effects were observed on reproduction and growth, inhibition of each parameter was less than 50% at the highest concentration tested and, therefore, the study did not determine a discrete EC 50 . Furthermore, accurate NOEC a nd LOEC values could not be identified because mean measured concentrations ( 0.14, 0.13, 0.18, 0.12, and 0.21 mg/L, respectively, for nominal loading rates of 1, 3.2, 10, 32, and 100 mg/L) did not consistently increase with increasing loading rate concentrations . The large variations in measured test substance concentrations prevent the interpretation of the biological data to characte rize the concentration

- response relationship. ASTM E1193

- 97, p. 217, Section 4 “Summary of Guide”, Sub

- section 4.1 states: “... Measurement end points obtained during the test include the concentration of the test material and final number alive, final weig ht, and number of progeny per daphnid. Then data are analyzed to determine the effect of the test material on survival, growth, and reproduction of D. magna .” ASTM E1193

- 97, p. 221, Section 10 “Test Material”, Sub

- section 10.3 “Test Concentration(s)”, ite m 10.3.1 states: “If the test is intended to provide a good estimate of the highest concentration that will not unacceptably affect the survival, growth, or reproduction of D. magna , the test concentrations (see 12.1.1.1) should bracket the best prediction of that concentration.”

Summary of Quoted Material: The primary objective of a chronic toxicity to Daphnia test is to derive concentration

- response data for the effects of the test substance on survival, growth rate, and reproduction, including determin ation of discrete values for the EC 50 , LOEC, and NOEC. Interpretation: If a study fails to derive concentration

- response data and fails to identify discrete EC 50 , LOEC, and NOEC values, the study does not provide useful information for the assessment of risk to Daphnia and other similar species.Addendum to HPV 3 Chemical Data Adequacy Review (Final ) CASRN 22527

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- 8 [B] Deviation 2: The test item was detected in control samples collected on Days 0, 2, 7, 12, 16, and 21, suggesting unreliable analyti cal techniques for measuring concentrations or inadequate quality control. Appropriate controls should not contain the test item. ASTM E1193

- 97, p. 217, Section 4 “Summary of Guide”, Sub

- section 4.1 states: “... A control consists of maintaining daphnids in dilution water to which no test material has been added to provide (1) a measure of the acceptability of the test by giving an indication of the quality of the test organisms and the suitability of the dilution water, food, test conditions, handling proce dures, and so forth, and (2) the basis for interpreting data obtained from the other treatments.” ASTM E1193

- 97, p. 225, Section 13 “Analytical Methodology”, Sub

- section 13.3 states: “Chemical and physical data should be obtained using appropriate ASTM sta ndards whenever possible. For those measurements for where [ sic ] ASTM standards do not exist or are not sufficiently sensitive enough, methods should be obtained from other reliable sources...” ASTM E1193

- 97, p. 225, Section 14 “Acceptability of Test”, Sub

- section 14.1 states: “A life

- cycle test with D. magna should be considered unacceptable if one or more of the following occurred.”; and item 14.1.3 states: “Appropriate dilution

- water controls (and solvent controls if necessary) were not included in the te st.” ASTM E1193

- 97, p. 226, Section 16 “Report”, Sub

- section 16.1 states: “The record of the results of an acceptable life

- cycle toxicity test with D. magna should include the following information either directly or by reference to available docu ments:”; and item 16.1.15 states: “Anything unusual about the test, any deviation from these procedures, and any other relevant information.”

Summary of Quoted Material: The control is intended to provide a baseline response of test organisms in the absence of th e test substance. The analytical method used for measurement of test substance concentrations should be conducted using appropriate ASTM standards or comparable reliable methods. A study should be deemed unacceptable if the appropriate dilution water (or solvent water) controls are not included. Any unusual findings (e.g., detection of test substance in control samples) should be addressed in the study report. Interpretation: The reliability of the analytical and/or quality control procedures used in a study is questionable if test substance is detected in dilution water or solvent water control samples.

[C] Deviation 3: Measured test substance concentrations among “fresh” WAF test solutions (sampled at the time of solution renewal) of the same nominal loading rate were highly variable at different measurement time points. During the study, measured concentrations of “fresh” test solutions varied by a factor of at least 15 (highest measured “fresh” value divided by lowest measured “fresh” value) for each treatment level. For loading rates of 1, 3.2, 10, 32, and 100 mg/L, individual measured concentrations of “fresh” test solutions varied by as much as 140, 180, 134, 144, an d 133% from the mean measured “fresh” concentration for that loading rate during the 21

- day exposure period. This variability raises concerns about the consistency and reproducibility of the methods used to prepare the test solutions during the test and/o r the analytical methods used to determine test substance concentrations. No explanations for the variability among measured concentrations of “fresh” test solutions were provided in the study report. T he study report did not provide a detailed descripti on of the procedures for storage and handling of WAF preparations. Missing information included, for example, whether the WAF was stored prior to use and, if so, for how long and under what conditions.Addendum to HPV 3 Chemical Data Adequacy Review (Final ) CASRN 22527

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- 9 ASTM E1193

- 97, p. 222, Section 12 “Procedure”, Sub

- section 12.1 “Experimental Design”, item 12.1.5 “Test Material Measurement” states: “ A general guide is that the highest values for a given treatment level divided by the lowest measured value for the same treatment level should not vary by more than a fac tor of 1.5 ...” ASTM E1193

- 97, p. 223, Section 12 “Procedure”, Sub

- section 12.6 “Beginning the Test”, 12.6.1 “Selecting the Test System and Preparing Test Solutions”, item 12.6.1.1 states: “For a renewal test, fresh test solutions containing appropriate amoun ts of test material and food should be prepared less than 4 h before each renewal .” ASTM E1193

- 97, p. 223, Section 12 “Procedure”, Sub

- section 12.6 “Beginning the Test”, 12.6.1 “Selecting the Test System and Preparing Test Solutions”, item 12.6.1.1 states: “... The test concentrations should vary less than ± 30% of the mean measured amounts over a 21

- day period...” ASTM E1193

- 97, p. 225, Section 12 “Procedure”, Sub

- section 12.11.3 “Test Material”, 12.11.3.3 states: “In each treatment, the highest of all the mea sured concentrations obtained during the test divided by the lowest must be less than two .” ASTM E1193

- 97, p. 225, Section 13 “Analytical Methodology”, Sub

- section 13.3 states: “Chemical and physical data should be obtained using appropriate ASTM standards whenever possible. For those measurements for where [ sic ] ASTM standards do not exist or are not sufficiently sensitive enough, methods should be obtained from other reliable sources...” ASTM E1193

- 97, p. 225, Section 14 “Acceptability of Test”, Sub

- section 14.1 states: “A life

- cycle test with D. magna should be considered unacceptable if one or more of the following occurred.”; and item 14.1.11 states: “The highest measured concentration of test material in a treatment was more than twice the low est in the same treatment.” ASTM E1193

- 97, p. 226, Section 16 “Report”, Sub

- section 16.1 states: “The record of the results of an acceptable life

- cycle toxicity test with D. magna should include the following information either directly or by reference to available documents:”; and item 16.1.15 states: “Anything unusual about the test, any deviation from these procedures, and any other relevant information.”; and item 16.2 states: “Published reports should contain enough information to clearly identify the procedures used and the quality of the results.”

Summary of Quoted Material: Measured concentrations should vary less than 30% from the mean measured concentration for a given treatment level over the 21

- day test period. The highest measured concentration value divided by lowest measured value for each treatment level should generally be ≤ 1.5, but absolutely must be < 2; otherwise, the test should be considered unacceptable. Any unusual findings (e.g., large variations in measured test conce ntrations of freshly prepared WAF test solutions) should be addressed in the study report. WAF preparations should not be stored more than 4 hours prior to use. The analytical method used for measurement of test substance concentrations should be conducted using appropriate ASTM standards or comparable reliable methods. The study report should provide a sufficient description of all procedures, including storage and handling of WAF preparations. Interpretat ion: High variability (> 2

- fold variation) in measured test substance concentrations suggests that unreliable procedures were used in the preparation, handling, storage, and/or analysis of test solutions, rendering the test unacceptable.Addendum to HPV 3 Chemical Data Adequacy Review (Final ) CASRN 22527

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- 10 [D] Deviation 4: S ubstantial declines in measured test substance concentrations were observed between test solution renewals. M easured concentrations of “old” test solutions (sampled prior to solution renewal) were up to 99% less than the corresponding measured “fres h” concentrations of the same solution. No explanation was provided in the study report for these declines and no analysis was conducted to determine the cause. During the study, measured concentrations of “old” test solutions varied by a factor of at le ast 3 (highest “old” measured value divided by lowest “old” measured value) from other “old” test solutions of the same treatment level. When measurements from “old” and “fresh” test solutions were combined, measured concentrations varied by a factor of a t least 144 (highest measured value divided by lowest measured value) for a given treatment level. For loading rates of 1, 3.2, 10, 32, and 100 mg/L, individual measured concentrations (“old” and “fresh” combined) varied by as much as 272, 311, 349, 374, and 350% from the mean measured concentration for that loading rate during the 21

- day exposure period. No data were provided to demonstrate stability of the test substance in the test media. ASTM E1193

- 97, p. 220, Section 10 “Test Material”, Sub

- section 1 0.1 “General” states: “... Before a test is begun, the following should be known about the test material:”; and item 10.1.2 states: “Solubility and stability in the dilution water and solvents.” ASTM E1193

- 97, p. 222, Section 12 “Procedure”, Sub

- section 12.1 “Experimental Design”, item 12.1.5 “Test Material Measurement” states: “ A general guide is that the highest values for a given treatment level divided by the lowest measured value for the same treatment level should not vary by more than a factor of 1.5 ...” ASTM E1193

- 97, p. 223, Section 12 “Procedure”, Sub

- section 12.6 “Beginning the Test”, item 12.6.1.1 states: “The test concentrations should vary less than ± 30% of the mean measured amounts over a 21

- day period. If test material concentrations decline by more than 30% over the longest interval between renewals, the beakers might be pre

- exposed (for example, preconditioned) to the test material to help maintain constant test concentrations. The test chambers can be preconditioned by allowing the appropria te test solutions to sit in the test chambers for at least 1 h at which time these solutions would be discarded. The test chambers would then be refilled with the appropriate test solutions.” ASTM E1193

- 97, p. 225, Section 12 “Procedure”, Sub

- section 12.1 1.3 “Test Material”, 12.11.3.3 states: “In each treatment, the highest of all the measured concentrations obtained during the test divided by the lowest must be less than two .” ASTM E1193

- 97, p. 225, Section 12 “Procedure”, Sub

- section 12.11.3 “Test Materi al”, item 12.11.3.4 states: “If the daphnids are possibly being exposed to substantial concentrations of one or more impurities or degradation or reaction products, measurement of the impurities and products is desirable.” ASTM E1193

- 97, p. 225, Section 14 “Acceptability of Test”, Sub

- section 14.1 states: “A life

- cycle test with D. magna should be considered unacceptable if one or more of the following occurred.”; and item 14.1.11 states: “The highest measured concentration of test material in a treatment was more than twice the lowest in the same treatment.” ASTM E1193

- 97, p. 226, Section 16 “Report”, Sub

- section 16.1 states: “The record of the results of an acceptable life

- cycle toxicity test with D. magna should include the following information either directly or by reference to available documents:”; and item 16.1.2 states: “Source of test material, its lot number, composition (identities and concentrations of major ingredients and maj or impurities), known chemical and physical properties , and the identityAddendum to HPV 3 Chemical Data Adequacy Review (Final ) CASRN 22527

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- 11 and concentration(s) of any solvent used.”; and item 16.1.15 states: “Anything unusual about the test, any deviation from these procedures, and any other relevant information.” OECD 2 3, p. 15, Section 1 “Preliminary assessment of stability of test substance” states:

“ Modifications to media preparation and exposure systems may be required where exposure concentrations of a test substance are likely to decline significantly over the test period. In OECD guidelines for aquatic toxicity testing a decline in concentration of 20% is considered sufficient to warrant consideration of measures to reduce the decline. Data providing an indication of the stability of the test substance under the test conditions should therefore be obt ained before commencing testing... ” OECD 23, pp. 16

- 17, Section 1.2 “Preliminary stability study” states: “The possibility of losses during sampling, sample treatment and analysis must be considered. Where samples are stored before analysis, losses during sample storage should also be assessed.” OECD 23, p. 37, Section 3.11 “Multi

- component substances”, Sub

- s ection entitled “Partially soluble” states: “... The method used to prepare the WAF should therefore be fully described in the test report and evidence of its compositional stability over time provided... Chemical specific analysis is required to demonstrate attainment of equilibrium in WAF preparation and stability during the test. Methods capable of identifying gross changes in the composition of WAFs with time are required...”

Summary of Quoted Material: Measured concentrations should vary less than 30% from the mean measured concentration for a given treatment level over the 21

- day test period and should
not decline by more than 30% between renewals . If the decline in measured concentrations is greater t han 30% , steps should be taken to reduce or minimize the change in test substance concentration. The highest measured value divided by lowest measured value for each treatment level should be generally be ≤ 1.5, but absolutely must be < 2; otherwise, the test should be considered unacceptable. Any unusual findings (e.g., large decreases in measured test concentrations between test solution renewals) should be addressed in the study report. The stability of the test item in the dilution water should be kn own prior to study initiation. Data on the compositional stability of the WAF over time should be provided. If the test substance is not stable under the test conditions, the degradation or reaction products should be identified before study initiation a nd measured during the test. The possibility that test substance was lost from solution during sampling, handling of samples, and analysis of samples must also be considered. Interpretation: If large, unexplained variations in measured test substance co ncentration occur for a given treatment level over the test period or large, unexplained declines in test substance concentration occur between test solution renewals, the reliability of the reported endpoint values for toxicity to Daphnia is uncertain

.
[ E] Deviation 5: There was no evidence that the solubility and stability of the test substance in the dilution water (culture medium) were determined prior to the initiation of the chronic daphnid test; the study report did not provide measurements of thes e physical

- chemical properties in the dilution water. ASTM E1193

- 97, p. 220, Section 10 “Test Material”, Sub

- section 10.1 “General” states: “... Before a test is begun, the following should be known about the test material:”; and item 10.1.2 states: “ Solubility and stability in the dilution water and solvents .” ASTM E1193

- 97, p. 226, Section 16 “Report”, Sub

- section 16.1 states: “The record of the results of an acceptable life

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- 12 information e ither directly or by reference to available documents:”; and item 16.1.2 states: “Source of test material, its lot number, composition (identities and concentrations of major ingredients and major impurities), known chemical and physical properties , and th e identity and concentration(s) of any solvent used.” OECD 23, p. 15, Section 1 “Preliminary assessment of stability of test substance” states: “Data providing an indication of the stability of the test substance under the test conditions should therefore be obtained before commencing testing.” OECD 23, p. 20, Section 3.1 “Poorly water

- soluble substances”, Sub

- section 3.1.1 “Preliminary experiment” states: “A preliminary experiment should be considered for all studies with poorly water

- soluble su bstances to determine the maximum dissolved concentration that can be achieved under test conditions, which is defined as the saturation concentration, and the preparation conditions that are required to achieve it. The results of the preliminary experime nt will form a basis of and justification for the media preparation procedures adopted for the toxicity tests and a reference point (i.e. water solubility under test conditions) against which the test results can be evaluated. In view of its importance th e preliminary experiment should be fully reported.”

Summary of Quoted Material: The solubility and stability of the test substance in the dilution water should be determined prior to study initiation and provided in the study report. Interpretation: It is important to know the solubility of the test substance in the dilution water so that the appropriateness of the media preparation procedures can be assessed. Data describing test substance solubility and stability in the growth medium that are gen erated prior to the study are useful in optimizing the study design; without such data, an optimal design is difficult to achieve.

[F] Deviation 6: The study did not include data on observations of first

- and second

- generation daphnids for abnormal devel opment or aberrant behavior. ASTM E1193

- 97, p. 224, Section 12 “Procedure”, Sub

- section 12.10 “Biological Data”, item 12.10.5 states: “Both first

- and second

- generation daphnids should be carefully and regularly observed during the test for abnormal develo pment and aberrant behavior , such as inability to maintain position in the water column, uncoordinated swimming, and cessation of feeding. Although developmental and behavioral effects are often difficult to quantify and might not provide suitable end poi nts, they might be useful for interpreting effects on survival and growth and for deciding whether the test should be extended beyond the minimum duration (see 12.8).”

Summary of Quoted Material: Data on abnormal development and aberrant behavior for fir st

and second

- generation daphnids should be collected and provided in the study report.

[G] Deviation 7: The 100 mg/L loading rate WAF was filtered through a glass wool plug on day 9 to remove microscopic particles of the test item in the water column. The use of glass wool for removal of undissolved test material is not recommended. Instead, the use of

cen trifugation or a separatory funnel is suggested. OECD 23, pp. 20

- 21, Section 3.1 “Poorly water

- soluble substances”, Sub

- section 3.1.1 “Preliminary experiment” states: “It should not be assumed that a clear test mediumAddendum to HPV 3 Chemical Data Adequacy Review (Final ) CASRN 22527

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- 13 indicates a true solution since cryst als, aggregates, micelles etc. cannot easily be detected by visual observation. Possible separation techniques include:

 Centrifugation

- the preferred separation method but there may be practical difficulties in applying the technique to large volumes of test medium...

 Filtration through a membrane filter

- less widely advocated because of the potential for losses due to adsorption onto the filter matrix... Adsorption of the test substance may be reduced to insignificant levels by preconditioning filters wit h solutions of the test substance prepared at the app ropriated test concentrations... A justification for, or validation of, the separation technique should always be provided in the test report.” OECD 23, pp. 36

- 37, Section 3.11 “Multi

- component substances” , Sub

- section “Partially soluble” states: “The toxicity of complex multi

- component substances, which are only partially soluble in water, can be determined by preparing water

- accommodated fractions (WAFs) of them... Generally, any non

- dissolved test materia l which has sedimented or precipitated out in the test vessels should be removed from the test media using, for example, a separating funnel.”

Summary of Quoted Material: Filters (e.g., glass wool plugs) are not the preferred method for separation of non

- dissolved test substance from test medium because dissolved test substance may adsorb to the filter, resulting in a lower initial concentration of test substance in solution (i.e., a concentration below the solubility limit). Centrifugation or a separatory funnel can be used for removal of undissolved test substance from a test solution, and poses a lower concern for loss of dissolved test substance from solution. The test report should provide a justification for, and/or validation of, the separ ation technique used during test solution preparation.

REFERENCE

Harris, S. 2013. Benzoflex 1046: Daphnia magna reproduction test. Harlan Laboratories Ltd.

, Shardlow , Derbyshire, UK, Project No. 41202706. Sponsor: Eastman Chemical Company, Kingsport, TN. Study report available online at http://www.regulations.gov/#!document Detail; D=EPA

- HQ

- OPPT

- 2009

- 0112

- 0145 as of January 7, 2014.