SRC TR-12-033

HPV3 TOXICITY TEST PLAN REVIEW (DRAFT)

Propanoic acid, 2-methyl-, 3-(benzoyloxy)-2,2,4-trimethylpentyl ester, CASRN 22527-63-5

Prepared by:

William L. Richards, Teresa Manyin, and Laura Morlacci

Chemical, Biological and Environmental Center

SRC, Inc.

7502 Round Pond Road

North Syracuse, NY 13212

Contract No. EP-W-09-027

Task FG007.2.009.006

Submitted to:

OCSPP/OPPT/RAD (7403M)

U.S. Environmental Protection Agency

1200 Pennsylvania Avenue

Washington, D.C. 20460

Debra Milligan, Project Officer

Meena Sonawane, Work Assignment Manager

David Brooks, Alternate Work Assignment Manager

March 9, 2012
Final HPV3 Toxicity Test Plan Review CASRN 22527-63-5

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HPV3 Toxicity Test Plan Review

Propanoic acid, 2-methyl-, 3-(benzoyloxy)-2,2,4-trimethylpentyl ester, CASRN 22527-63-5

The test rule, “Testing of Certain High Production Volume Chemicals; Third Group of 

Chemicals” (76 FR 65385, October 21, 2011), requires the following toxicity testing for

propanoic acid, 2-methyl-, 3-(benzoyloxy)-2,2,4-trimethylpentyl ester, CASRN 22527-63-5: C1 

(aquatic toxicity), D (mammalian toxicity – acute), E1 (bacterial reverse mutation test), E2

(chromosomal aberration or micronucleus test) and F1 (mammalian toxicity – repeated 

dose/reproduction/developmental). Eastman Chemical Company (Eastman) submitted a test plan 

on February 20, 2011 to address the endpoints required by the test rule. The adequacy of the test 

plan is evaluated below.

1. General Comments:

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, for toxicity testing, Eastman proposes the 

use of a bulk reaction product of the following purity: 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). EPA approves Eastman’s plan to conduct aquatic and 

mammalian toxicity tests of this mixture (“the bulk reaction product”) based on the following: 

(1) Eastman states 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.”

(2) In commerce, the bulk reaction product that is formed through an esterification 

reaction between benzoic acid (CASRN 65-85-0) and 2,2,4-trimethyl-1,3-pentanediol 

diisobutyrate (CASRN 6846-50-0) results in three chemicals (CASRNs 22527-63-5, 

68052-23-3, and 6846-50-0). These chemicals, the diesters of 2,2,4-trimethyl-1,3-

pentanediol with benzoic acid and 2-methylpropanoic acid (isobutyric acid), can be 

considered close analogs.

(3) The major constituent 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.

For all toxicity tests, the final study report should include the analyzed composition of the test 

substance, including identities and concentrations of major ingredients and of impurities. 
Final HPV3 Toxicity Test Plan Review CASRN 22527-63-5

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2. Aquatic Toxicity

Under testing requirement C1 of the test rule, a determination of whether to perform aquatic 

toxicity testing under Test Group 1 or Test Group 2 is based on a consideration of the measured 

log Kow for the test substance. EPA approves Eastman’s plan to conduct a Chronic Toxicity to 

Daphnia test of the bulk reaction product using ASTM E 1193-97 (Reapproved 2004) if the 

measured log Kow is ≥ 4.2 or an Acute Toxicity to Daphnia test using ASTM E 729-96 

(Reapproved 2007) if the measured log Kow is < 4.2. 

In addition to daphnid testing, the following studies of the bulk reaction product must be 

conducted to fulfill testing requirement C1 of the test rule: Toxicity to Plants (Algae) using 

ASTM E 1218-04 (required for all chemicals regardless of log Kow value); and Acute Toxicity to 

Fish using ASTM E 729-96 (Reapproved 2007) if the measured log Kow is < 4.2. Note that 

Eastman failed to include these required tests in the submitted test plan.

3. Mammalian Toxicity – Acute

EPA approves Eastman’s plan to conduct an Acute Oral Toxicity – Up-and-Down Procedure test

of the bulk reaction product using OECD TG 425 to fulfill testing requirement D.

4. Bacterial Reverse Mutation Test

EPA approves Eastman’s plan to conduct a Bacterial Reverse Mutation Test using 40 CFR 

799.9510 of the bulk reaction product to fulfill testing requirement E1.

5. Chromosomal Aberration/Micronucleus Test

EPA approves Eastman’s plan to conduct an In vitro Mammalian Chromosomal Aberration Test 

of the bulk reaction product using 40 CFR 799.9537 to fulfill testing requirement E2.

6. Mammalian Toxicity – Repeated Dose/Reproduction/Developmental

For the following reasons, EPA does not approve Eastman’s request to fulfill testing requirement 

F1 “through the use of data already available on the three primary metabolites of the test 

substance: namely benzoic acid, 2,2,4-trimethyl-1,3-pentanediol (TMPD) and isobutyrate.”: 

(1) For the diesters of 2,2,4-trimethyl-1,3-pentanediol (CASRNs 22527-63-5, 68052-23-3

and 6846-50-0) that are present in the bulk reaction product that Eastman proposes for 

testing for the physical/chemical properties, environmental fate, aquatic toxicity, acute 

mammalian toxicity and genotoxicity endpoints, Eastman has not provided data to show 

that non-enzymatic or enzymatic hydrolysis of these esters will occur fast enough to 

justify testing of only the presumptive hydrolysis products/metabolites for addressing the 

mammalian repeated-dose, reproductive, and developmental toxicity endpoints. 

Eastman cites an oral gavage study of diethyleneglycol dibenzoate, which showed “that 

the ester linkages are completely hydrolyzed with no evidence of parent compound in the 
Final HPV3 Toxicity Test Plan Review CASRN 22527-63-5

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urine”. However, the rate and extent of non-enzymatic or enzymatic hydrolysis of the 

three diesters present in Eastman’s bulk reaction product (CASRNs 22527-63-5, 68052-

23-3 and 6846-50-0) may be inhibited compared with diethyleneglycol dibenzoate 

because of greater steric hindrance by the branched 2,2,4-trimethylpentyl moiety than by 

the diethylene glycol moiety.

(2) Eastman does not present a rationale to show that data from separate repeated-dose, 

reproductive, and developmental toxicity studies of individual hydrolysis 

products/metabolites of the bulk reaction product would be adequate to characterize 

toxicity of the mixture of these substances expected to occur in vivo. Although data for 

the individual hydrolysis products/metabolites are useful to address the overall toxicity of 

the bulk reaction product, the data for these individual hydrolysis products/metabolites 

would not address the potential for interactions (e.g., additivity, synergism or 

antagonism) among these degradatation products, mono-ester intermediates, and the 

residual diesters of the bulk reaction product. The likelihood for such interactions could 

vary with exposure route since the acidic environment of the stomach during oral 

exposure could result in higher rates of nonenzymatic ester hydrolysis than for exposures 

by the inhalation or dermal routes.

Based on the preceding discussion, testing requirement F1 should be fulfilled by conducting a 

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity

Screening Test (40 CFR 799.9365) of the bulk reaction product; OR the 

Reproduction/Developmental Toxicity Screening Test (40 CFR 799.9355) AND the Repeated 

Dose 28-Day Oral Toxicity Study in Rodents Test (40 CFR 799.9305) of the bulk reaction 

product.

Conclusion: The submitted test plan for toxicity testing is adequate to satisfy test rule 

requirements C1, D, E1, E2, and F1 with the exceptions and stipulations listed above.