SRC TR-13-060

HPV3 TEST RULE DATA ADEQUACY REVIEW (REVISED FINAL):

TOXICITY TO ALGAE, CHRONIC TOXICITY TO DAPHNIA, AND 

CHROMOSOMAL ABERRATIONS

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, Alternate Work Assignment Manager

January 7, 2014
Final HPV3 Chemical Data Adequacy Review (January 2014) CASRN 22527-63-5

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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-1
Final HPV3 Chemical Data Adequacy Review (January 2014) CASRN 22527-63-5

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A. SOURCE MATERIAL USED FOR ASSESSMENT

This document evaluates toxicity tests for propanoic 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-OPPT2009-0112-0086 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 

Kow < 4.2 or chronic toxicity to Daphnia and toxicity to algae testing if log Kow ≥ 4.2), D (acute 

mammalian toxicity test), E1 (bacterial 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 Kow of CASRN 22527-63-5 

has been experimentally determined to be ≥ 5.4 (based on measured log Kow values of 5.4, 5.5, 

and 5.8 for the three main components of the test item)1, which is > 4.2, the C1 aquatic toxicity 

testing requirement is considered to include chronic toxicity to Daphnia and toxicity to algae 

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: Daphnia 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 summarized 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 standard 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 Kow 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/#!documentDetail;D=EPA-HQ-OPPT-2009-0112-0145 as of January 7, 2014).
Final HPV3 Chemical Data Adequacy Review (January 2014) CASRN 22527-63-5

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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-OPPT2009-0112-0107 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). 

Eastman’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 substance 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 benzoate 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 

(CASRN 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. 

Campanella (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 chromosomal 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 chronic toxicity to Daphnia for Benzoflex 1046, 

supplied on June 20, 2013 by Eastman, are not considered adequate to satisfy the C1 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 EC50 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 EL50 (based on WAF loading rates) was greater than 100 mg/L and that the 

EC50 (based on mean measured concentrations) was greater than 0.19 mg/L. The highest test 
Final HPV3 Chemical Data Adequacy Review (January 2014) CASRN 22527-63-5

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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. 

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 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 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 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 study report 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 96-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 samples 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 nature 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 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. 

The chronic toxicity to Daphnia study is considered inadequate because the test failed to identify 

a discrete EC50 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 EC50. 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 HPV3 Chemical Data Adequacy Review (January 2014) CASRN 22527-63-5

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variations in measured test substance concentrations prevent the interpretation of the biological 

data to characterize the concentration-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 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” 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/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 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. The inconsistencies among test item concentrations 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. Measured concentrations of “old” test solutions (sampled 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 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 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 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, < LOQ0.529, 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, 0.12, and 0.21 mg/L, respectively, for nominal loading rates of 1, 3.2, 10, 32, 
Final HPV3 Chemical Data Adequacy Review (January 2014) CASRN 22527-63-5

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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-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 funnel is suggested.

Therefore, the 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 intended 

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 concentrations of fresh test solutions at 

renewal. In addition, stability trials should be conducted to determine if the test substance 

significantly degrades during the intended testing period (algae) or renewal cycle (Daphnia). It 

is recommended that test concentration measurements be conducted more frequently (every 24 

hours) to better characterize exposure concentrations during the test period. The study reports 

should contain detailed procedures for storage and handling conditions of prepared WAFs prior 

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 tests. 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 test rule.

Remaining HPV3 Test Rule Toxicity Testing Requirements:

In an email dated December 18, 2013 (from L.W. Velsor [Eastman] to J. Schaeffer [EPA]), 

Eastman supplied data from a combined repeated dose toxicity study with the 

reproduction/developmental toxicity screening test 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 aberrations 
Final HPV3 Chemical Data Adequacy Review (January 2014) CASRN 22527-63-5

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endpoints. Data submitted by Eastman for the repeated-dose, reproductive, and developmental 

toxicity endpoints are currently under review.
Final HPV3 Chemical Data Adequacy Review (January 2014) CASRN 22527-63-5

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D. DATA FOR SIDS ENDPOINTS

D-1. Toxicity to Aquatic Plants

Summary of endpoint: Not Adequate

Study 1: Algal growth inhibition toxicity test in Pseudokirchneriella subcapitata (Vryenhoef, 

2013)

Title: Benzoflex 1046: Algal growth inhibition test

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 discrete EC50

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 EL50

(based on WAF loading rates) was greater than 100 mg/L and that the EC50 (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 SUBSTANCE 

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% propanoic 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 850.5400 (“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-OPPT-2009-0112-0145 as of January 7, 2014).

2 The certificate of analysis provided in the study report describes the composition 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 “diester” was synonymous 

with propanoic 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-trimethyl-1,3-pentanediol diisobutyrate (CASRN 6846-50-0).
Final HPV3 Chemical Data Adequacy Review (January 2014) CASRN 22527-63-5

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Test type: Toxicity to plants (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 medium was 0.15 mmol/L (= 15 mg/L as CaCO3).” [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. 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. 

Range-Finding Test Procedure: 

In the range-finding test, Pseudokirchneriella subcapitata cells were exposed to a series of 

nominal loading rates of 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 polyurethane foam bungs to reduce evaporation. Two replicate 

flasks were prepared for each control and test concentration.” [Vryenhoef 2013, p. 12]
Final HPV3 Chemical Data Adequacy Review (January 2014) CASRN 22527-63-5

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“Amounts of test item (20 and 200 mg) were each separately added to the surface of 2 liters of 

culture medium 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 mixtures [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 showed 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 104cells/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 were 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 were 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 

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 mixture [was] 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 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 floating 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 oily 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 HPV3 Chemical Data Adequacy Review (January 2014) CASRN 22527-63-5

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“Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell 

density of 3.30 x 105cells per mL. Inoculation of 1 liter of test medium with 30.3 mL of this 

algal suspension gave an initial nominal cell density of 1 x 104cells per mL...” [Vryenhoef 

2013, p. 13]

“The flasks were plugged with polyurethane foam bungs and incubated ... at 24 ± 1 °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. 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 temperatures within the incubator 

were recorded hourly.

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

R1 – R6 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 occasion and 

stored at approximately -20 °C for further analysis if necessary.” [Vryenhoef 2013, p. 14]

Test concentrations were analyzed using high performance liquid chromatography (HPLC) with 

UV detection (wavelength of 230 nm). The limit of quantitation [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 test 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, an 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). 
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RESULTS

Endpoint value(s): 

The following results were based on WAF loading rates: 

96-hour EL50 (growth rate, yield, and biomass) > 100 mg/L

96-hour NOEL1(growth rate, yield, and biomass) = 100 mg/L 

96-hour LOEL2(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 EC50 (growth rate, yield, and biomass) > 0.19 mg/L

96-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 inhibition 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 control 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 loading 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 

concentration 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 significant 

differences (p ≥ 0.05) between the control and 100 mg/L loading rate WAF test group were

 

1 NOEL = No Observed Effect Loading Rate

2 LOEL = Lowest Observed Effect Loading Rate
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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 2013, 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 rates; the 96-hour NOEC was 0.19 

mg/L based on mean measured concentrations. The 96-hour EL50 values for growth rate, yield, 

and biomass were > 100 mg/L based on WAF loading rates; the 96-hour EC50 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 solubility limit of the test substance and the test failed to 

identify a discrete EC50 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 EL50 (based on WAF loading rates) was greater than 100 mg/L and that the 

EC50 (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.

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 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 

 

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 HPV3 Chemical Data Adequacy Review (January 2014) CASRN 22527-63-5

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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 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 study report 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 96-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 samples 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 nature 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 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 http://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).
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D-2. Chronic Toxicity to Aquatic Invertebrates

Summary of endpoint: Not adequate

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

Title: Benzoflex 1046: Daphnia magna reproduction test

This study is: Adequate Not Adequate

This study is not considered adequate because the test failed to identify a discrete EC50 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 EC50. 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 relationship.

TEST SUBSTANCE 

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% propanoic 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 ASTM Standard 

E1193-97 (“Standard Guide for Conducting Daphnia magna Life-Cycle Toxicity Tests”)

Test type: Chronic toxicity to Daphnia – Semi-Static

GLP compliant?: Yes

 

1 The certificate of analysis provided in the study report describes the composition 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 “diester” was synonymous 

with propanoic 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-trimethyl-1,3-pentanediol diisobutyrate (CASRN 6846-50-0).
Final HPV3 Chemical Data Adequacy Review (January 2014) CASRN 22527-63-5

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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 days

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, 0.12, and 

0.21 mg/L1

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 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. 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 liters 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 by 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 stirring 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 micro-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.
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“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. 13]

Daphnia magna (10 replicates per test concentration, each containing a single daphnid) 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 solutions 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 vessels 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 parental 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, P1) and filial (F1) 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 

media 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 temperature 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 Days 0, 5, 9, 14 and 

19 and of the expired test preparations on Days 2, 7, 12, 16 and 21. Samples were stored at 

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

approximately -20 °C for further analysis if necessary.” [Harris 2013, p. 14]
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“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 was considered valid if the following criteria were met:

 Control mortality was ≤ 30%.

 Dissolved oxygen content was > 3 mg O2/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 were born before a maximum of nine days.

RESULTS

Endpoint value(s):

The following values are based on WAF loading rates:

21-day EL50 (mortality/immobility) > 100 mg/L 

21-day EL50 (reproduction) > 100 mg/L 

21-day EL50 (growth) > 100 mg/L 

21-day NOEL (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 LOEL (reproduction) = 32 mg/L 

21-day LOEL (growth) = 3.2 mg/L

MATC (growth) = 1.79 mg/L (loading rate)
Final HPV3 Chemical Data Adequacy Review (January 2014) CASRN 22527-63-5

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The following values are based on mean measured concentrations that correspond with nominal 

loading rates:

21-day EC50 (mortality/immobility) > 0.21 mg/L 

21-day EC50 (reproduction) > 0.21 mg/L 

21-day EC50 (growth) > 0.21 mg/L 

21-day NOEC (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) = 0.12 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 LOEC values, by definition, must be greater than NOEC values, the LOEC, NOEC, and 

MATC values identified above are not considered accurate. The 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), 0.12 (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 Substance

Time point 

(day)

Nominal concentration (mg/L)

0 1 3.2 10 32 100

0 (fresh)

0.00414/ 

< LOQa 0.521 0.529 0.814 0.546 0.927

2 (old)

0.00372/

< LOQa 0.301 0.330 0.0163 0.0102 0.0441

5 (fresh) < LOQ 0.14 0.0475 0.509 0.0168 0.0522

7 (old)

0.00441/

< LOQa < LOQ < LOQ 0.00517 < LOQ < LOQ

9 (fresh) < LOQ < LOQ < LOQ 0.0870 0.061 0.489

12 (old)

0.0115/

< LOQa < LOQ < LOQ 0.0209 0.00371 0.00486

14 (fresh) < LOQ 0.0888 0.121 0.281 0.151 0.467
Final HPV3 Chemical Data Adequacy Review (January 2014) CASRN 22527-63-5

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Table 1. Measured Concentrations of Test Substance

Time point 

(day)

Nominal concentration (mg/L)

0 1 3.2 10 32 100

16 (old) 

0.0192/

< LOQa < LOQ < LOQ 0.018 0.0123 0.00943

19 (fresh) < LOQ 0.331 0.242 0.0513 0.342 0.0573

21 (old)

0.0180/

< LOQa < LOQ < LOQ 0.0114 0.00588 < LOQ

Mean measured 

concentration (mg/L)b 0.14 0.13 0.18 0.12 0.21

a Duplicate sample stored at -20°C prior to analysis

b Mean of combined fresh and old samples; 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.

LOQ = Limit of Quantitation (0.0036 mg/L)

“Analysis 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 length 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 loading rate WAF test 

concentrations.” [Harris 2013, p. 18]

Mean daphnid 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 HPV3 Chemical Data Adequacy Review (January 2014) CASRN 22527-63-5

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17.7, 106.0 ± 18.8, and 99.9 ± 12.2, respectively. Live young per adult, expressed as percentages 

of control values, were 111%, 112%, 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 all control and treatment 

groups surviving to maturation.” [Harris 2013, p. 19]

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 O2/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 water hardness, conductivity, and alkalinity measurements in the 

control and 100 mg/L nominal loading rate treatments during the study were 216-260 mg 

CaCO3/L, 680-833 μS/cm, and 39.6-51.6 mg CaCO3/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 O2/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 rates. The 21-day EL50 values for adult mortality/immobility, 

reproduction, and growth were > 100 mg/L based on WAF loading rates. The corresponding 21-

day EC50 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

EC50 or accurate LOEC and NOEC values for chronic effects on survival, reproduction, and 
Final HPV3 Chemical Data Adequacy Review (January 2014) CASRN 22527-63-5

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 EC50. 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 

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” 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/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 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. The inconsistencies among test item concentrations 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. Measured concentrations of “old” test solutions (sampled 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 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 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 concentrations (“old” and “fresh” 
Final HPV3 Chemical Data Adequacy Review (January 2014) CASRN 22527-63-5

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) were < LOQ-0.521, < LOQ0.529, 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, 0.12, 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, 

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 funnel is suggested.

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/#!documentDetail;

D=EPA-HQ-OPPT-2009-0112-0145 as of January 7, 2014.

Text in quotes is taken directly from Harris (2013).
Final HPV3 Chemical Data Adequacy Review (January 2014) CASRN 22527-63-5

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: Clear colorless liquid; Batch No. V046212201; Composition: 68.8% propanoic 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: Compatible with OECD TG 473 (“In Vitro Mammalian 

Chromosome Aberration Test”); Method B10 of Commission Regulation (EC) No. 440/2008

Test type: In vitro mammalian chromosome aberration test

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 

formulation 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 describes the composition 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 “diester” was synonymous 

with propanoic 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-trimethyl-1,3-pentanediol diisobutyrate (CASRN 6846-50-0).
Final HPV3 Chemical Data Adequacy Review (January 2014) CASRN 22527-63-5

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*, 80*, 120*, 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 sulfoxide

Test method/conditions remarks: Human lymphocytes were collected from a blood sample 

isolated from the peripheral circulation of a screened volunteer. Approximately 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, therefore, 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 mOsm.” [Bowles 2013, p. 9]

The S9 homogenate was produced from homogenized liver samples collected from male rats that 

had received three daily oral doses of a phenobarbitone (80 mg/kg-bw/day) and βnaphthoflavone (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 that 
Final HPV3 Chemical Data Adequacy Review (January 2014) CASRN 22527-63-5

25

test item precipitate observations could be made. Precipitate observations were recorded at the 

beginning and end of the exposure periods.” [Bowles 2013, p. 12]

Chromosome Aberrations Assay:

Experiment 1: Lymphocytes were exposed to the test item or control for 4 hours without or with 

S9 (2%), followed by a 20-hour incubation period. Concentrations without S9 activation

selected for metaphase analysis were 0 (vehicle control), 5, 10, and 20 μg Benzoflex 1046/mL. 

Concentrations 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. Concentrations without S9 activation selected for metaphase analysis were 0 

(vehicle control), 20, 40, and 60 μg Benzoflex 1046/mL. Concentrations with S9 activation

selected for metaphase analysis were 0 (vehicle control), 20, 40, and 80 μg Benzoflex 1046/mL. 

Lymphocytes were arrested in mitosis with demecolcine (Colcemid, 0.1 μg/mL) two hours 

before scheduled harvest. The cells were treated with 0.075M hypotonic KCl, fixed with 

methanol/glacial acetic acid (3:1 v/v), mounted on microscope slides, and stained with 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 and 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-spread 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]ells 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 frequency 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 HPV3 Chemical Data Adequacy Review (January 2014) CASRN 22527-63-5

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-hour exposures without S9: 78.13 μg/mL; 4-hour

exposures with S9: 156.25 μg/mL (based on ≥ 50% inhibition of mitotic index in the preliminary 

toxicity test)

Results remarks: Positive and negative control responses were appropriate.

Preliminary Toxicity Test Results:

Precipitate 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 μg/mL in the absence of S9 and 156.25 μg/mL 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 

S9 [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. 17] 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 indices were inhibited 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 80 

μg/mL without S9 and at 80 μg/mL with S9. However, “[t]he toxicity observed at 80 μg/ml in 

the absence of S9 [i.e., a 77% inhibition of the mitotic index compared with the control] was 
Final HPV3 Chemical Data Adequacy Review (January 2014) CASRN 22527-63-5

27

considered to be excessive and precluded the dose from chromosome analysis.” [Bowles 2013, 

p. 18] 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/mL 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, did not induce chromosomal aberrations in human peripheral 

blood lymphocytes in vitro.

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. 2013. Benzoflex 1046: Chromosome aberration test in human lymphocytes in vitro. 

Harlan Laboratories Ltd., Shardlow, Derbyshire, UK, Project No. 41201918. 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 HPV3 Chemical Data Adequacy Review (January 2014) CASRN 22527-63-5

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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 Kow of CASRN 22527-63-5 is < 4.2.

2 Data submitted by Eastman on December 18, 2013 are currently under review.
Addendum to HPV3 Chemical Data Adequacy Review (Final) CASRN 22527-63-5

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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 report), 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 reviewer.

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 

ought 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 EC50 or IC50 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 EL50 (based on WAF loading rates) was 

greater than 100 mg/L and that the EC50 (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 the 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 be 
Addendum to HPV3 Chemical Data Adequacy Review (Final) CASRN 22527-63-5

A-2

necessary if the highest chemical concentration 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, and 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 information 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 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 solubility 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.”

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 EC50 or IC50 value (EL50 or IL50 value if expressed as loading rate). The highest 

concentration tested should be equivalent to the water solubility limit. Interpretation: If a 

discrete EC50 or IC50 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 determined 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 is required.
Addendum to HPV3 Chemical Data Adequacy Review (Final) CASRN 22527-63-5

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 solubility 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.”

ASTM 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 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 experiment 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 the 

preliminary experiment should be fully reported.”

Summary of Quoted Material: 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 report 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 the study are useful in optimizing the study design; without such data, an 

optimal design is difficult to achieve. 
Addendum to HPV3 Chemical Data Adequacy Review (Final) CASRN 22527-63-5

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 reported:”; 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, sorption, 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 should 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 documents:”; 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

and 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 for 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 standards. Interpretation: If highly-variable test 
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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 algae 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 test 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: “... 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, 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 relevant 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 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 

obtained 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. Methods 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 

during 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 be 
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considered. Interpretation: If test substance concentrations decline 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 growth 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 decrease 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 test (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 growth 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 

cultures 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, either 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 exponential 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

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 http://www.regulations.gov/#!documentDetail;

D=EPA-HQ-OPPT-2009-0112-0145 as of January 7, 2014.
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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).” Subsection 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 EC50 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 

EC50. 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 

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 weight, 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)”, item 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 determination of discrete values for the EC50, LOEC, and 

NOEC. Interpretation: If a study fails to derive concentration-response data and fails to identify 

discrete EC50, LOEC, and NOEC values, the study does not provide useful information for the 

assessment of risk to Daphnia and other similar species.
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[B] Deviation 2: 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. 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 procedures, 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 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 14.1.3 states: “Appropriate dilution-water controls (and 

solvent controls if necessary) were not included in the test.”

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.”

Summary of Quoted Material: The control is intended to provide a baseline response of test 

organisms in the absence of the 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, and 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/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 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. 
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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”, 

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 amounts 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 measured 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 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 16.1.15 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 concentrations 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. 

Interpretation: 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. 
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[D] Deviation 4: 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 corresponding measured “fresh” 

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 least 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 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. 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 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. 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 appropriate 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 12.11.3 “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 Material”, 

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 major impurities), known chemical and physical properties, and the identity 
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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 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 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 

obtained 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. 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 than 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 known 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 and 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 concentration 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 these 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-cycle toxicity test with D. magna should include the following 
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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 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 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 experiment 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 the 

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 generated 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 development 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 development 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 points, 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 firstand 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 

centrifugation 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 medium 
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indicates a true solution since crystals, 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 with solutions of the 

test substance prepared at the appropriated 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 material 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 separation 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/#!documentDetail;

D=EPA-HQ-OPPT-2009-0112-0145 as of January 7, 2014.