SRC TR-13-050

HPV3 TEST RULE DATA ADEQUACY REVIEW (FINAL):

ACUTE TOXICITY AND GENE MUTATION

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

Prepared by:

Justine Von Runnen, Julie Klotzbach, 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 FG007.3.009.006

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

July 11, 2013
Final HPV3 Chemical Data Adequacy Review 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 ........ 1

D. DATA FOR SIDS ENDPOINTS.............................................................................................. 3

D-1. Acute Toxicity.................................................................................................................... 3

D-2. Genetic Toxicity – Gene Mutation..................................................................................... 5

E. DATA ADEQUACY SUMMARY TABLE .......................................................................... 11
Final HPV3 Chemical Data Adequacy Review CASRN 22527-63-5

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A. SOURCE MATERIAL USED FOR ASSESSMENT

This document evaluates a submission from Eastman Chemical Company (dated February 26, 

2013; available online at http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT2009-0112-0138 as of July 8, 2013) received by EPA in response to the final test rule, “Testing 

of Certain High Production Volume Chemicals; Third Group of Chemicals” (76 FR 65385, 

October 21, 2011; referred to herein as HPV3; available online at http://www.regulations.gov/

#!documentDetail;D=EPA-HQ-OPPT-2009-0112-0086 as of July 8, 2013) for propanoic acid, 2-

methyl-, 3-(benzoyloxy)-2,2,4-trimethylpentyl ester (CASRN 22527-63-5). In this submission, 

Eastman Chemical Company (“Eastman”) supplied data for the acute mammalian toxicity (D) 

and gene mutation (E1) testing required by the HPV3 test rule. These newly submitted data are 

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

In addition to the D and E1 testing requirements, the HPV3 test rule requires testing with 

CASRN 22527-63-5 for the following toxicity endpoints: 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), E2 (chromosomal aberration or 

micronucleus test) and F1 (combined repeated dose toxicity study with the 

reproduction/developmental toxicity screening test).

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. Studies for a 

common endpoint are grouped together in the document and an endpoint adequacy assessment is 

provided near the top of the first page of the summaries for that endpoint. 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 on the last page 

of this document (Section E).

C. SUMMARY OF DATA ADEQUACY FOR TESTING REQUIRED BY TEST RULE

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 July 8, 2013), Eastman proposed 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). 

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 
Final HPV3 Chemical Data Adequacy Review CASRN 22527-63-5

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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. 

Acute mammalian toxicity and gene mutation test results for Benzoflex 1046, supplied on 

February 26, 2013 by Eastman, are adequate to satisfy the D and E1 test requirements 

promulgated in the final HPV3 test rule. 

In a separate submission to EPA, Eastman provided the following additional studies of 

Benzoflex 1046:

 Daphnia magna Reproduction Test

 Algae Growth Inhibition Test

 Chromosome Aberration Test in Human Lymphocytes in vitro

Data from the above tests will be evaluated for adequacy in a separate report.

Data have not yet been submitted for the following toxicity testing requirement: combined 

repeated dose toxicity study with the reproduction/developmental toxicity screening test (F1 

testing). In a letter dated December 21, 2012 (available online at http://www.regulations.gov/

#!documentDetail;D=EPA-HQ-OPPT-2009-0112-0131 as of July 11, 2013), Eastman provided 

an update on testing status. At that time, Eastman expected initiation of a definitive combined 

repeated dose toxicity study with the reproduction/developmental toxicity screening test in the 

second quarter of 2013.
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D. DATA FOR SIDS ENDPOINTS

D-1. Acute Toxicity

Summary of endpoint: Adequate

Study 1: Acute oral toxicity study in rats (Sanders, 2012)

Title: Benzoflex 1046: acute oral toxicity in the rat-up-and-down procedure

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: OECD TG 425

Test type: Acute Oral Toxicity – Up-and-Down Procedure

GLP compliant?: Yes

Year study performed: 2012

Test species: Female Wistar (RccHanTM:WIST) rats; supplied by Harlan Laboratories UK Ltd., 

Oxon, UK; at test initiation, animals were 8-12 weeks of age and weighed 161-177 g.

Number of animals/sex/dose: 3 females

Route of administration: Oral gavage

 

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).
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Vehicle: None

Doses/concentrations tested: 2000 mg Benzoflex 1046/kg-bw

Statistical methods used: Not applicable

Test method/conditions remarks: One fasted animal was administered undiluted test substance 

at a dose level of 2000 mg/kg-bw of Benzoflex 1046 mixture. Based upon no observed mortality 

in this animal, two additional animals were treated sequentially at the same dose level. 

Sufficient time was allowed between dosing of each individual animal to confirm the survival of 

the previously dosed animal. Following treatment, animals were observed for mortality and 

systemic signs of toxicity at 0.5, 1, 2, and 4 hours post-dose and subsequently once daily for 14 

days. Body weights were recorded prior to dosing and on days 7 and 14. Following the 14-day 

observation period, all animals were sacrificed and subjected to gross pathological examination.

RESULTS

Endpoint value(s): LD50 > 2000 mg Benzoflex 1046/kg-bw

Results remarks: No deaths, signs of systemic toxicity, or effects on body weights were 

observed. No abnormal findings were noted at necropsy.

CONCLUSIONS

The oral LD50 for female rats was > 2000 mg Benzoflex 1046/kg-bw.

STUDY RELIABILITY

[1] Reliable without restrictions. 

REFERENCE

Sanders, A. 2012. Benzoflex 1046: Acute Oral Toxicity in the Rat – Up-and-Down Procedure. 

Harlan Laboratories Ltd., Project No. 41201919. Sponsor: Eastman Chemical Company.

Available online at http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-

0112-0138 as of July 8, 2013.
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D-2. Genetic Toxicity – Gene Mutation

Summary of endpoint: Adequate

Study 1: Mutagenicity study in Salmonella typhimurium and Escherichia coli (Thompson, 

2012)

Title: Benzoflex 1046: Reverse mutation assay “Ames test” using Salmonella typhimurium and 

Escherichia coli

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: OECD TG 471; Japanese Regulatory Authorities (METI, MHLW 

and MAFF) guidelines; Method B13/14 of Commision Regulation (EC) number 440/2008; EPA 

(TSCA) OPPTS harmonized guidelines

Test type: Bacterial Reverse Mutation Test

GLP compliant?: Yes (with the exception of analysis for concentration, homogeneity and 

stability of the test item formulations)

Year study performed: 2012

Test species: Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and

Escherichia coli strain WP2 uvrA

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).
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Concentrations tested: 0 (negative control), 0 (vehicle control), 50, 150, 500, 1500 and 5000 

μg Benzoflex 1046/plate 

Statistical methods used: Statistical analysis of data was determined by UKEMS (Mahon et al.,

1989; Analysis of data from microbial colony assays, In: Statistical Evaluation of Mutagenicity 

Test Data, UKEMS sub-committee on guidelines for mutagenicity testing, Kirkland, D.J., Ed., 

Cambridge University Press Report, 26-65).

Vehicle: Dimethyl sulfoxide

Test method/conditions remarks: 

Preparation of Test Items:

“The test item [Benzoflex 1046 mixture] was immiscible in sterile distilled water at 50 mg/ml 

but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks 

performed in-house. Dimethyl sulphoxide was selected as the vehicle.” [Thompson, 2012, p. 9]

“The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl 

sulphoxide by mixing on a vortex mixer and sonication for 10 minutes at 40°C on the day of 

each experiment. All formulations were used within four hours of preparation and were assumed 

to be stable for this period. Analysis for concentration, homogeneity and stability of the test item 

formulations is not a requirement of the test guidelines and was, therefore, not determined. This 

is an exception with regard to GLP and has been reflected in the GLP compliance statement. 

Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium 

alumino-silicate pellets with a nominal pore diameter of 4 x 10-4 microns.” [Thompson 2012, p. 

9]

A vehicle control group was treated with the solvent. The following positive controls were used 

without S-9 mix:

N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG): 2 μg/plate for WP2 uvrA

N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG): 3 μg/plate for TA100

N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG): 5 μg/plate for TA1535

9-Aminoacridine (9AA): 80 μg/plate for TA1537

4-Nitroquinoline-1-oxide (4NQO): 0.2 μg/plate for TA98

The following positive controls were used in the presence of S-9 mix:

2-Aminoanthracene (2AA): 1 μg/plate for TA100

2-Aminoanthracene (2AA): 2 μg/plate for TA1535 and TA1537

2-Aminoanthracene (2AA): 10 μg/plate for WP2 uvrA

Benzo(a)pyrene (BP): 5 μg/plate for TA98
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Preliminary Toxicity Test:

“In order to select appropriate dose levels for use in the main test, a preliminary assay was 

carried out to determine the toxicity of the test item [Benzoflex 1046 mixture]. The 

concentrations [of test item] tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 

μg/plate. The assay was performed by mixing 0.1 ml of bacterial culture (TA100 orWP2 uvrA),

0.1 ml of test item formulation, 0.5 ml of S9-mix or phosphate buffer and 2 ml of molten, trace 

histidine or tryptophan supplemented, top agar and overlaying onto sterile plates of VogelBonner Minimal agar (30 ml/plate). Ten concentrations of the test item and a vehicle control 

(dimethyl sulphoxide) were tested. In addition, 0.1 ml of the maximum concentration of the test

item and 2 ml of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto 

a sterile nutrient agar plate in order to assess the sterility of the test item. After approximately 48 

hours incubation at 37°C the plates were assessed for numbers of revertant colonies using a 

Domino colony counter and examined for effects on the growth of the bacterial background 

lawn.” [Thompson 2012, p. 11]

Mutation Test - Experiment 1 (Range-finding Test):

“Five concentrations of the test item (50, 150, 500, 1500 and 5000 μg/plate) were assayed in 

triplicate against each tester strain, using the direct plate incorporation method.” [Thompson 

2012, p. 12]

“Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes 

followed by 2 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of the 

vehicle, test item formulation or positive control and either 0.5 ml of S9-mix or phosphate buffer. 

The contents of each test tube were mixed and equally distributed onto the surface of VogelBonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for 

each bacterial strain and for each concentration of test item both with and without S9-mix.” 

[Thompson 2012, p. 12]

“All of the plates were incubated at 37°C for approximately 48 hours and the frequency of 

revertant colonies assessed using a Domino colony counter.” [Thompson 2012, p. 12]

Mutation Test - Experiment 2 (Main Test):

“The second experiment was performed using fresh bacterial cultures, test item and control 

solutions. The test item dose range was the same as the range-finding test (50 to 5000 

μg/plate).” [Thompson 2012, p. 12]

“As it is good scientific practice to alter one condition in the replicate assay, the exposure

condition was changed from plate incorporation to pre-incubation. The test item formulations 

and vehicle control were therefore dosed as follows:

Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes 

followed by 0.5 ml of S9-mix or phosphate buffer and 0.1 ml of the vehicle or test item 

formulation and incubated for 20 minutes at 37°C with shaking at approximately 130 rpm prior 

to the addition of 2 ml of molten, trace histidine or tryptophan supplemented, top agar. The 

contents of the tube were then mixed and equally distributed on the surface of Vogel-Bonner 

Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each 
Final HPV3 Chemical Data Adequacy Review CASRN 22527-63-5

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bacterial strain and for each concentration of test item both with and without S9-mix. The 

positive and untreated controls were dosed using the standard plate incorporation method 

described [above].” [Thompson 2012, p. 12] 

“All of the plates were incubated at 37°C for approximately 48 hours and the frequency of

revertant colonies assessed using a Domino colony counter.” [Thompson 2012, p. 13]

Acceptance Criteria: 

According to the study report, the following criteria must be met for the assay to be considered 

valid:

“All bacterial strains must have demonstrated the required characteristics as determined by their 

respective strain checks... All tester strain cultures should exhibit a characteristic number of 

spontaneous revertants per plate in the vehicle and untreated controls... All tester strain cultures 

should be in the range of 0.9 to 9 x 109bacteria per ml. Diagnostic mutagens (positive control 

chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to 

mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in 

the study should induce marked increases in the frequency of revertant colonies, both with or

without metabolic activation... There should be a minimum of four non-toxic test item dose 

levels. There should be no evidence of excessive contamination.” [Thompson 2012, p. 13]

Evaluation Criteria:

“There are several criteria for determining a positive result. Any, one, or all of the following can 

be used to determine the overall result of the study:

1. A dose-related increase in mutant frequency over the dose range tested....

2. A reproducible increase at one or more concentrations.

3. Biological relevance against in-house historical control ranges.

4. Statistical analysis of data as determined by UKEMS...

5. Fold increase greater than two times the concurrent solvent control for any tester strain 

(especially if accompanied by an out-of-historical range response).” [Thompson, 2012, 

p. 13-14]

“A test item will be considered non-mutagenic (negative) in the test system if the above criteria 

are not met.” [Thompson 2012, p. 14]

“Although most experiments will give clear positive or negative results, in some instances the 

data generated will prohibit making a definite judgement about test item activity. Results of this 

type will be reported as equivocal.” [Thompson 2012, p. 14]

RESULTS

Genotoxicity result: Negative with and without metabolic activation

Cytotoxic concentration (if applicable): > 5000 μg Benzoflex 1046/plate, with and without S9 mix
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Results remarks: 

Preliminary Toxicity Test: 

“The test item was non-toxic to the strains of bacteria used (TA100 and WP2 uvrA). The test 

item formulation and S9-mix used in this experiment were both shown to be sterile.” [Thompson 

2012, p. 15] 

Mutation Tests:

“Prior to use, the master strains were checked for characteristics, viability and spontaneous 

reversion rate (all were found to be satisfactory). These data are not given in the report. The 

amino acid supplemented top agar and the S9-mix used in both experiments was shown to be 

sterile.” [Thompson 2012, p. 15]

Results for the negative (untreated) and vehicle controls were acceptable.

“The test item caused no visible reduction in the growth of the bacterial background lawn at any 

dose level and was, therefore, tested up to the maximum recommended dose level of 5000 

μg/plate. A test item precipitate (oily in appearance) was noted at and above 1500 μg/plate, this

observation did not prevent the scoring of revertant colonies.” [Thompson 2012, p. 16]

“No significant increases in the frequency of revertant colonies were recorded for any of the 

bacterial strains, with any dose of the test item, either with or without metabolic activation or 

[using either] exposure method.” [Thompson 2012, p. 16]

“All of the positive control chemicals used in the test induced marked increases in the frequency 

of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the 

bacterial strains.” [Thompson 2012, p. 16]

CONCLUSIONS

The test chemical, Benzoflex 1046, did not induce gene mutations in bacteria either with or 

without metabolic activation.

STUDY RELIABILITY

[2] Reliable with restrictions. The second of two assays, which used the preincubation method, 

did not include positive or untreated controls tested by that method. Instead, “[t]he positive and 

untreated controls [run concurrently with cells exposed to the test substance using the 

preincubation method in Experiment 2] were dosed using the standard plate incorporation

method...” [Thompson 2012, p. 12]
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REFERENCE

Thompson, P.W. 2012. Benzoflex 1046: Reverse Mutation Assay “Ames Test” Using 

Salmonella typhimurium and Escherichia coli. Harlan Laboratories Ltd., Project No. 41201917. 

Sponsor: Eastman Chemical Company. Available online at http://www.regulations.gov

/#!documentDetail;D=EPA-HQ-OPPT-2009-0112-0138 as of July 8, 2013.

Text in quotes is taken directly from Thompson (2012).
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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

Yes2– TBD

Toxicity to Aquatic Plants Yes2– TBD

Health Effects

Acute Toxicity Yes Yes No

Repeated-Dose Toxicity No3– Yes

Gene Mutation Yes Yes No

Chromosomal Aberrations Yes2– TBD

Reproductive Toxicity No3– Yes

Developmental Toxicity No3– Yes

1 Acute toxicity to fish and acute toxicity to aquatic invertebrate testing are not required if the 

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

2 Data were received from Eastman via email and will be evaluated for adequacy in a separate 

report.

3 According to Eastman’s letter to EPA dated December 21, 2012, Eastman expected initiation of 

a definitive combined repeated dose toxicity study with the reproduction/developmental toxicity 

screening test in the second quarter of 2013.

TBD = To be determined