SRC TR-13-076

HPV1/HPV2 TEST RULE DATA ADEQUACY REVIEW: DECEMBER 2013 UPDATE

(DRAFT)

Phenol, 4,4’-methylenebis[2,6-bis(1,1-dimethylethyl)-, CASRN 118-82-1

Prepared by:

Justine Von Runnen, Julie Klotzbach, Fernando Llados, Ashlee Aldridge, Amy Nguyen, 

Jennifer Rayner, William Richards, and Teresa Manyin

Defense and Environmental Solutions Division

SRC, Inc.

7502 Round Pond Road

North Syracuse, NY 13212

Contract No. EP-W-09-027

Task FG007.4.009.004

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

December 5, 2013
Draft HPV1/HPV2 Test Rule Data Adequacy Review (December 2013) CASRN 118-82-1

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TABLE OF CONTENTS

A. SOURCE MATERIAL USED FOR ASSESSMENT .............................................................. 1

B. EVALUATING DATA ADEQUACY ..................................................................................... 5

C. DATA FOR SIDS ENDPOINTS .............................................................................................. 6

C-1. Acute Toxicity to Fish........................................................................................................ 6

C-2. Acute Toxicity to Aquatic Invertebrates.......................................................................... 12

C-3. Chronic Toxicity to Aquatic Invertebrates....................................................................... 18

C-4. Toxicity to Aquatic Plants................................................................................................ 25

C-5. Acute Toxicity.................................................................................................................. 34

C-6. Repeated-Dose Toxicity................................................................................................... 46

C-7. Genetic Toxicity – Gene Mutation................................................................................... 63

C-8. Genetic Toxicity – Chromosomal Aberrations ................................................................ 77

C-9. Reproductive/Developmental Toxicity ............................................................................ 83

D. SUPPLEMENTAL DATA (NON-SIDS ENDPOINTS)...................................................... 104

D-1. Carcinogenicity .............................................................................................................. 104

D-2. Skin Irritation ................................................................................................................. 108

D-3. Eye Irritation .................................................................................................................. 114

E. DATA ADEQUACY SUMMARY TABLE......................................................................... 116
Draft HPV1/HPV2 Test Rule Data Adequacy Review (December 2013) CASRN 118-82-1

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A. SOURCE MATERIAL USED FOR ASSESSMENT

In this document, the toxicity of phenol, 4,4'-methylenebis[2,6-bis(1,1-dimethylethyl)- (CASRN

118-82-1) is assessed based on toxicological data for CASRN 118-82-1 and the supporting 

chemical, 2,6-di-tert-butylphenol (CASRN 128-39-2); available data are summarized and 

evaluated for data adequacy. This document represents an update of the data adequacy review 

written previously for this chemical: EPA. 2007. Data Adequacy Review and Robust Study 

Summary Adequacy Requirements: 4,4’-Methylenebis(2,6-di-tert-butylphenol) (CAS No. 118-

82-1) prepared by Oak Ridge National Laboratory, Oak Ridge, TN. October 18, 2007. 

This document includes data submitted in response to HPV TSCA Section 4 Test Rules: 

(1) Testing of Certain High Production Volume Chemicals; Final Rule, 71 FR 13708, March 

16, 2006; available online at http://www.regulations.gov/#!documentDetail;D=EPA-HQOPPT-2005-0033-0197 as of December 4, 2013; referred to herein as HPV1; and

(2) Testing of Certain High Production Volume Chemicals; Second Group of Chemicals; 

Final Rule, 76 FR 1067, January 7, 2011; available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2007-0531-0579 as of 

December 4, 2013; referred to herein as HPV2. 

The HPV1 test rule required the following toxicological tests for CASRN 118-82-1: D (acute 

mammalian toxicity), E1 (bacterial reverse mutation test), E2 (chromosomal aberration or 

micronucleus test), and F2 (reproduction/developmental toxicity screening test). 

From July 14, 2006 to May 17, 2007, Albemarle Corporation, the sponsor for CASRN 118-82-1,

submitted the following existing information on acute mammalian toxicity, gene mutation, and 

reproductive/developmental toxicity endpoints in response to the D, E1, and F2 testing 

requirements. In addition, the July 14, 2006 letter indicated that Albemarle “will conduct the 

testing as required for E2” (chromosomal aberration or micronucleus test).

Letter to Document Control Office, Office of Pollution Prevention and Toxics, U.S. EPA, 

Attn: TSCA Section 4, from Kim Boudreaux, Regulatory Advisor, Albemarle 

Corporation, Baton Rouge, Louisiana, dated July 14, 2006, with three attachments. 

Available online at http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT2005-0033-0181 as of December 4, 2013. 

Brooks, TM, and D.E. Wiggins. 1987. Microbial Mutagenicity Studies with IONOX 

220. Shell Research Limited, Sittingbourne Research Center. Submitted to EPA on May 

17, 2007. Available online at http://www.regulations.gov/#!documentDetail;D=EPAHQ-OPPT-2005-0033-0250 as of December 4, 2013.

Following review of this existing information for CASRN 118-82-1, EPA determined that the 

data submitted for acute mammalian toxicity and bacterial reverse mutation were adequate to 

satisfy the D and E1 requirements of the Test Rule. However, the existing studies submitted for 

the reproductive/developmental toxicity endpoint (conducted with CASRN 118-82-1) were not 

considered to be adequate to satisfy the F2 testing requirement. 
Draft HPV1/HPV2 Test Rule Data Adequacy Review (December 2013) CASRN 118-82-1

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In a letter dated May 16, 2007 (available online at http://www.regulations.gov/#!document

Detail;D=EPA-HQ-OPPT-2005-0033-0247 as of December 4, 2013), Albemarle Corporation 

proposed the use of 2,6-di-tert-butylphenol (CASRN 128-39-2) as a supporting chemical for 

characterizing the effects of phenol, 4,4'-methylenebis[2,6-bis(1,1-dimethylethyl)- (CASRN 118-

82-1) on human health. EPA considers CASRN 128-39-2 to be an appropriate supporting 

chemical for CASRN 118-82-1 based on structural and functional similarity. Therefore, EPA 

accepts the use of toxicological data from studies with CASRN 128-39-2 for the purposes of 

characterizing the effects of CASRN 118-82-1 on human health. 

EPA identified the following gene mutation and reproductive/developmental toxicity studies,

conducted with the acceptable supporting chemical (CASRN 128-39-2), which satisfy the F2 

testing requirement (reproduction/developmental toxicity screening test) and provide mammalian 

cell mutagenicity data that supplement the bacterial mutagenicity data for CASRN 118-82-1,

submitted by Albemarle Corporation for the E1 testing requirement.

Sandoz Chemicals Ltd. 1991. Unpublished CCR Report [on a mammalian cell gene 

mutation assay] - Sandoz, 243617. Muttenz, Switzerland. Summarized in the online 

ECHA (http://echa.europa.eu) entry for CASRN 128-39-2 (study indicated as Exp Key 

Genetic toxicity in vitro.001). Secondary reference: OECD. 2002. SIDS/IRPTC Data 

Profile for 2,6-Di-tert-butylphenol (CASRN 128-39-2). UNEP Publications. 2002. 

Available online at http://www.chem.unep.ch/irptc/sids/OECDSIDS/128392.pdf as of 

December 4, 2013.

Sandoz Chemicals Ltd. 1992. Unpublished CCR Report [on a 

reproduction/developmental toxicity test] - Sandoz, 321794. Muttenz, Switzerland. 

Summarized in the online ECHA (http://echa.europa.eu) entry for CASRN 128-39-2

(study indicated as Exp Key Toxicity to reproduction.001). Secondary reference: 

OECD. 2002. SIDS/IRPTC Data Profile for 2,6-Di-tert-butylphenol (CASRN 128-39-

2). UNEP Publications. 2002. Available online at http://www.chem.unep.ch/irptc/sids

/OECDSIDS/128392.pdf as of December 4, 2013.

Due to the identification of adequate studies on acute mammalian toxicity and gene mutation for 

the test rule chemical (CASRN 118-82-1) and on reproductive/developmental toxicity for an 

acceptable supporting chemical (CASRN 128-39-2), EPA revoked the D, E1, and F2 testing 

requirements for CASRN 118-82-1 (Revocation of TSCA Section 4 Testing Requirements for 

Certain High Production Volume Chemical Substances; Direct Final Rule, 77 FR 15609, March 

16, 2012; available online at http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT2005-0033-0402 as of December 4, 2013). 

To address the E2 testing requirement, Albemarle Corporation contracted TNO Laboratories to 

conduct a chromosomal aberration study and submitted the following report on June 28, 2007; 

EPA reviewed this chromosomal aberration study and considered it adequate to satisfy the E2 

testing requirement promulgated in the HPV1 test rule: 
Draft HPV1/HPV2 Test Rule Data Adequacy Review (December 2013) CASRN 118-82-1

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de Vogel, N. and Usta, B. 2007. Chromosomal Aberration test with AN-2 in Cultured 

Chinese Hamster Ovary (CHO) Cells. TNO Laboratories, Zeist, Netherlands, Project No. 

031.11113/01.42, Study No. 6802/24, May 9, 2007. Submission documented online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2005-0033-0257 as of 

December 4, 2013.

The HPV2 test rule required C1 aquatic toxicity testing for CASRN 118-82-1. The log Kow

value of CASRN 118-82-1 was experimentally determined to be > 6.50 by SafePharm 

Laboratories,

1

indicating that testing under Test Group 2, which applies to chemicals with log 

Kow ≥ 4.2, is required. Test Group 2 includes the following tests: (1) Chronic Toxicity to 

Daphnia (ASTM E 1193-97) and (2) Toxicity to Plants (Algae; ASTM E 1218-04). 

Albemarle submitted comments (available online at http://www.regulations.gov/#!document

Detail;D=EPA-HQ-OPPT-2007-0531-0068 as of December 4, 2013) in response to the proposed 

HPV2 test rule on October 21, 2008, suggesting that the following study by Mattock and Girling 

(1988) would satisfy the toxicity to plants testing requirement; EPA reviewed this study and 

considered it to be inadequate to satisfy any component of the C1 testing requirement:

Mattock, S.D. and Girling, A.E. 1988. IONOX 220: Acute Toxicity (Salmo gairdneri, 

Daphnia magna and Selenastrum capricornutum) and n-octanol/water partition 

coefficient. Sittingbourne Research Centre, Shell Research Limited. Submitted by Shell 

Oil Co. to EPA in 1992 as part of a TSCA 8(e) submission (EPA Doc. No. 88-

920003444, Fiche No. OTS0537403) entitled “Initial submission: acute toxicity of 

IONOX 220 in Salmo gairdneri, Daphnia magna and Selenastrum capricornutum and noctanol/water partition coefficient w-letter dated 053092”.

The following existing aquatic toxicity data, submitted by Albemarle Corporation on July 14, 

2006, were also reviewed by EPA and considered to be inadequate to satisfy the C1 testing 

requirement:

Stonybrook Laboratories Inc. 1995. Study No. 66125, conducted for Ethyl Corporation. 

Source: Ethyl Petroleum Additives International, Bracknell, Berkshire. (Summarized in 

IUCLID Dataset for CASRN 118-82-1, European Chemical Bureau, 2000). Robust 

summary available online at http://www.regulations.gov/#!documentDetail;D=EPA-HQOPPT-2005-0033-0181 as of December 4, 2013.

Stonybrook Laboratories Inc. 1995. Study No. 66126, conducted for Ethyl Corporation. 

Source: Ethyl Petroleum Additives International, Bracknell, Berkshire. (Summarized in 

IUCLID Dataset for CASRN 118-82-1, European Chemical Bureau, 2000). Robust 

summary available online at http://www.regulations.gov/#!documentDetail;D=EPA-HQOPPT-2005-0033-0181 as of December 4, 2013.

 

1 Study submitted to EPA by Albemarle Corporation on May 12, 2006, available online at http://www.regulations.

gov/#!documentDetail;D=EPA-HQ-OPPT-2005-0033-0163 as of December 4, 2013; “4,4’-Methylene bis(2,6-ditert-butylphenol): Determination of Water Solubility and Partition Coefficient”, SPL Project Number 1193/011, 

study completed on July 18, 2005.
Draft HPV1/HPV2 Test Rule Data Adequacy Review (December 2013) CASRN 118-82-1

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Albemarle Corporation contracted Wildlife International, Ltd. to conduct chronic toxicity to 

Daphnia and toxicity to algae studies and submitted the following studies on June 29, 2012 to 

address the C1 - Test Group 2 testing requirement; EPA considers these studies adequate to 

satisfy the C1 testing requirement promulgated in the HPV2 test rule:

Minderhout, T., Kendall, T.Z., Gallagher, S.P. and Krueger, H.O. 2012. AN-2: A flowthrough life-cycle toxicity test with the cladoceran (Daphnia magna). Final Report. 

Wildlife International, Ltd., Easton, MD. Project No. 471A-120. Study report available 

online at http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2007-0531-

0678 as of December 4, 2013.

Arnie, J.R., Porch, J.R., Kendall, T.Z. and Krueger, H.O. 2012. AN-2: A 96-hour 

toxicity test with the freshwater alga (Pseudokirchneriella subcapitata). Wildlife 

International, Ltd., Easton, MD. Project No. 471A-119. Study report available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2007-0531-0678 as of 

December 4, 2013.

Although the test rules did not require repeated-dose toxicity testing, the following repeated-dose 

studies were included herein to provide a complete assessment of toxicological SIDS endpoints:

Jolley, W.P., Stemmer, K.L., Grande, F., Richmond, J., and Pfitzer, E.A. 1969. The 

effects exerted upon beagle dogs during a period of two years by the introduction of 4,4'-

methylenebis (2,6-ditertiary butylphenol) into their daily diets. Kettering Laboratory, 

University of Cincinnati, OH. Submitted to EPA in 1992 as part of a TSCA 8(e)

submission (EPA Doc. No. 88-920001353S, Fiche No. OTS0536073) entitled “Initial 

sub: the effect exerted upon beagle dogs during a period of 2-yrs by the introduction of 

4,4'-methylenebis (2,6-ditertaryl butylphenol) into their daily diets (sanitized)”.

Jolley, W.P., Stemmer, K.L., Pfitzer, E.A., Grande, F., and Richmond, J. 1975. The 

effects upon beagle dogs following the ingestion of 4,4'-methylenebis (2,6-ditertiary 

butylphenol) for two years including the changes after an additional seven months on 

control diet. Kettering Laboratory, University of Cincinnati, OH. Submitted to EPA in 

1992 as part of a TSCA 8(e) submission (EPA Doc. No. 88-920001353S, Fiche No. 

OTS0536073) entitled “Initial sub: the effect exerted upon beagle dogs during a period of 

2-yrs by the introduction of 4,4'-methylenebis (2,6-ditertaryl butylphenol) into their daily 

diets (sanitized)”. 

ECHA. Oral repeated-dose toxicity in rats (Exp WoE Repeated dose toxicity: oral.003).

Study summary available from the ECHA website at

http://apps.echa.europa.eu/registered/data/dossiers/DISS-dcee47d2-62ad-0171-e044-

00144f67d031/AGGR-bf547f31-7fe6-4a5f-a06a-e41a1a9a2ab0_DISS-dcee47d2-62ad0171-e044-00144f67d031.html#AGGR-bf547f31-7fe6-4a5f-a06a-e41a1a9a2ab0 as of 

December 4, 2013. The data source is referred to as a “study report”, with a report date 

of 1969-07-08. No other reference information is provided. 
Draft HPV1/HPV2 Test Rule Data Adequacy Review (December 2013) CASRN 118-82-1

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Data from all studies submitted by Albemarle Corporation, as well as other available data for 

SIDS toxicity endpoints (including data evaluated in the previous data adequacy review [EPA, 

20071]), are evaluated for adequacy in Section C of this document. Additional data for nonSIDS toxicity endpoints (“supplemental” data), including skin and eye irritation, are reviewed in 

Section D. Sections C and D incorporate information from the High Production Volume 

Information System (HPVIS) and the European Chemicals Agency (ECHA) website

(http://echa.europa.eu/). 

B. EVALUATING DATA ADEQUACY

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 at 

the end of the summary document. 

 

1 EPA. 2007. Data Adequacy Review and Robust Study Summary Adequacy Requirements: 4,4’-

Methylenebis(2,6-di-tert-butylphenol) (CAS No. 118-82-1) prepared by Oak Ridge National Laboratory, Oak Ridge, 

TN. October 18, 2007.
Draft HPV1/HPV2 Test Rule Data Adequacy Review (December 2013) CASRN 118-82-1

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C. DATA FOR SIDS ENDPOINTS

C-1. Acute Toxicity to Fish

Summary of endpoint: Not Adequate. Testing of acute toxicity to fish is not required for the 

chemical because the log Kow is > 4.2.

1

Study 1: Acute toxicity study in rainbow trout (Mattock and Girling, 1988) 

Title: IONOX 220: Acute toxicity (Salmo gairdneri, Daphnia magna, and Selenastrum 

capricornutum) and n-octanol/water partition coefficient

This study is: Adequate Not Adequate

This study is not adequate because analytical monitoring was not conducted and the test 

concentrations (10–1000 mg/L) exceeded the water solubility limit of the test substance 

(experimentally determined to be < 0.01 mg/L)2by many orders of magnitude, although a 

vehicle was not used. In addition, the study is non-GLP, a test method/guideline was not 

specified, and the temperature varied by more than 2°C.

TEST SUBSTANCE 

Identity: Phenol, 4,4’-methylenebis[2,6-bis(1,1-dimethylethyl)-, CASRN 118-82-1; referred to 

as IONOX 220 in study report

Remarks: 99.04% purity (by GC); pale yellow powder; obtained from San Celoni, Barcelona, 

Spain

METHOD

Method/guideline followed: Not specified

Test type: Acute Toxicity to Fish (Semi-Static)

GLP compliant?: No

Year study was performed: 1988

 

1The log Kow value of CASRN 118-82-1 was experimentally determined to be > 6.50 by SafePharm Laboratories 

(study submitted to EPA by Albemarle Corporation on May 12, 2006, available online at http://www.regulations.

gov/#!documentDetail;D=EPA-HQ-OPPT-2005-0033-0163 as of December 4, 2013; “4,4’-Methylene bis(2,6-ditert-butylphenol): Determination of Water Solubility and Partition Coefficient”, SPL Project Number 1193/011, 

study completed on July 18, 2005).

2The water solubility limit of CASRN 118-82-1 was experimentally determined to be < 0.01 mg/L by SafePharm 

Laboratories (study submitted to EPA by Albemarle Corporation on May 12, 2006, available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2005-0033-0163 as of December 4, 2013; “4,4’-

Methylene bis(2,6-di-tert-butylphenol): Determination of Water Solubility and Partition Coefficient”, SPL Project 

Number 1193/011, study completed on July 18, 2005).
Draft HPV1/HPV2 Test Rule Data Adequacy Review (December 2013) CASRN 118-82-1

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Test species: Rainbow trout (Salmo gairdneri); obtained from Itchin Valley Trout Farm, 

Hampshire; mean length of 3.6 cm and mean weight of 0.81g (n = 10)

Analytical monitoring: No

Exposure duration: 96 hours

Concentrations tested: 0 (control), 10, 30, 100, 300, and 1000 mg/L

Test method/conditions remarks: Test solutions were renewed daily.

Ten fish per concentration were placed in glass aquaria filled with 10 L of filtered dechlorinated 

main water. Fish were not fed during exposure. 

Test Conditions:

Temperature: 13–17°C

pH: 7.7–8.3 

Hardness: 242–260 mg CaCO3/L

Dissolved Oxygen: 9.6–10.4 mg/L

Statistics: The 72- and 96-hour LC50 values were calculated using probit analysis after log 

transformation of the concentrations.

RESULTS

Endpoint value(s): Note that analytical monitoring was not conducted and nominal 

concentrations (10-1000 mg/L) were many orders of magnitude greater than the water solubility 

limit of the test substance (experimentally determined to be < 0.01 mg/L)1. Therefore, it is not 

possible to interpret the reported toxicity values.

72-hour LC50 = 870 mg/L; 96-hour LC50 = 820 mg/L, based on nominal concentrations.

Results remarks: Of the ten fish exposed to the highest concentration (1000 mg/L), three had 

died within 24 hours, five had died within 48 hours, and six had died within 72–96 hours. One 

death occurred in the 300 mg/L dose group at 96-hours.

Page 2 of the test report, which included studies for fish, Daphnia, and green algae, provided a 

general comment that the test substance “appeared to be not wholly soluble at concentrations 

greater than” 10 mg/L. 

 

1 The water solubility limit of CASRN 118-82-1 was experimentally determined to be < 0.01 mg/L by SafePharm 

Laboratories (study submitted to EPA by Albemarle Corporation on May 12, 2006, available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2005-0033-0163 as of December 4, 2013; “4,4’-

Methylene bis(2,6-di-tert-butylphenol): Determination of Water Solubility and Partition Coefficient”, SPL Project 

Number 1193/011, study completed on July 18, 2005).
Draft HPV1/HPV2 Test Rule Data Adequacy Review (December 2013) CASRN 118-82-1

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CONCLUSIONS

The 96-hour LC50 for rainbow trout is 820 mg/L (95% CI, 490–2600 mg/L) based on nominal 

concentrations. 

STUDY RELIABILITY

[3] Not reliable. Non-GLP study. Test method/guideline was not specified, analytical 

monitoring was not conducted, the temperature varied by more than 2°C, and the test 

concentrations exceeded the water solubility limit of the test substance (experimentally 

determined to be < 0.01 mg/L)1by many orders of magnitude, although a vehicle was not used. 

REFERENCE

Mattock, S.D. and Girling, A.E. 1988. IONOX 220: Acute Toxicity (Salmo gairdneri, Daphnia 

magna and Selenastrum capricornutum) and n-octanol/water partition coefficient. Sittingbourne 

Research Centre, Shell Research Limited. Submitted by Shell Oil Co. to EPA in 1992 as part of 

a TSCA 8(e) submission (EPA Doc. No. 88-920003444, Fiche No. OTS0537403) entitled 

“Initial submission: acute toxicity of IONOX 220 in Salmo gairdneri, Daphnia magna and

Selenastrum capricornutum and n-octanol/water partition coefficient w-letter dated 053092”.

 

1 The water solubility limit of CASRN 118-82-1 was experimentally determined to be < 0.01 mg/L by SafePharm 

Laboratories (study submitted to EPA by Albemarle Corporation on May 12, 2006, available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2005-0033-0163 as of December 4, 2013; “4,4’-

Methylene bis(2,6-di-tert-butylphenol): Determination of Water Solubility and Partition Coefficient”, SPL Project 

Number 1193/011, study completed on July 18, 2005).
Draft HPV1/HPV2 Test Rule Data Adequacy Review (December 2013) CASRN 118-82-1

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Study 2: Acute toxicity study in sheepshead minnow (Stonybrook Laboratories Inc., 1995) 

Title: Acute/Prolonged Toxicity to Fish

This study is: Adequate Not Adequate

Important study details missing from this report include test substance purity, test 

method/guideline followed, GLP compliance, whether or not analytical monitoring was 

conducted, length, weight and source of test species, control treatment, and detailed numerical 

results. Concentrations of the test substance (10–1000 mg/L) exceeded the water solubility limit 

of the test substance (experimentally determined to be < 0.01 mg/L)1by many orders of 

magnitude and a vehicle was not used. Furthermore, the study report did not provide any 

information regarding evaluations for physical evidence of insolubility of the test substance in 

the media (e.g., visible precipitate or residue). Additionally, the temperature varied by more than 

2°C, only three concentrations were tested, and units of salinity measurements were not reported.

TEST SUBSTANCE 

Identity: Phenol, 4,4’-methylenebis[2,6-bis(1,1-dimethylethyl)-, CASRN 118-82-1 

Remarks: Purity not provided

METHOD

Method/guideline followed: Not specified

Test type: Acute Toxicity to Fish (Static)

GLP compliant?: Not specified

Year study was performed: 1995

Test species: Sheepshead Minnow (Cyprinodon variegatus)

Analytical monitoring: Not specified

Exposure duration: 96 hours

Concentrations tested: 10, 100, and 1000 mg/L

 

1 The water solubility limit of CASRN 118-82-1 was experimentally determined to be < 0.01 mg/L by SafePharm 

Laboratories (study submitted to EPA by Albemarle Corporation on May 12, 2006, available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2005-0033-0163 as of December 4, 2013; “4,4’-

Methylene bis(2,6-di-tert-butylphenol): Determination of Water Solubility and Partition Coefficient”, SPL Project 

Number 1193/011, study completed on July 18, 2005).
Draft HPV1/HPV2 Test Rule Data Adequacy Review (December 2013) CASRN 118-82-1

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Test method/conditions remarks: The tests were carried out with a dispersion of the test 

material in water under static conditions. The test volume was 30 L.

Experimental Conditions:

Salinity: 21–22 (units not specified)

Temperature: 19.2–22.3°C

Dissolved oxygen: 6.7–7.6 mg/L

pH: 8.55

No. of replicates: 2

No. of fish per replicate: 10 

RESULTS

Endpoint value(s): Note that analytical monitoring was not conducted and nominal 

concentrations (10–1000 mg/L) were many orders of magnitude greater than the water solubility 

limit (experimentally determined to be < 0.01 mg/L)1for this compound. Therefore, it is not 

possible to interpret the reported toxicity values.

96-hour LC50 > 1000 mg/L, based on nominal concentrations.

Results remarks: No mortalities were observed. No additional information was provided in 

study summary. 

CONCLUSIONS

The 96-hour LC50 for sheepshead minnow is > 1000 mg/L, based on nominal concentrations. 

STUDY RELIABILITY

[3] Not reliable. Important study details missing from this report include test substance purity, 

test method/guideline followed, GLP compliance, whether or not analytical monitoring was 

conducted, length, weight and source of test species, control treatment, and detailed numerical 

results. Test material concentrations exceeded the water solubility limit of the test substance 

(experimentally determined to be < 0.01 mg/L)1by many orders of magnitude and a vehicle was 

not used. Furthermore, the study report did not provide any information regarding evaluations 

for physical evidence of insolubility of the test substance in the media (e.g., visible precipitate or 

residue). Additionally, the temperature varied by more than 2°C, only three concentrations were 

tested, and units of salinity measurements were not reported.

 

1 The water solubility limit of CASRN 118-82-1 was experimentally determined to be < 0.01 mg/L by SafePharm 

Laboratories (study submitted to EPA by Albemarle Corporation on May 12, 2006, available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2005-0033-0163 as of December 4, 2013; “4,4’-

Methylene bis(2,6-di-tert-butylphenol): Determination of Water Solubility and Partition Coefficient”, SPL Project 

Number 1193/011, study completed on July 18, 2005).
Draft HPV1/HPV2 Test Rule Data Adequacy Review (December 2013) CASRN 118-82-1

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REFERENCE

Stonybrook Laboratories Inc. 1995. Study No. 66126, conducted for Ethyl Corporation. 

Source: Ethyl Petroleum Additives International, Bracknell, Berkshire. (As summarized in 

IUCLID Dataset for CASRN 118-82-1, European Chemical Bureau, 2000). Robust summary 

available online at http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2005-

0033-0181 (on p. 66 of the document “End_Point_D – Mammalian_Toxicity_Acute.pdf”) as of 

December 4, 2013.
Draft HPV1/HPV2 Test Rule Data Adequacy Review (December 2013) CASRN 118-82-1

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C-2. Acute Toxicity to Aquatic Invertebrates1

Summary of endpoint: Not adequate. Testing of acute toxicity to Daphnia is not required for 

the chemical because the log Kow is > 4.2. 

Study 1: Acute toxicity study in Daphnia (Mattock and Girling, 1988) 

Title: IONOX 220: Acute toxicity (Salmo gairdneri, Daphnia magna, and Selenastrum 

capricornutum) and n-octanol/water partition coefficient

This study is: Adequate Not Adequate

This study is not adequate because concentrations of test material (500–1000 mg/L) exceeded the 

water solubility limit of the test substance (experimentally determined to be < 0.01 mg/L)2by 

many orders of magnitude and a vehicle was not used. Furthermore, the study report did not 

provide any information regarding evaluations for physical evidence of insolubility of the test 

substance in the media (e.g., visible precipitate or residue). In addition, the study was not 

conducted under GLP, the test method/guideline followed and statistical methods used were not 

specified, only two concentrations were tested, and the temperature varied by more than 2°C.

TEST SUBSTANCE 

Identity: Phenol, 4,4’-methylenebis[2,6-bis(1,1-dimethylethyl)-, CASRN 118-82-1; referred to 

as IONOX 220 in study report

Remarks: 99.04% purity (by GC); pale yellow powder; obtained from San Celoni, Barcelona, 

Spain

METHOD

Method/guideline followed: Not specified

Test type: Acute Toxicity to Daphnia (Static)

GLP compliant?: No

Year study was performed: 1988

Test species: Water flea (Daphnia magna); < 24 hours old; obtained from ICI Brixham 

Laboratory

 

1 Although acute toxicity testing in Daphnia is not required, test requirement C1 under the Final HPV2 Test Rule 

dated January 7, 2011 requires chronic toxicity testing in Daphnia according to the ASTM E 1193 guideline.

2 The water solubility limit of CASRN 118-82-1 was experimentally determined to be < 0.01 mg/L by SafePharm 

Laboratories (study submitted to EPA by Albemarle Corporation on May 12, 2006, available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2005-0033-0163 as of December 4, 2013; “4,4’-

Methylene bis(2,6-di-tert-butylphenol): Determination of Water Solubility and Partition Coefficient”, SPL Project 

Number 1193/011, study completed on July 18, 2005).
Draft HPV1/HPV2 Test Rule Data Adequacy Review (December 2013) CASRN 118-82-1

13

Analytical monitoring: No

Exposure duration: 48 hours

Concentrations tested: 0 (control), 500, and 1000 mg/L 

Test method/conditions remarks: Ten Daphnia were allocated to 150 mL glass crystallizing 

dishes that contained 100 mL of reconstituted fresh water. Test substance was added to duplicate 

sets of dishes resulting in two concentrations of 500 and 100 mg/L. The two control dishes did 

not receive test substance. 

Test Conditions:

Temperature: 18–22°C

pH: 7.8–8.0

Hardness: 166 mg CaCO3/L

Dissolved Oxygen: 9.0–9.2 mg/L

Statistics: None specified

RESULTS

Endpoint value(s): Note that analytical monitoring was not conducted and nominal 

concentrations (500–1000 mg/L) were many orders of magnitude greater than the water 

solubility limit (experimentally determined to be < 0.01 mg/L)1for the test compound. 

Therefore, it is not possible to interpret the reported toxicity value.

48-hour EC50 > 1000 mg/L, based on nominal concentrations. 

Results remarks: Only 1 out of 20 daphnids was immobilized at 1000 mg/L. Page 2 of the test 

report, which included studies for fish, Daphnia, and green algae, provided a general comment 

that the test substance “appeared to be not wholly soluble at concentrations greater than”

10 mg/L. 

CONCLUSIONS

The 48-hour EC50 for Daphnia magna is > 1000 mg/L, based on nominal concentrations.

 

1 The water solubility limit of CASRN 118-82-1 was experimentally determined to be < 0.01 mg/L by SafePharm 

Laboratories (study submitted to EPA by Albemarle Corporation on May 12, 2006, available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2005-0033-0163 as of December 4, 2013; “4,4’-

Methylene bis(2,6-di-tert-butylphenol): Determination of Water Solubility and Partition Coefficient”, SPL Project 

Number 1193/011, study completed on July 18, 2005).
Draft HPV1/HPV2 Test Rule Data Adequacy Review (December 2013) CASRN 118-82-1

14

STUDY RELIABILITY

[3] Not reliable. Non-GLP study. Test method/guideline followed and statistical methods used 

were not specified, analytical monitoring was not conducted, only two concentrations were 

tested, the temperature varied by more than 2°C, the test concentrations (500–1000 mg/L) 

exceeded the water solubility limit of the test substance (experimentally determined to be 

< 0.01 mg/L)1by many orders of magnitude, and a vehicle was not used. 

REFERENCE

Mattock, S.D. and Girling, A.E. 1988. IONOX 220: Acute Toxicity (Salmo gairdneri, Daphnia 

magna and Selenastrum capricornutum) and n-octanol/water partition coefficient. Sittingbourne 

Research Centre, Shell Research Limited. Submitted by Shell Oil Co. to EPA in 1992 as part of 

a TSCA 8(e) submission (EPA Doc. No. 88-920003444, Fiche No. OTS0537403) entitled 

“Initial submission: acute toxicity of IONOX 220 in Salmo gairdneri, Daphnia magna and

Selenastrum capricornutum and n-octanol/water partition coefficient w-letter dated 053092”.

 

1 The water solubility limit of CASRN 118-82-1 was experimentally determined to be < 0.01 mg/L by SafePharm 

Laboratories (study submitted to EPA by Albemarle Corporation on May 12, 2006, available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2005-0033-0163 as of December 4, 2013; “4,4’-

Methylene bis(2,6-di-tert-butylphenol): Determination of Water Solubility and Partition Coefficient”, SPL Project 

Number 1193/011, study completed on July 18, 2005).
Draft HPV1/HPV2 Test Rule Data Adequacy Review (December 2013) CASRN 118-82-1

15

Study 2: Acute toxicity study in mysid shrimp (Stonybrook Laboratories Inc., 1995) 

Title: Acute Toxicity to Aquatic Invertebrates

This study is: Adequate Not Adequate

Important study details missing from the report include test substance purity, test 

method/guideline followed, GLP compliance, age and source of test species, and detailed 

numerical results. Additionally, only three concentrations were tested, and units of salinity 

measurements were not reported. Concentrations of test material (10–1000 mg/L) exceeded the 

water solubility limit of the test substance (experimentally determined to be < 0.01 mg/L)1by 

many orders of magnitude and a vehicle was not used. Furthermore, the study report did not 

provide any information regarding evaluations for physical evidence of insolubility of the test 

substance in the media (e.g., visible precipitate or residue). 

TEST SUBSTANCE 

Identity: Phenol, 4,4’-methylenebis[2,6-bis(1,1-dimethylethyl)-, CASRN 118-82-1

Remarks: Purity not provided

METHOD

Method/guideline followed: Not specified

Test type: Mysid acute toxicity test (static)

GLP compliant?: Not specified

Year study was performed: 1995

Test species: Mysid shrimp (Mysidopsis bahia)

Analytical monitoring: No

Exposure duration: 96 hours

Concentrations tested: 0, 10, 100, and 1000 mg/L

Test method/conditions remarks: The tests were carried out with water-accommodated 

fractions (WAFs) under static conditions. The test volume was 30 L.

 

1 The water solubility limit of CASRN 118-82-1 was experimentally determined to be < 0.01 mg/L by SafePharm 

Laboratories (study submitted to EPA by Albemarle Corporation on May 12, 2006, available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2005-0033-0163 as of December 4, 2013; “4,4’-

Methylene bis(2,6-di-tert-butylphenol): Determination of Water Solubility and Partition Coefficient”, SPL Project 

Number 1193/011, study completed on July 18, 2005).
Draft HPV1/HPV2 Test Rule Data Adequacy Review (December 2013) CASRN 118-82-1

16

Experimental Conditions:

Salinity: 20–22 (units not specified)

Temperature: 19.0–20.9°C

Dissolved oxygen: 6.0–7.1 mg/L

pH: 8.26–8.32

No. of replicates: 2

No. of mysids per replicate: 10

RESULTS

Endpoint value(s): Note that analytical monitoring was not conducted and nominal 

concentrations (10–1000 mg/L) were many orders of magnitude greater than the reported water 

solubility limit (experimentally determined to be < 0.01 mg/L)1for this compound. Therefore, it 

is not possible to interpret reported toxicity values.

96-hour LC50 > 1000 mg/L, based on nominal concentrations. 

Results remarks: No mortalities were observed. No additional information was provided in 

study summary. 

CONCLUSIONS

The 96-hour LC50 for Mysidopsis bahia is > 1000 mg/L, based on nominal concentrations.

STUDY RELIABILITY

[3] Not reliable. Important study details missing from the report include test substance purity, 

test method/guideline followed, GLP compliance, and detailed numerical results. Concentrations

of the test material exceeded the water solubility limit of the test substance (experimentally 

determined to be < 0.01 mg/L)1by many orders of magnitude and a vehicle was not used. 

Furthermore, the study report did not provide any information regarding evaluations for physical 

evidence of insolubility of the test substance in the media (e.g., visible precipitate or residue). 

Additionally, only three concentrations were tested, rather than five or more concentrations, and 

units of salinity measurements were not reported.

 

1 The water solubility limit of CASRN 118-82-1 was experimentally determined to be < 0.01 mg/L by SafePharm 

Laboratories (study submitted to EPA by Albemarle Corporation on May 12, 2006, available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2005-0033-0163 as of December 4, 2013; “4,4’-

Methylene bis(2,6-di-tert-butylphenol): Determination of Water Solubility and Partition Coefficient”, SPL Project 

Number 1193/011, study completed on July 18, 2005).
Draft HPV1/HPV2 Test Rule Data Adequacy Review (December 2013) CASRN 118-82-1

17

REFERENCE

Stonybrook Laboratories Inc. 1995. Study No. 66125, conducted for Ethyl Corporation. 

Source: Ethyl Petroleum Additives International, Bracknell, Berkshire. (Summarized in 

IUCLID Dataset for CASRN 118-82-1, European Chemical Bureau, 2000). Robust summary 

available online at http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2005-

0033-0181 (on p. 66 of the document “End_Point_D – Mammalian_Toxicity_Acute.pdf”) as of 

December 4, 2013. 
Draft HPV1/HPV2 Test Rule Data Adequacy Review (December 2013) CASRN 118-82-1

18

C-3. Chronic Toxicity to Aquatic Invertebrates

Summary of endpoint: Adequate

Study 1: Chronic toxicity study in Daphnia (Minderhout et al., 2012) 

Title: AN-2: A flow-through life-cycle toxicity test with the cladoceran (Daphnia magna)

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 4,4’-methylenebis (2,6-di-tert-butyl[U14C]phenol), Batch number CFQ41115, from 

Quotient Bioresearch. Test substance was a 14C-labeled form of CASRN 118-82-1; referred to 

as AN-2 in study report. 

Remarks: Properties of Quotient Research Batch number CFQ41115 were: Liquid, 99.8% 

radiochemical purity by high performance liquid chromatography (HPLC), specific activity of 

60 mCi/mmol by mass spectrometry and 55 mCi/mmol by gravimetric analysis, MW 426.6 at 

this specific activity. “The material co-chromatographs with client supplied material in the 

above chromatographic [HPLC] system. The mass spectrum is consistent with the proposed 

structure and a non labelled reference. The 1H-NMR [nuclear magnetic resonance] spectrum is 

consistent with the proposed structure and with a non labelled reference.” [Test substance 

properties and quoted material are from the Certificate of Analysis for Batch number CFQ41115 

presented in Appendix 2, p. 44 of: Arnie, J.R., Porch, J.R., Kendall, T.Z. and Krueger, H.O. 

2012. AN-2: A 96-Hour Toxicity Test with the Freshwater Alga (Pseudokirchneriella 

subcapitata). Wildlife International, Ltd. Project No. 471A-119. Submitted to Albemarle 

Corporation. Available online at http://www.regulations.gov/#!documentDetail;D=EPA-HQOPPT-2007-0531-0678 as of December 4, 2013.]

METHOD

Method/guideline followed: OECD TG 211, EPA OPPTS 850.1300, and ASTM E 1193-97

Test type: Chronic Toxicity to Daphnia (Flow-Through)

GLP compliant?: Yes

Year study was performed: 2012

Test species: Water flea (Daphnia magna); neonates (< 24 hours old at initiation); obtained 

from in-house cultures at Wildlife International, Ltd., Easton, MD.

Analytical monitoring: Yes

Exposure duration: 21 days
Draft HPV1/HPV2 Test Rule Data Adequacy Review (December 2013) CASRN 118-82-1

19

Concentrations tested: Nominal: 0 (negative control), 0 (solvent control), 1.6, 3.1, 6.3, 13, 25,

and 50 ng/L; mean measured concentrations based on 14C: < LOQ1, < LOQ, 1.4, 2.4, 5.5, 12, 21,

and 43 ng/L. Note: Measurement of 14C does not distinguish between test substance and 

degradation products; mean measured concentrations of the intact test substance were not 

reported.

Vehicle: HPLC-grade dimethylformamide (DMF)

Test method/conditions remarks: “Daphnids were exposed to a geometric series of six test 

concentrations, a negative control (dilution water) and a solvent control (0.025 mL/L HPLCgrade dimethylformamide). Two replicate test chambers were tested for each treatment and 

control group. Each replicate contained two compartments with five daphnids, resulting in a 

total of 20 daphnids in each treatment and control group. Nominal test concentrations were 

selected in consultation with the Sponsor based on exploratory range finding toxicity data and 

the solubility limit in the test system. The nominal test concentrations were 1.6, 3.1, 6.3, 13, 25 

and 50 ng AN-2/L. Mean measured test concentrations [based on measurement of 14C] were 

determined from samples of test water collected from each treatment and control group at test 

initiation, at approximately weekly intervals during the test and at test termination.” 

[Minderhout et al. (2012), p. 10]

“Delivery of the test substance to the test chambers was initiated seven days prior to the 

introduction of the daphnids to the test water in order to achieve equilibrium of the test substance 

in the test chambers. At test initiation, neonate daphnids were impartially assigned to exposure 

chambers. First-generation daphnids were observed daily during the test for mortality, the onset 

of reproduction, and clinical signs of toxicity. Following the onset of reproduction, the numbers 

of second-generation daphnids were counted three times per week (i.e., Monday, Wednesday and 

Friday), and at test termination (Day 21). Body lengths and dry weights of the surviving firstgeneration daphnids were measured at the end of the exposure period. Observations of the 

effects of AN-2 on survival, reproduction and growth were used to determine the no-observedeffect concentration (NOEC), the lowest-observed-effect concentration (LOEC), and the 

maximum acceptable toxicant concentration (MATC). EC50 values were determined based on 

reproduction and on first-generation immobility at test termination.” [Minderhout et al. (2012), 

pp. 10-11]

Preparation of Test Concentrations: “Individual stock solutions were prepared for each of the 

six concentrations tested, and were prepared once during the study. Each test stock solution was 

prepared by mixing a calculated amount of radiolabelled AN-2 into HPLC-grade 

dimethylformamide (DMF) at a nominal concentration of 0.064, 0.124, 0.252, 0.52, 1.0 and 2.0 

μg/mL. The stock solutions were mixed by inversion. The stock solutions at 0.064 to 1.0 μg/mL 

appeared clear and colorless, while the stock solution at 2.0 μg/mL appeared clear with a slight 

purple tint. Stock solutions were stored refrigerated in glass amber bottles, and aliquots of each 

stock were placed in the syringe pump every 4 to 7 days during the study.” [Minderhout et al. 

(2012), p. 14]

 

1 LOQ = 0.873 ng/L
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20

“The five test substance stock solutions were injected into the diluter mixing chambers at a rate 

of 2.5 μL/minute where they were mixed with dilution water delivered at a rate of 

100 mL/minute to achieve the desired test concentrations. The negative control received dilution 

water only. The solvent control was prepared by delivering HPLC-grade DMF to the mixing 

chamber for the solvent control. The concentration of DMF in the solvent control and all AN-2 

treatment groups was 0.025 mL/L.” [Minderhout et al. (2012), p. 14]

Analytical Sampling: “Samples were collected from each treatment and control group five days 

prior to the start of the test after conditioning the diluter for two days. Water samples also were 

collected from alternating replicate test chambers in each treatment and control group at the 

beginning of the test, at approximately weekly intervals during the test and at the end of the test 

to measure concentrations of the test substance. Samples of the stock solutions being delivered 

to the diluter were collected for analysis on Day –11 to confirm the stock concentrations, after 

the stocks were prepared. Additional samples of test solutions were collected on Day 22 to 

confirm concentration of the 1.6 ng/L treatment level when the analytical result of this treatment 

level on Day 21 was suspect. All test solution samples were collected from mid-depth, placed in 

separatory funnels, and processed immediately for analysis by LSC [liquid scintillation 

counting].” [Mindehout et al. (2012), p. 14]

Analytical Method: “The concentrations of the test substance in freshwater samples were verified 

by liquid scintillation counting (LSC). Samples were extracted two times with dichloromethane, 

evaporated to dryness and reconstituted using acetonitrile. Extracts were transferred into 

scintillation vials containing 13 mL of Ultima Gold XR scintillation cocktail and submitted for 

analysis by LSC. A Packard 2500TR Liquid Scintillation Analyzer was used to determine 

disintegrations per minute (dpm). In addition to the test samples, a 13 mL sample of Ultima 

Gold XR scintillation cocktail was analyzed at each interval to determine background

radioactivity.” [Mindehout et al. (2012), p. 15]

Biological Observations and Measurements: “Observations of each first-generation daphnid 

were made daily during the test. At these times, the numbers of immobile daphnids were 

recorded along with any clinical signs of toxicity (e.g., inability to maintain position in the water 

column, uncoordinated swimming or cessation of feeding). Immobility was defined as a lack of 

movement, except for minor spontaneous random movement of the appendages. The presence of 

eggs in the brood pouch, aborted eggs, males or ephippia also were recorded daily. With the 

onset of reproduction, neonates produced by the first-generation daphnids were counted and then 

discarded every Monday, Wednesday and Friday during the test. The body length and the dry 

weight of each surviving first-generation daphnid were measured at the end of the test.” 

[Minderhout et al. (2012), p. 16]

Statistical Analyses: “Test endpoints analyzed statistically for first-generation daphnids were 

survival, reproduction, and growth (length and dry weight). Reproduction was based on the 

number of live young produced per reproductive day. Reproductive days were defined as the 

number of days that the adult daphnid was alive from the day the first brood was released from 

any adult daphnid in the test until test termination. If an adult daphnid died, the number of 

reproductive days, for that adult, ended on the last day it was alive.” [Minderhout et al. (2012), p. 

16]
Draft HPV1/HPV2 Test Rule Data Adequacy Review (December 2013) CASRN 118-82-1

21

“Negative control and solvent control data for each parameter were compared using an 

appropriate statistical test. No significant differences between the control groups were found for 

any parameter tested (p > 0.05). Therefore, the control data for all parameters were pooled for 

comparison with the treatment groups.” [Minderhout et al. (2012), p. 17]

“Survival data was considered to be discrete-variable data, while reproduction and growth data 

were considered continuous-variable data. Discrete-variable data were analyzed using Chisquare and Fisher’s Exact test to identify treatment groups that showed a statistically significant 

difference (p ≤ 0.05) from the control. All continuous-variable data were evaluated for normality 

using Shapiro-Wilk’s and for homogeneity of variance using Levene’s (α = 0.01). When the data 

passed the assumptions of normality and homogeneity, those treatments that were significantly 

different from the control means were identified using Dunnett’s test (α = 0.05). All statistical 

tests were performed using a personal computer with SAS software.” [Minderhout et al. (2012), 

p. 17]

“The results of the statistical analyses were used to aid in the determination of the NOEC, LOEC 

and MATC. However, scientific judgment was used to determine if statistical differences were 

biologically meaningful, and if the data followed a concentration-dependent response. The 

NOEC was defined as the highest test concentration that produced no significant treatmentrelated effects on survival, reproduction or growth. The LOEC was defined as the lowest test 

concentration that produced a significant treatment-related effect on survival, reproduction or 

growth. The MATC was calculated as the geometric mean of the NOEC and LOEC. EC50 

values were determined based on reproduction and on the immobility observed in the firstgeneration daphnids at the end of the test.” [Minderhout et al. (2012), p. 17]

Test Conditions:

Temperature: 19.6–20.4°C

pH: 8.0–8.2 

Hardness: 134–141 mg CaCO3/L

Dissolved Oxygen: 5.6–8.6 mg/L

Dilution water TOC: < 1mg C/L

Light Intensity: 171 lux

RESULTS

Endpoint value(s): Based on mean measured concentrations of 14C, the 21-day EC50 for 

immobility, reproduction, and growth is greater than 43 ng/L; the 21-day NOEC for immobility,

reproduction, and growth is 43 ng/L based on no significant effects at the highest concentration 

tested. Note: Measurement of 14C does not distinguish between test substance and degradation 

products; mean measured concentrations of the intact test substance were not reported.
Draft HPV1/HPV2 Test Rule Data Adequacy Review (December 2013) CASRN 118-82-1

22

Results remarks: Test solutions appeared clear and colorless with no evidence of precipitation.

Measurement of Test Concentrations: “Samples of the stock solutions being delivered to the 

diluter system had measured [

14C] concentrations that ranged from 100 to 105% of nominal 

concentrations, confirming the concentrations being delivered to the diluter system .... The 

measured [

14C] concentrations of samples collected to verify the diluter system prior to the test 

ranged from 76.9 to 96.7% of nominal concentrations .... Samples of the test solutions collected 

during the test had measured concentrations [of 14C] that ranged from 66.0 to 177% of nominal 

concentrations .... Since the analytical result of the sample [for the] 1.6 ng/L treatment group on 

Day 21 was suspect (the recovery was 177%), [an] additional sample was collected from this 

treatment group to confirm the test concentration on Day 22. The results of the sample collected 

on Day 22 confirmed the test concentration; the result of the sample for the 1.6 ng/L treatment 

group collected on Day 21 was excluded from the calculation of mean measured test 

concentration. When the measured concentrations of test solution samples collected on Days 0, 

7, 14, 21 and 22 of the test were averaged for each treatment group, the mean measured test 

concentrations [based on concentrations of 14C] were 1.4, 2.4, 5.5, 12, 21 and 43 ng/L, which 

represented 88, 77, 87, 92, 84 and 86% of nominal concentrations, respectively. The results of 

the study were based on the mean measured concentrations [of 14C].” [Minderhout et al. (2012), 

p. 18]

Survival and Clinical Observations: “After 21 days of exposure, survival in the negative control 

and solvent control groups were 75 and 95%, respectively. There was statistically no significant 

difference in survival between the negative and solvent control groups. Therefore, the control 

data were pooled for comparison with the treatment groups. Survival in the 1.4, 2.4, 5.5, 12, 21 

and 43 ng/L treatment groups at test termination was 90, 90, 100, 95, 90 and 90%, respectively. 

Fisher’s Exact test indicated that no statistically significant decrease in survival was detected in 

any of the AN-2 treatment groups in comparison to the pooled control (p > 0.05). Consequently, 

the NOEC for survival was 43 ng/L and the LOEC was greater than 43 ng/L. Based on the 

immobilities observed in the treatment groups, the 21-day EC50 value was greater than 43 ng/L, 

the highest concentration tested.” [Minderhout et al. (2012), pp. 18-19]

“Daphnids in the 1.4, 2.4, 5.5, 12, 21 and 43 ng/L treatment groups that survived to test 

termination generally appeared normal. During the test pale, small and/or lethargic daphnids 

were observed in the 1.4, 2.4, 12, 21 and 43 ng/L treatment groups. However, the numbers of 

sublethal effects noted in the 1.4, 12 and 21 ng/L treatment groups were infrequent. At test 

termination, approximately 33% of surviving first-generation daphnids in the 43 ng/L treatment 

group appeared pale in comparison to the control organisms. While the discoloration of the 

daphnids in the 43 ng/L treatment group were not analyzed statistically, it is important to note 

that the condition appeared to follow the dose-response pattern. All surviving daphnids in the 

2.4 and 5.5 ng/L treatment groups were normal in appearance at test termination.” [Minderhout 

et al. (2012), p. 19]
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23

Reproduction: “The first day of brood production in the negative and solvent control replicates 

and in all AN-2 treatment replicates was Day 7, 8 or 9 of the test, indicating that there was no 

apparent delay in the onset of production at any AN-2 concentration tested. With the exception 

of two immobile neonates in the negative control and solvent control groups and three immobile 

neonates in the 43 ng/L treatment group on Day 9 or 11 of the test, no immobile neonates were 

noted in the 1.4, 2.4, 5.5, 12 and 21 ng/L treatment groups during the test (Appendix 9 [of the 

study report]). No aborted or shed eggs were present in any of the control or treatment groups. 

No males or ephippia were produced during the test.” [Minderhout et al. (2012), p. 19]

“Adult daphnids in the negative and solvent control groups produced an average of 8.1 and 8.7 

live young per reproductive day, respectively. There was no significant difference in 

reproduction between the negative and solvent control groups. Therefore, the control data were 

pooled for comparison with the treatment groups. Adult daphnids in the 1.4, 2.4, 5.5, 12, 21 and 

43 ng/L treatment groups produced an average of 8.9, 6.7, 8.1, 6.9, 7.7 and 8.3 live young per 

reproductive day, respectively. Dunnett’s test indicated there were no statistically significant 

decreases in mean neonate production in any of the AN-2 treatment groups in comparison to the 

pooled control (p > 0.05). Consequently, the NOEC for reproduction was 43 ng/L and the LOEC 

was greater than 43 ng/L. Based on the reproduction observed in the treatment groups, the 

21-day EC50 value was greater than 43 ng/L, the highest concentration tested.” [Minderhout et 

al. (2012), p. 20]

Growth: “Daphnids in the negative and solvent control groups averaged 4.6 mm in length and 

0.63 mg in dry weight, while daphnids in the solvent control group averaged 4.5 mm in length, 

and 0.69 mg in dry weight, respectively. There were no significant differences in the growth 

parameters between the negative and solvent control groups. Therefore, the control data were 

pooled for comparison with the treatment groups. Daphnids in the 1.4, 2.4, 5.5, 12, 21 and 43 

ng/L treatment groups had mean lengths of 4.6, 4.3, 4.4, 4.4, 4.4 and 4.5 mm, respectively, and 

mean dry weights of 0.76, 0.60, 0.74, 0.76, 0.63 and 0.60 mg, respectively. Dunnett’s test 

indicated there were no statistically significant decreases in mean dry weight in any of the AN-2 

treatment groups in comparison to the pooled control (p > 0.05) but statistically significant 

reductions were noted in mean total length in the 2.4, 5.5, 12 and 21 ng/L treatment groups when 

compared to the pooled controls (p ≤ 0.05). Since the reductions in mean total length of 

daphnids in the 2.4, 5.5, 12 and 21 ng/L treatment groups did not follow a dose response pattern, 

the slight differences noted were not considered to be biologically meaningful. In addition, the 

mean length of the daphnids in the 2.4, 5.5, 12 and 21 ng/L treatment groups were within the 

ranges of the mean length of daphnids in the historical control data (3.6 to 6.3 mm). 

Consequently, the NOEC for growth (length and dry weight) was 43 ng/L, and the LOEC was 

greater than 43 ng/L.” [Minderhout et al. (2012), p. 20]

CONCLUSIONS

“The cladoceran, Daphnia magna, was exposed to AN-2 at mean measured concentrations 

[based on concentrations of 14C] of 1.4, 2.4, 5.5, 12, 21 and 43 ng/L under flow-through 

conditions for 21 days. There were no statistically significant treatment-related effects on 

survival, reproduction or growth at concentrations ≤ 43 ng/L. Consequently, the NOEC, based 

on survival, reproduction and growth, was 43 ng/L. The LOEC was greater than 43 ng/L and the 
Draft HPV1/HPV2 Test Rule Data Adequacy Review (December 2013) CASRN 118-82-1

24

MATC was calculated to be greater than 43 ng/L. The 21-day EC50 values for adult immobility 

and reproduction [and growth] were both greater than 43 ng/L, the highest concentration tested.” 

[Minderhout et al. (2012), p. 21]

STUDY RELIABILITY

[2] Reliable with restrictions. Following the preparation of test solutions containing 

radiolabelled test substance [4,4’-methylenebis (2,6-di-tert-butyl[U14C]phenol)], the method 

used for monitoring test solutions over the course of the study for the presence of radiolabel 

(extraction of organic components from the aqueous samples into dichloromethane, evaporation 

of the dichloromethane, reconstitution of dried material in acetonitrile, and use of liquid 

scintillation counting [LSC] to analyze total 14C radioactivity) did not distinguish between the 

intact test substance and degradation products. However, this deficiency does not appear to 

affect the validity of the EC50, NOEC, LOEC, and MATC values that were derived from the data 

of this study. 

REFERENCE

Minderhout, T., Kendall, T.Z., Gallagher, S.P. and Krueger, H.O. 2012. AN-2: A flow-through 

life-cycle toxicity test with the cladoceran (Daphnia magna). Final Report. Wildlife 

International, Ltd., Easton, MD. Project No. 471A-120. Study report available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2007-0531-0678 as of 

December 4, 2013.

Text in quotes is taken directly from Minderhout et al., 2012.
Draft HPV1/HPV2 Test Rule Data Adequacy Review (December 2013) CASRN 118-82-1

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C-4. Toxicity to Aquatic Plants

Summary of endpoint: Adequate 

Study 1: Toxicity study in green algae (Arnie et al., 2012) 

Title: AN-2: A 96-hour toxicity test with the freshwater alga (Pseudokirchneriella subcapitata)

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 4,4’-methylenebis (2,6-di-tert-butyl[U14C]phenol), Batch number CFQ41115, from 

Quotient Bioresearch. Test substance was a 14C-labeled form of CASRN 118-82-1; referred to 

as AN-2 in study report. 

Remarks: Properties of Quotient Research Batch number CFQ41115: Liquid, 99.8% 

radiochemical purity by high performance liquid chromatography (HPLC), specific activity of 

60 mCi/mmol by mass spectrometry and 55 mCi/mmol by gravimetric analysis, MW 426.6 at 

this specific activity. “The material co-chromatographs with client supplied material in the 

above chromatographic [HPLC] system. The mass spectrum is consistent with the proposed 

structure and a non labelled reference. The 1H-NMR [nuclear magnetic resonance] spectrum is 

consistent with the proposed structure and with a non labelled reference.” [Arnie et al., 2012, 

Certificate of Analysis, Appendix 2, p. 44].

METHOD

Method/guideline followed: EPA OPPTS 850.5400; OECD TG 201; ASTM E1218-04

Test type: Toxicity to Green Algae (Static)

GLP compliant?: Yes

Year study was performed: 2012

Test species: Freshwater green algae (Pseudokirchneriella subcapitata); original algal cultures 

were obtained from the University of Toronto Culture Collection, and had been maintained in 

culture medium in-house at Wildlife International, Ltd., Easton, Maryland.

Analytical monitoring: Yes 

Exposure duration: 96 hours

Concentrations tested: Nominal: 0 (negative control), 0 (solvent control), 2, 4, 8, 16, and 

32 ng/L; Initial (day 0) measured concentrations based on 14C: < LOQ1(negative control), 

 

1 LOQ = 0.582 ng/L
Draft HPV1/HPV2 Test Rule Data Adequacy Review (December 2013) CASRN 118-82-1

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< LOQ (solvent control), 1.50, 3.35, 6.84, 12.9, and 26.5 ng/L. Note: Measurement of 14C does 

not distinguish between test substance and degradation products; mean measured concentrations 

of the intact test substance were not reported.

Vehicle: Dimethylformamide (DMF)

Test method/conditions remarks: 

“The green alga, Pseudokirchneriella subcapitata, was exposed to five test concentrations, a 

solvent control (0.1 mL N,N-dimethylformamide per L) and a negative control (culture medium) 

for 96 hours. Nominal test concentrations were selected in consultation with the Sponsor and 

were based upon the results of an exploratory range-finding toxicity test. Nominal test 

concentrations selected were 2.0, 4.0, 8.0, 16.0 and 32.0 nanograms per liter (ng/L). Measured 

concentrations [based on measurement of 14C] were determined from samples of test medium 

collected from each treatment and control group at 0 and 96 hours of the test.” [Arnie et al. 

(2012), p. 11]

“Three replicate test chambers were maintained in each treatment group and six replicates were 

maintained in the negative control group. At test initiation, an inoculum of the algal cells was 

added to each test chamber to achieve a nominal concentration of approximately 

10,000 cells/mL. Samples were collected from each replicate test chamber at approximately 

24-hour intervals during the test to determine cell densities, yield and growth rates. Cell 

densities, yields and growth rates were used to calculate percent inhibition values relative to the 

control over the 96-hour exposure period. EC50 and ErC50 values (i.e., the theoretical toxicant 

concentrations that would produce a 50% reduction in cell density and growth rate, respectively) 

were calculated, when possible, for each 24-hour interval of the exposure period. EyC50 values 

(i.e., the theoretical toxicant concentrations that would produce a 50% reduction in yield) were 

calculated, when possible, at 72 and 96 hours of exposure. No-observed-effect concentrations 

(NOEC) were determined at 72 and 96 hours through statistical evaluation of cell densities, 

yields and growth rates, as well as examination of the concentration-response pattern.” [Arnie et 

al. (2012), pp. 11-12]

Test Apparatus: “Test chambers were sterile, 250-mL Erlenmeyer flasks plugged with foam 

stoppers, and contained 100 mL of test or control medium. The test chambers were labeled with 

the project number, test concentration and replicate, and were indiscriminately positioned daily 

on a mechanical shaker in an environmental chamber designed to maintain the desired test 

temperature throughout the test. The test flasks were shaken continuously at 100 rpm.” [Arnie 

et al. (2012), p. 13]

Culture Medium: “The algal cells were cultured and tested in freshwater AAP algal medium. 

Stock nutrient solutions were prepared by adding reagent-grade chemicals to purified well water 

(NANOpure® water). The test medium was then prepared by adding appropriate volumes of the 

stock nutrient solutions to NANOpure® water... The pH of the medium was adjusted to 7.5 ± 

0.1 using 10% hydrochloric acid and was sterilized by filtration (0.22 μm) prior to use.” [Arnie et 

al. (2012), p. 13] Analyses performed to measure selected contaminants (pesticides, organics, 

and metals) in the well water used to prepare the algal medium showed acceptable 

concentrations.
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Environmental Conditions: “Test chambers were held in an environmental chamber at a 

temperature of 24 ± 2°C. The temperature of a container of water adjacent to the test chambers 

in the environmental chamber was recorded twice daily during the test using a liquid-in-glass 

thermometer. In addition, temperature in the environmental chamber was measured 

continuously using a minimum-maximum thermometer. The algae were held under continuous 

cool-white fluorescent lighting throughout the test. The target light intensity was 6,000 lux ± 

10%. Light intensity was measured at test solution level at five locations surrounding the test 

flasks at test initiation using a SPER Scientific 840006C light meter. The pH of the medium in 

each treatment and control group was measured at test initiation and exposure termination using 

a Thermo Orion Model 525 APlus. At test initiation, pH was measured in the individual batches 

of test solution prepared for each treatment and control group. At exposure termination, pH was 

measured in pooled samples of test solution collected from each of the replicates of each 

treatment and control group.” [Arnie et al. (2012), p. 13]

Preparation of Test Concentrations: “A primary stock solution was prepared by dissolving 

0.213 mL of radiolabelled AN-2 in 50 mL of N,N-diemethylformamide to achieve a nominal 

concentration of 32.0 μg/mL. The primary stock was mixed by inversion and appeared clear and 

purple in color upon preparation. A secondary stock was prepared by diluting 1.0 mL of 32.0 

μg/mL primary stock in 100 mL of N,N-dimethylformamide to achieve a nominal concentration 

of 0.319 μg/mL. Additional stocks were prepared at nominal concentrations of 0.16, 0.08, 0.04, 

0.02 μg/mL in N,N-dimethylformamide by serial dilution. The 2.0, 4.0, 8.0, 16.0 and 32.0 ng/L 

test solutions were prepared by diluting 100 μL of each respective stock solution in 1000 mL of 

freshwater AAP medium. The solvent control solution was prepared by diluting 100 μL of N,Ndimethylformamide in 1000 mL of freshwater AAP medium to achieve a nominal concentration 

of 0.1 mL N,N-dimethylformamide per liter, equivalent to the solvent concentration in all of the 

test solutions except the negative control. The negative control solution consisted of freshwater 

AAP algal medium without test substance or solvent added. All test solutions were mixed by 

inversion, and appeared clear and colorless and were otherwise unremarkable after mixing.” 

[Arnie et al. (2012), p. 14]

Analytical Sampling: “Samples of the test solutions were collected at approximately 0 and 96 

hours to measure concentrations of the test substance. Samples at test initiation were collected 

from the individual batches of test solution prepared for each treatment and control group after 

distribution into the test chambers. At exposure termination, samples were collected from the 

pooled replicates from each treatment and control group. Samples were processed immediately 

for LSC [liquid scintillation counting] analysis.” [Arnie et al. (2012), p. 14]

Analytical Method: “The concentrations of the test substance in freshwater algal medium

samples were verified by liquid scintillation counting (LSC). Samples were extracted two times 

with dichloromethane, rotary evaporated to dryness and reconstituted using acetonitrile. Extracts 

were quantitatively transferred into scintillation vials containing 10 mL of Ultima Gold XR 

scintillation cocktail and submitted for analysis by LSC. A Packard 2500TR Liquid Scintillation 

Analyzer was used to determine disintegrations per minute (dpm). In addition to the test 

samples, a 10 mL sample of Ultima Gold XR scintillation cocktail was analyzed at each interval 

to determine background radioactivity.” [Arnie et al. (2012), p. 14]
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Algal Growth Measurements and Observations: “Test medium samples were collected from 

each replicate of the treatment and control groups for the determination of algal cell densities. 

Samples were collected at approximately 24-hour intervals during the 96-hour exposure and 

were held for a maximum of three days under refrigerated conditions sufficient to inhibit growth 

until cell counts could be performed.” [Arnie et al. (2012), p. 15]

“Samples of test solution were collected from each replicate at the end of the test. These samples 

were pooled within their respective treatments, and sub-samples were removed and examined 

microscopically for atypical cell morphology (e.g., changes in cell shape, size or color). Cells in 

the replicate test chambers also were assessed for aggregation or flocculation of cells, and 

adherence of the cells to the test chamber.” [Arnie et al. (2012), p. 15]

Statistical Analyses: “The calculation of cell densities, yield, growth rates and percent inhibition 

values, as well as all statistical analyses, were conducted using ‘The SAS System for Windows, 

Version 8.2’. Growth rate was calculated for each replicate of the control and treatment 

groups...” [Arnie et al. (2012), p. 15]

“Yield was calculated for each replicate of the control and treatment groups as the final biomass 

(cell density) in the exposure period minus the initial biomass (cell density). Inhibition values 

were calculated for each treatment group as the percent reduction in cell density and growth rate 

relative to the negative control replicates for each 24-hour interval, and yield at 72 and 96 hours 

of exposure.” [Arnie et al. (2012), p. 16]

“EC50 values in this study represent the theoretical test concentrations that would produce a 50% 

reduction in a variable of interest relative to the negative control. The EyC50 and ErC50 values 

and their 95% confidence intervals were calculated, when possible, using non-linear regression 

with replicate data (cell density, yield or growth rate) and day 0 measured test concentrations.” 

[Arnie et al. (2012), p. 16]

“The negative and solvent control data was compared using a student’s t-test (α = 0.05). No 

statistically significant differences were detected between the negative and solvent control data. 

The treatment group responses were compared to the negative control response for statistical 

comparisons and EC50 calculations, when possible. The 72 and 96-hour cell density, yield and 

growth rate data were evaluated for normality and homogeneity of variance (α = 0.01) using 

Shapiro-Wilk’s and Levene’s tests, respectively. All data met assumptions or normal 

distribution and equal variance; therefore, the treatment groups were compared to the negative 

control using Dunnett’s one-tailed t-test (α = 0.05). The results of the statistical analyses, as well 

as an evaluation of the concentration-response pattern, were used to determine the NOEC 

relative to each parameter at 72 and 96 hours.” [Arnie et al. (2012), p. 16]

Test Conditions:

Temperature: 22.4–25.5°C

pH: 7.1–8.5

Light Intensity: 5870–6430 lux
Draft HPV1/HPV2 Test Rule Data Adequacy Review (December 2013) CASRN 118-82-1

29

RESULTS

Endpoint value(s): Based on initial [day 0] measured concentrations of 14C: 96-hr EC50 (cell 

density, growth rate, and yield) > 26.5 ng/L; NOEC (cell density, growth rate, and yield) = 

26.5 ng/L

Results remarks: 

Measurement of Test Concentrations: “The test solutions appeared clear and colorless at the 

time of preparation, and there was no evidence of surface slicks or precipitation at exposure 

termination. Measured concentrations [of 14C] on Day 0 ranged from 75 to 86% of nominal. 

Measured concentrations [of 14C] declined by Day 4, and ranged from 16 to 24% of nominal. 

The results of the study [i.e., the EC50 values and NOEC] are based on day 0 measured 

concentrations [of 14C] of 1.50, 3.35, 6.84, 12.9 and 26.5 ng/L, representing 75, 84, 86, 81 and 

83% of the target nominal test concentrations [of 14C]. Measured concentrations of 14C AN-2 by 

LSC analysis in the negative and solvent controls were less than the limit of quantitation (LOQ) 

at both sampling intervals.” [Arnie et al. (2012), pp. 16-17]

Observations and Measurements: “After 72 hours of exposure, inhibition of cell density in the 

1.50, 3.35, 6.84, 12.9 and 26.5 ng/L treatment groups was 15, 8, 49, -9 and -3%, respectively, 

relative to the negative control. Inhibition of yield in the 1.50, 3.35, 6.84, 12.9 and 26.5 ng/L 

treatment groups was 15, 8, 49, -9 and -3[%], relative to the negative control. Inhibition of 

growth rate in the 1.50, 3.35, 6.84, 12.9 and 26.5 ng/L treatment groups was 4, 1, 13, -2 and -1%, 

respectively, relative to the negative control. Dunnett’s test indicated that there were significant 

reductions (p ≤ 0.05) in cell density, yield and growth rate in the 6.8 ng/L treatment group. Since 

this reduction was not dose-responsive and appeared analogous to the data collected from the 

other treatment groups, it was not considered to be treatment related. Consequently, the 72-hour 

NOEC for cell density, yield and growth rate was determined to be 26.5 ng/L based on 

evaluation of the dose-response.” [Arnie et al. (2012), pp. 17-18]

“After 96 hours of exposure, inhibition of cell density in 1.50, 3.35, 6.84, 12.9 and 26.5 ng/L 

treatment groups was -23, -22, 12, -10 and 5%, respectively, relative to the negative control. 

Inhibition of yield in 1.50, 3.35, 6.84, 12.9 and 26.5 ng/L treatment groups was -24, -22, 12, -10 

and -5%, respectively, relative to the negative control. Inhibition of growth rate in the 1.50, 

3.35, 6.84, 12.9 and 26.5 ng/L treatment groups was -3, -4, 2, -2 and -1%, respectively, relative 

to the negative control. No significant reductions in cell density, yield or growth rate were 

detected (Dunnett’s test, p > 0.05) when the responses of the treatment groups were compared to 

the negative control response. Consequently, the 96-hour NOEC for cell density, yield and 

growth rate was 26.5 ng/L.” [Arnie et al. (2012), p. 18]

“After 96 hours of exposure, cells present in the 1.50, 3.35, 6.84, 12.9 and 26.5 ng/L treatment 

groups appeared normal when compared to cells present in the negative and solvent control 

groups during microscopic observations of test solution samples. Adherence to the test chambers 

and flocculation or aggregation of cells were not observed in any of the experimental groups.” 

[Arnie et al. (2012), p. 18]
Draft HPV1/HPV2 Test Rule Data Adequacy Review (December 2013) CASRN 118-82-1

30

Validity of the Test: The mean cell density in the negative control replicates increased by a 

factor of 189 within 72 hours and by a factor of 382 within 96 hours. The coefficient of variation 

of average specific growth rates in the control replicates was 5.45% within 72 hours, and the 

mean coefficient of variation for section-by-section specific growth rates (days 0–1, 1–2, and 2–

3) was 26.3%. These values conformed to the OECD and ASTM criteria for validity of an algae 

test.

CONCLUSIONS

The 96-hr EC50 for cell density, growth rate, and yield is > 26.5 ng/L (based on initial [day 0] 

measured concentrations of 14C); the NOEC is 26.5 ng/L. 

STUDY RELIABILITY 

[2] Reliable with restrictions. Following the preparation of test solutions containing 

radiolabelled test substance [4,4’-methylenebis (2,6-di-tert-butyl[U14C]phenol)], the method 

used for monitoring test solutions at 0 and 96 hours for the presence of radiolabel (extraction of 

organic components from the aqueous samples into dichloromethane, evaporation of the 

dichloromethane, reconstitution of dried material in acetonitrile, and use of liquid scintillation 

counting [LSC] to analyze total 14C radioactivity) did not distinguish between test substance and 

degradation products. Furthermore, the test report did not provide an explanation of why the 

concentrations of 14C were not stable over the 4-day exposure period. Measured concentrations

of 14C on Day 0 ranged from 75 to 86% of nominal, declining to 16 to 24% of nominal on Day 4. 

There is uncertainty about whether this decline represents an actual loss of test substance (or its 

degradates) from the test vessels or only an apparent loss, that is possibly due to: (1) the 

interference of increasing algae biomass with the extraction of the test substance or its degradates 

from aqueous solution into dichloromethane (e.g., does test substance radioactivity remain in the 

water phase with the algae cells rather than being extracted into the dichloromethane phase?), or 

(2) interference with the quantitation of radioactivity by LSC (e.g., does color from the algae 

cause a reduction of counting efficiency [i.e., quenching] by LSC?). However, these deficiencies 

do not appear to affect the validity of the EC50 values and NOEC values, which were calculated 

using day 0 “measured” values that are based on radioactivity measurements. 

REFERENCE

Arnie, J.R., Porch, J.R., Kendall, T.Z. and Krueger, H.O. 2012. AN-2: A 96-hour toxicity test 

with the freshwater alga (Pseudokirchneriella subcapitata). Wildlife International, Ltd., Easton, 

MD. Project No. 471A-119. Study report available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2007-0531-0678 as of 

December 4, 2013.

Text in quotes is taken directly from Arnie et al., 2012.
Draft HPV1/HPV2 Test Rule Data Adequacy Review (December 2013) CASRN 118-82-1

31

Study 2: Toxicity study in green algae (Mattock and Girling, 1988) 

Title: IONOX 220: Acute toxicity (Salmo gairdneri, Daphnia magna, and Selenastrum 

capricornutum) and n-octanol/water partition coefficient

This study is: Adequate Not Adequate

This study is not adequate because the test concentrations exceeded the water solubility limit of 

the test substance (experimentally determined to be < 0.01 mg/L)1by many orders of magnitude. 

Furthermore, this is a non-GLP study, the test method/guideline followed and statistical methods 

used were not specified, analytical monitoring was not conducted, water chemistry parameters 

were not provided (hardness), and control treatment and control responses were not specified.

TEST SUBSTANCE 

Identity: Phenol, 4,4’-methylenebis[2,6-bis(1,1-dimethylethyl)-, CASRN 118-82-1; referred to 

as IONOX 220 in study report

Remarks: 99.04% purity (by GC); pale yellow powder; obtained from San Celoni, Barcelona, 

Spain

METHOD

Method/guideline followed: Not specified

Test type: Toxicity test to freshwater algae

GLP compliant?: No

Year study was performed: 1988

Test species: Freshwater green algae (Selenastrum capricornutum); from the axenic laboratory 

culture obtained from the American Type Culture Collection, Maryland, U.S.A.

Analytical monitoring: No

Exposure duration: 96 hours

Concentrations tested: 0 (control), 1, 2.2, 4.6, 10, 22, 46, 100, 220, 460, and 1000 mg/L

 

1 The water solubility limit of CASRN 118-82-1 was experimentally determined to be < 0.01 mg/L by SafePharm 

Laboratories (study submitted to EPA by Albemarle Corporation on May 12, 2006, available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2005-0033-0163 as of December 4, 2013; “4,4’-

Methylene bis(2,6-di-tert-butylphenol): Determination of Water Solubility and Partition Coefficient”, SPL Project 

Number 1193/011, study completed on July 18, 2005).
Draft HPV1/HPV2 Test Rule Data Adequacy Review (December 2013) CASRN 118-82-1

32

Test method/conditions remarks: IONOX 220 in Analar acetone was added to Erlenmeyer 

flasks containing 50 mL of culture medium to give an approximately logarithmically spaced 

series of concentrations ranging from 1 to 1000 mg/L. The concentration of Analar acetone in 

all test flasks, including the controls, was adjusted to 0.1 mL/L. Each flask was inoculated with 

S. capricornutum to give an initial concentration of 500 cells/mL. Flasks were then incubated 

under constant illumination at 22–27.6°C for 4 days. Following the 4-day incubation period, the 

algal biomass in each flask was estimated based on the concentration of Chlorophyll a.

The pH of the test solutions during the test was 7.6–7.9. 

Statistics: None specified

RESULTS

Endpoint value(s): Note that analytical monitoring was not conducted and nominal 

concentrations (1–1000 mg/L) were many orders of magnitude greater than the water solubility 

limit (experimentally determined to be < 0.01 mg/L)1for this compound. Therefore, it is not 

possible to interpret reported toxicity values.

96-hour EC50 (Chlorophyll a concentrations) > 1000 mg/L, based on nominal concentrations. 

Results remarks: The Chlorophyll a concentrations in all of the test solutions, except for 

4.6 mg/L, were higher than the mean concentration in the controls. Page 2 of the test report, 

which included studies for fish, Daphnia, and green algae, provided a general comment that the 

test substance “appeared to be not wholly soluble at concentrations greater than” 10 mg/L.

CONCLUSIONS

The 96-hr EC50 is > 1000 mg/L (nominal concentration) based on Chlorophyll a concentrations.

STUDY RELIABILITY 

[3] Not reliable. Non-GLP study. Test method/guideline followed and statistical methods used 

were not specified, analytical monitoring was not conducted, water chemistry parameters were 

not provided (hardness), control treatment and control responses were not specified, and the test 

concentrations exceeded the water solubility limit of the test substance (experimentally 

determined to be < 0.01 mg/L)1by many orders of magnitude. 

 

1 The water solubility limit of CASRN 118-82-1 was experimentally determined to be < 0.01 mg/L by SafePharm 

Laboratories (study submitted to EPA by Albemarle Corporation on May 12, 2006, available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2005-0033-0163 as of December 4, 2013; “4,4’-

Methylene bis(2,6-di-tert-butylphenol): Determination of Water Solubility and Partition Coefficient”, SPL Project 

Number 1193/011, study completed on July 18, 2005).
Draft HPV1/HPV2 Test Rule Data Adequacy Review (December 2013) CASRN 118-82-1

33

REFERENCE

Mattock, S.D. and Girling, A.E. 1988. IONOX 220: Acute Toxicity (Salmo gairdneri, Daphnia 

magna and Selenastrum capricornutum) and n-octanol/water partition coefficient. Sittingbourne 

Research Centre, Shell Research Limited. Submitted by Shell Oil Co. to EPA in 1992 as part of 

a TSCA 8(e) submission (EPA Doc. No. 88-920003444, Fiche No. OTS0537403) entitled 

“Initial submission: acute toxicity of IONOX 220 in Salmo gairdneri, Daphnia magna and

Selenastrum capricornutum and n-octanol/water partition coefficient w-letter dated 053092”.
Draft HPV1/HPV2 Test Rule Data Adequacy Review (December 2013) CASRN 118-82-1

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C-5. Acute Toxicity

Summary of endpoint: Adequate

Study 1: Acute oral toxicity study in rats (Ethyl Petroleum Additives International, 1994)

Title: Acute Oral Toxicity Study

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Phenol, 4,4’-methylenebis[2,6-bis(1,1-dimethylethyl)-, CASRN 118-82-1

Remarks: Purity not provided

METHOD

Method/guideline followed: OECD 401

Test type: Acute Oral - Rat

GLP compliant?: Yes

Year study performed: 1994

Test species: Rats (strain not specified)

Number of animals/sex/dose: 5

Route of administration: Oral gavage

Vehicle: Not specified

Doses/concentrations tested: 5000 mg/kg-bw

Statistical methods used: None specified

Test method/conditions remarks: Ten rats (5/sex) were administered a single limit dose of 

5000 mg/kg-bw by gavage, according to OECD TG 401. Animals were observed for 14 days 

post-dosing. 

RESULTS

Endpoint value(s): LD50 > 5000 mg/kg-bw
Draft HPV1/HPV2 Test Rule Data Adequacy Review (December 2013) CASRN 118-82-1

35

Results remarks: No animals died. Signs of toxicity included diarrhea and yellow urine. No 

visible lesions were noted. No additional details were available from this study summary.

CONCLUSIONS

The oral LD50 is > 5000 mg/kg-bw.

STUDY RELIABILITY

[2] Reliable with restrictions. Although this is an OECD guideline study, important study details 

were missing from the study summary, including test substance purity and use of a vehicle. Data 

are considered adequate based on weight of evidence.

REFERENCE

Ethyl Petroleum Additives International. 1994. Study No. PH-402-ET-005-94 for Ethyl 

Corporation, Pharmakon Research International, Waverly, PA. (Cited in IUCLID Dossier, 

European Chemical Bureau, 2000). Submitted by Albemarle Corporation. Robust summary 

located at http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2005-0033-0181

(on p. 5 of the document “End_Point_D – Mammalian_Toxicity_Acute.pdf”) as of December 4, 

2013.
Draft HPV1/HPV2 Test Rule Data Adequacy Review (December 2013) CASRN 118-82-1

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Study 2: Acute oral toxicity study in rats (Carter and Dharmarajan, 1976)

Title: A Report on the Acute Toxicity of AN-702

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Phenol, 4,4’-methylenebis[2,6-bis(1,1-dimethylethyl)-, CASRN 118-82-1

Remarks: Purity not provided

METHOD

Method/guideline followed: Not indicated

Test type: Acute Oral - Rat

GLP compliant?: Not indicated

Year study performed: 1976

Test species: Sprague-Dawley rats

Number of animals/sex/dose: 10 males/dose

Route of administration: Oral gavage

Vehicle: Peanut Oil

Doses/concentrations tested: 3000 mg/kg-bw

Statistical methods used: None specified

Test method/conditions remarks: Ten male Sprague-Dawley rats (weight of 200–300 g) were 

administered by gavage a single dose of 3000 mg/kg-bw of the test substance as a 10% w/v 

solution in peanut oil and were observed for 14 days following dosing. At termination, the 

survivors were sacrificed and necropsied.

RESULTS

Endpoint value(s): LD50 > 3000 mg/kg-bw

Results remarks: No animals died or exhibited any signs of toxicity. Extensive dark areas 

were observed on the liver. Treatment with the test material did not affect body weight gain 

during the observation period. No additional details were available from this study summary.
Draft HPV1/HPV2 Test Rule Data Adequacy Review (December 2013) CASRN 118-82-1

37

CONCLUSIONS

The oral LD50 is > 3000 mg/kg-bw.

STUDY RELIABILITY

[2] Reliable with restrictions. Important study details were missing from the study summary,

including test substance purity, method/guideline followed, and GLP compliance. Data are 

considered adequate based on weight of evidence.

REFERENCE

Carter, G. and V. Dharmarajan, “A Report on the Acute Toxicity of AN-702,” Tulane University 

School of Medicine, for Ethyl Corporation, 1976. Submitted by Albemarle Corporation. Study 

report available online at http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT2005-0033-0181 (on pp. 3, 7-13 of the document “End_Point_D –

Mammalian_Toxicity_Acute.pdf”) as of December 4, 2013.
Draft HPV1/HPV2 Test Rule Data Adequacy Review (December 2013) CASRN 118-82-1

38

Study 3: Acute oral toxicity study in guinea pigs (Haring and Robbins, 1965)

Title: Acute Oral Toxicity of SCH 12150 in Guinea Pigs

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Phenol, 4,4’-methylenebis[2,6-bis(1,1-dimethylethyl)-, CASRN 118-82-1; referred to 

as SCH 12150 in study report; Batch No. 3004-95

Remarks: Purity not provided

METHOD

Method/guideline followed: Not indicated

Test type: Acute Oral - Guinea Pig

GLP compliant?: N/A (Pre-GLP)

Year study performed: 1965

Test species: Albino guinea pigs; from Gordon’s Biological Supply Company; average weight 

of 282 g

Number of animals/sex/dose: 6 males/group

Route of administration: Oral gavage

Vehicle: Methyl Cellulose

Doses/concentrations tested: 0 (control), 1000, 1780, 3170, 5630, or 10,000 mg/kg-bw

Statistical methods used: None specified

Test method/conditions remarks: Single doses of the test substance were administered by 

gavage as a 20% suspension in 0.5% methyl cellulose to groups of 6 male guinea pigs. Animals 

were observed for 14 days following dosing. Following the observation period, all surviving 

animals were autopsied and gross observations were recorded. Liver sections were retained for 

microscopic examination. Other tissues which showed gross pathology were preserved.

RESULTS

Endpoint value(s): LD50 > 10,000 mg/kg-bw
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Results remarks: Two deaths occurred in treated animals: one in the 1000 mg/kg-bw group 

(day 10) and one in the highest-dose group (day 12). Cause of death in both cases was reported 

as “secondary infection”. One control animal died on day 11, and pneumonia was present on 

autopsy. Pneumonia was found randomly in all groups. No treatment-related clinical signs were 

observed. Reduced weight gain (approximately 16% compared to control; statistical significance 

not reported) occurred in the highest-dose group. Absolute liver weights were not significantly 

different in treated versus control animals. Portions of the liver from five animals in each dose 

group, as well as any gross lesions, were processed for microscopic examination. No significant 

treatment-related changes were seen in microscopic examination of the liver; cellular fat and 

liver glycogen content appeared normal. Results of a follow-up study conducted in 1966 were 

included in this study report. The follow-up study was a repeat of the original experiment using 

two groups of animals at the high-dose level. One group was given chlortetracycline in the 

drinking water to ensure that endemic infection was not present. There were no differences in 

the two groups with respect to infection or body weight gain.

CONCLUSIONS

The oral LD50 is > 10,000 mg/kg-bw.

STUDY RELIABILITY

[2] Reliable with restrictions. Important study details missing from the study summary included 

test substance purity, method/guideline followed, statistical methods used and detailed numerical 

findings. Data are considered adequate based on weight of evidence.

REFERENCE

Haring, G.D. and G.R. Robbins, “Acute Oral Toxicity of SCH 12150 In Guinea Pigs,” Schering 

Laboratories, 1965. Submitted by Albemarle Corporation. Study report available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2005-0033-0181 (on pp. 4-5, 

41-55 of the document “End_Point_D – Mammalian_Toxicity_Acute.pdf”) as of December 4, 

2013.
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Study 4: Acute dermal toxicity study in rabbits (Carter and Dharmarajan, 1976)

Title: A Report on the Acute Toxicity of AN-702

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Phenol, 4,4’-methylenebis[2,6-bis(1,1-dimethylethyl)-, CASRN 118-82-1; referred to 

as AN-702 in study report

Remarks: Purity not provided

METHOD

Method/guideline followed: Not specified

Test type: Acute Dermal - Rabbit

GLP compliant?: Not specified

Year study performed: 1976

Test species: New Zealand White rabbits; 2.3–3.0 kg

Number of animals/sex/dose: 4 (sex not specified)

Route of administration: Dermal, occluded

Vehicle: Peanut Oil

Doses/concentrations tested: 1500 mg/kg-bw

Statistical methods used: No information available

Test method/conditions remarks: The test substance (10% w/v solution in peanut oil) was 

injected under a plastic sleeve wrapped around the clipped trunks of four rabbits (two with intact 

skin and two with abraded skin) for an exposure period of 24 hours. Evaluations of erythema 

and edema were made 24 and 72 hours after application of the test material. After removal of 

the sleeves, the animals were observed for 14 days.

RESULTS

Endpoint value(s): LD50 > 1500 mg/kg-bw
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Results remarks: No mortalities occurred. Well defined erythema was observed on the first 

day of observation.

CONCLUSIONS

The dermal LD50 is > 1500 mg/kg-bw.

STUDY RELIABILITY

[2] Reliable with restrictions. Important study details that were missing from the study summary 

included test substance purity, methods/guidelines followed, GLP compliance, sex of test 

animals, statistical methods, detailed findings, and numerical results. Data are considered 

adequate based on weight of evidence.

REFERENCE

Carter, G. and V. Dharmarajan, “A Report on the Acute Toxicity of AN-702,” Tulane University 

School of Medicine, for Ethyl Corporation, 1976. Submitted by Albemarle Corporation. Study 

summary available online at http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT2005-0033-0181 (on pp. 7-13 of the document “End_Point_D –

Mammalian_Toxicity_Acute.pdf”) as of December 4, 2013.
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Study 5: Acute oral toxicity study in rats (Cleveland and Witherup, 1962)

Title: The Immediate Oral Toxicity of 4,4'-Methylenebis(2,6-Di-tert-Butylphenol)

This study is: Adequate Not Adequate

The post-dosing observation period was less than 14 days. Also, important study details were 

missing from the study summary, including test substance purity, method/guideline followed, 

and statistical methods used.

TEST SUBSTANCE 

Identity: Phenol, 4,4’-methylenebis[2,6-bis(1,1-dimethylethyl)-, CASRN 118-82-1

Remarks: Purity not provided

METHOD

Method/guideline followed: Not indicated

Test type: Acute Oral - Rat

GLP compliant?: N/A (Pre-GLP)

Year study performed: 1962 

Test species: Rats (strain not specified)

Number of animals/sex/dose: 10 males/dose (definitive study); 1–2 males/dose (preliminary 

study)

Route of administration: Oral gavage

Vehicle: Peanut Oil

Doses/concentrations tested: 180, 1000, 1600, 2400, 3200, 4700, or 10,000 mg/kg-bw 

(preliminary study); 3200, 4700, 7000, 10,000, 16,000, or 24,000 mg/kg-bw (definitive study)

Statistical methods used: None specified

Test method/conditions remarks: In a preliminary study, male rats (10–14 weeks old) were 

administered the test substance as a 10% suspension in peanut oil by gavage at doses ranging 

from 180 to 10,000 mg/kg-bw. In the definitive study, the rats received various amounts of a 

suspension containing 30 g of the test substance diluted to 100 mL with peanut oil; this provided 

doses of the test substance ranging from 3200 to 24,000 mg/kg-bw. At the end of the
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observation period, animals were necropsied and microscopic examination of the viscera was 

conducted.

RESULTS

Endpoint value(s): LD50 > 24,000 mg/kg-bw

Results remarks: The duration of the observation period was not specified, but it appears to 

have been 4 weeks in the preliminary study and 5 days in the definitive study (from Tables in the 

report). No animals died in either study. No signs of intoxication were observed, and only mild 

transient diarrhea was observed in animals administered the higher doses (not specified). 

Postmortem examination revealed no gross pathology findings; no histopathological changes to 

the viscera were observed. No additional details were available from this study summary.

CONCLUSIONS

The oral LD50 is > 24,000 mg/kg-bw. 

STUDY RELIABILITY

[3] Not reliable. The post-dosing observation period was less than 14 days. Also, important 

study details were missing from the study summary, including test substance purity, 

method/guideline followed, and statistical methods used.

REFERENCE

Cleveland, F.P. and S. Witherup, “The Immediate Oral Toxicity of 4,4'-Methylenebis(2,6-Ditert-Butylphenol),” The Kettering Laboratory in the Department of Preventive Medicine and 

Industrial Health, College of Medicine, University of Cincinnati, Cincinnati, Ohio, for Ethyl 

Corporation, 1962. (Submitted as part of Food Additive Petition No. 4B 1469 and Food 

Additive Petition 1397, Food and Drug Administration). Study report available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2005-0033-0181 (on pp. 3, 

14-17 of the document “End_Point_D – Mammalian_Toxicity_Acute.pdf”) as December 4, 

2013.
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Study 6: Acute oral toxicity study in dogs (Vymetal and Robbins, 1965)

Title: Acute Oral Toxicity of SCH 12150 in Dogs

This study is: Adequate Not Adequate

The number of animals per group (one male and one female) is not sufficient to evaluate acute 

toxicity. Furthermore, statistical methods could not be used because of the small number of 

animals per group. Important study details missing from the study summary included test 

substance purity and method/guideline followed. 

TEST SUBSTANCE 

Identity: Phenol, 4,4’-methylenebis[2,6-bis(1,1-dimethylethyl)-, CASRN 118-82-1; referred to 

as SCH 12150 in study report; Batch No. 3004-96; supplied as a white powder which was not 

soluble in water

Remarks: Purity not provided

METHOD

Method/guideline followed: Not indicated

Test type: Acute Oral - Dogs

GLP compliant?: N/A (Pre-GLP)

Year study performed: 1965

Test species: Beagle Dogs; 8–16 months old; obtained from Ubrian Farm Kennels

Number of animals/sex/dose: 1

Route of administration: Oral - capsule

Vehicle: No

Doses/concentrations tested: 0 (control), 100, 251, 631, 1585, and 2512 mg/kg-bw

Statistical methods used: Not applicable given the use of one dog/sex in each dose group.

Test method/conditions remarks: Groups of one male and one female beagle were 

administered a single dose of the test substance in gelatin capsules via the oral route and were 

observed for 14 days post-administration. Endpoints monitored included clinical signs, food 

consumption, body weight, as well as hematology, clinical chemistry, and urinalysis. At 
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termination, the dogs were necropsied and the liver, kidneys, heart, gall bladder, pancreas, 

adrenals, and pituitary were prepared for microscopic examination.

RESULTS

Endpoint value(s): LD50 > 2512 mg/kg-bw

Results remarks: There were no adverse clinical signs during the study. There were no 

treatment-related changes in any biochemical parameters measured (blood glucose, BUN, SGPT

[ALT], SGOT [AST], alkaline phosphatase, icteric index) with the exception of an insignificant 

temporary rise in BUN in the male dog at the 631 mg/kg dose level. No treatment-related 

hematological effects were noted (hematocrit, hemoglobin, total WBC and differential counts, 

prothrombin time and platelet count). Urinalysis revealed no treatment-related findings. No 

significant pathology was found at necropsy and microscopic examination of the selected organs. 

CONCLUSIONS

The oral LD50 is > 2512 mg/kg-bw.

STUDY RELIABILITY

[3] Not reliable. The number of animals per group (one male and one female) is not sufficient to 

evaluate acute toxicity. Furthermore, statistical methods could not be used because of the small 

number of animals per group. Important study details missing from the study summary included 

test substance purity and method/guideline followed. 

REFERENCE

Vymetal, R. and G.R. Robbins, “Acute Oral Toxicity of SCH 12150 in Dogs,” Schering 

Laboratories, 1965. Submitted by Albemarle Corporation. Study report available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2005-0033-0181 (on pp. 4, 

18-40 of the document “End_Point_D – Mammalian_Toxicity_Acute.pdf”) as of December 4, 

2013.
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C-6. Repeated-Dose Toxicity

Summary of endpoint: Adequate

Study 1: 2-Year repeated-dose oral toxicity study in dogs (Jolley et al., 1969)

Title: The effects exerted upon beagle dogs during a period of two years by the introduction of 

4,4'-methylenebis (2,6-ditertiary butylphenol) into their daily diets

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Phenol, 4,4’-methylenebis[2,6-bis(1,1-dimethylethyl)-, CASRN 118-82-1

Remarks: 99% pure; Sample No. 718-65; Appearance: fine, pale yellow colored, crystalline 

solid.

METHOD

Method/guideline followed: Not specified

Test type: 2-Year Oral Repeated-Dose Toxicity 

GLP compliant?: N/A (pre-GLP)

Year study performed: 1969

Test species: Beagle dogs; obtained from Atlantic Kennels of Zionville, Indiana; 6.5–

8.5 months old at study initiation

Number of animals/sex/dose: 3/sex in each of the treated groups, 6/sex in the control group

Route of administration: Oral - diet

Vehicle: Corn oil (prior to incorporation into diet at a level of 5% corn oil by weight)

Exposure duration: 2 years

Doses/concentrations tested: 0, 15, 60, 100, 500, and 3000 ppm (0, 0.395, 1.40, 2.52, 14.0, and 

96.9 mg/kg-bw/day, respectively, for males and 0, 0.372, 1.74, 2.57, 16.3, and 99.7 mg/kgbw/day, respectively, for females; estimated by the investigators based on mean body weight and 

food consumption throughout the study) 

Statistical methods used: None specified
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Test method/conditions remarks: Beagle dogs were fed diets containing the test substance at 

various concentrations for a period of 2 years. Dogs were observed daily for clinical signs of 

toxicity. Individual body weights and food consumption were recorded weekly. Analyses of 

hematological (packed cell volume, hemoglobin concentration, total leukocyte count, percentage 

of differentiated leukocytes, and prothrombin time) and clinical chemistry parameters (total 

serum cholesterol, total lipids, alkaline phosphatase, and serum glutamic-pyruvic transaminase 

[SGPT]) as well as urinalysis (color, appearance, specific gravity, pH, albumin, sugar, acetone, 

and microscopic assessment) were conducted prior to treatment (baseline; two measurements per 

dog), after 2 weeks, and after 1, 6, 12, 18, and 24 months of feeding on the test diets. 

Measurements of alkaline phosphatase, SGPT, and prothrombin time were not performed at the 

2-week feeding period but were performed at all other time points. Additional tests (serum 

bilirubin, blood urea nitrogen, bromsulfalein retention, calcium, inorganic phosphate, total 

protein, albumin, uric acid, glucose, and glutaminic-oxalacetic transaminase [SGOT]) were 

conducted to assess the possible effects on liver function at 12, 18, and/or 24 months. At 

termination, all dogs were subjected to gross examination and the major organs (liver, heart, 

lungs, kidneys, spleen, brain, prostate, testes, ovaries, pituitary, adrenals, and thyroids) were 

weighed. These organs, as well as the aorta, gall bladder, stomach, duodenum-pylorus, small 

intestine (ileum), large intestine (colon), mesenteric lymph nodes, urinary bladder, uterus, 

pancreas, thymus, spinal cord, eye, bone marrow, bone, skeletal muscle, and skin were processed 

for microscopic examination. 

Examination of feces at 6 months revealed hookworms, roundworms, and whip-worms present. 

Although not all of the dogs had these parasites, all were treated with the same worming 

treatment. 

RESULTS

Endpoint value(s): LOAEL = 500 ppm (14.0 and 16.3 mg/kg-bw/day for males and females, 

respectively) based on reduced body weight gains (in the absence of a treatment-related effect on 

food consumption), increased relative liver weights, and histopathological changes in the liver 

(increased fat in hepatic cells [males] and increased prominence of Glisson’s capsule [females]); 

NOAEL = 100 ppm (2.52 and 2.57 mg/kg-bw/day for males and females, respectively).

Results remarks: Note that the study authors did not report group means or percent change 

from baseline (either for individual dogs or for treatment groups). Therefore, quantitative results 

reported as percent change from baseline or percent change from control were calculated by the 

EPA reviewers for this review. If baseline data were reported, the percent change relative to the 

baseline (mean of the two baseline values for each dog) for individual dogs was determined, and 

then group means for the percent change were calculated for each treatment group. If baseline 

data were not reported, group means for treatment groups were calculated and compared to 

control means for each time point.

At 3000 ppm, one male died during week 40 and another was sacrificed moribund during week 

57. Both deaths were considered to be due to liver cirrhosis resulting from dietary administration 

of the test substance. No deaths occurred at dietary concentrations of 500 ppm and lower in 

males, and no deaths occurred in females.
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Dogs from all groups, including the control group, exhibited one or more clinical signs including 

patches of alopecia, enlarged nipples and/or mammary glands, excess of tartar, undershot jaw, 

and tonsillitis. The study authors did not consider any of these signs to be treatment-related; 

however, no incidence data were provided to support this claim. 

Body weight loss was observed in two dogs. After 2 years of treatment, body weight of the 

surviving male in the 3000 ppm group was 22% lower and body weight of one female in the 

500 ppm was 5.3% lower, compared to body weights at the start of treatment. Body weight gain 

in males was decreased by 46% in the 500 ppm group, compared to the control group. Body 

weight gain in females in the 500 and 3000 ppm groups was decreased by 54 and 66%, 

respectively, relative to female controls. No adverse treatment-related effects on body weight 

gain were apparent in males or females at dietary concentrations of 15, 60, or 100 ppm. Food 

consumption did not appear to be affected by treatment. 

Evaluation of hematological parameters showed decreased packed cell volume and hemoglobin 

concentration in males in the 3000 ppm group, compared to baseline, but not at lower dietary 

concentrations or in any group for females. Packed cell volume in males was decreased by 

approximately 26 (n=2), 34 (n=1), 33 (n=1), and 33% (n=1) after 12, 14 (early death), 18, and 

24 months of treatment, respectively. Hemoglobin concentration in males was decreased by 

approximately 35 (n=2), 41 (n=1), 38 (n=1), and 31% (n=1) after 12, 14 (early death), 18, and 

24 months, respectively. Due to early deaths of 2 out of 3 males, all measurements after 

12 months were for only 1 dog; therefore, it is difficult to draw conclusions regarding the 

potential for treatment-related effects on packed cell volume and hemoglobin concentration. 

Study authors stated that these hematological changes were not clinically significant based on the 

absence of reticulocytosis. No treatment-related effects were observed on other hematologic 

parameters. Urinalysis revealed no treatment-related findings.

Serum cholesterol was decreased, compared to baseline, throughout the treatment period in 

males and females in the 3000 ppm group. Decreases were 47, 53, 72, 71, 86, and 66% in males 

and 38, 54, 61, 60, 76, and 66% in females after 2 weeks and after 1, 6, 12, 18, and 24 months of 

treatment, respectively. Other treatment groups displayed more modest decreases in serum 

cholesterol; these changes were not consistently observed throughout the study period and their 

relationship to treatment was unclear. Total serum lipids were decreased (relative to baseline) in 

males and females in the 3000 ppm group, although changes were not consistent in males over 

the 2-year treatment period. In males, lipids were decreased by 33, 41, 21, 43, and 9% after 1, 6, 

12, 18, and 24 months of treatment, respectively. In females, lipids were decreased by 32, 32, 

36, and 39% after 1, 6, 18, and 24 months of treatment, respectively, with no decrease at 

12 months. Total serum lipids were not affected in any other treatment group. 

Study authors stated that a “questionable lowering” of serum total protein was observed in dogs 

in the 3000 ppm group after 24 months of treatment (not measured at earlier times), compared to 

controls (no baseline measurement reported). In the one male dog surviving until 24 months, 

protein was decreased by 13%. In female dogs, there was no apparent treatment-related effect on 

serum total protein, with a decrease of only 8% at 24 months. There were variations in total 
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protein values among animals from other groups but they were very slight and did not show a 

dose-response relationship. 

Clinically significant increases (≥ 3-fold) in serum alkaline phosphatase levels were observed in 

males and females in the 3000 ppm group, relative to baseline, at several time points. In males, 

alkaline phosphatase levels were increased by 4.1-, 6.9-, and 5.2-fold at 6, 12, and 24 months, 

respectively, with a smaller increase of 2.5-fold after 1 and 18 months of exposure. In females, 

levels were increased by 3.0-, 4.3-, and 3.7-fold after 6, 12, and 18 months of exposure, 

respectively, with smaller increases of 1.7- and 2.2-fold after 1 and 24 months of exposure. In 

the 500 ppm group, no clinically significant increases in alkaline phosphatase were observed; the 

highest increases were 1.6-fold in males at 24 months and 2.3-fold in females at 12 months. 

Alkaline phosphatase levels were not increased at dietary concentrations ≤ 100 ppm. Increases 

in mean SGPT levels were observed at all concentrations at varying time points throughout the 

2-year study period; however, these increases showed no clear, consistent dose-response 

relationship and were less than 3-fold compared to baseline. 

No treatment-related effects were observed for serum bilirubin, blood urea nitrogen, 

bromsulfalein retention, calcium, inorganic phosphate, albumin, uric acid, glucose, or SGOT.

At the end of treatment (upon early death or after 24 months on treatment), liver weights were 

increased in dogs fed diets containing 500 and 3000 ppm compared to controls. In the 500 ppm 

group, relative liver weights were increased by 32 and 28% in males and females, respectively, 

while in the 3000 ppm group, relative liver weights were increased by 92 and 61% in males and 

females, respectively. In the 500 ppm group, absolute liver weights were increased by 20 and 

7% in males and females, respectively; similar increases were observed at lower doses and 

absolute liver weights did not show a dose-response relationship at dietary concentrations 

≤ 500 ppm. In the 3000 ppm group, absolute liver weights were increased by 53 and 31% in 

males and females, respectively. The study authors considered the increase in relative liver 

weight in the 500 ppm group to be of questionable toxicological significance. However, since 

histopathological findings in the liver also were observed in the 500 ppm group, the EPA 

reviewers consider the increase in relative liver weight to be toxicologically significant. 

Increased absolute and relative kidney weights were observed in male dogs in the 3000 ppm 

group (37% increase in absolute weight and 72% increase in relative weight, compared to 

controls), with a smaller increase in females in the 3000 ppm group (10% increase in absolute 

kidney weight and 31% increase in relative kidney weight, compared to controls). The 

toxicological significance of this finding is questionable, as no treatment-related changes in renal 

function or histopathological effects in the kidney were observed. No consistent dose-related 

changes were observed for weights of other organs. 

Post-mortem examination of all dogs receiving the 3000 ppm diet revealed marked cirrhosis of 

the liver and hyperplasia of the gall bladder mucosa. In the 500 ppm group, histopathological 

changes in the liver were observed in one male dog (increased fat in the hepatic cells) and one 

female dog (increased prominence of the Glisson’s capsule). Study authors described severity of 

these findings as “slight”. These changes in the liver were not observed in control animals. In 

the kidney, vacuolation of the epithelial cells of the Loop of Henle was noted in one female dog 

at 500 ppm and two female dogs at 3000 ppm, as well as one female dog in the control group; 
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the toxicological significance of this finding is uncertain. There were no treatment-related 

histopathological findings at concentrations less than 500 ppm.

CONCLUSIONS

The LOAEL is 500 ppm (14.0 and 16.3 mg/kg-bw/day for males and females, respectively) 

based on reduced body weight gains (in the absence of a treatment-related effect on food 

consumption), increased relative liver weights, and histopathological changes in the liver 

(increased fat in hepatic cells [males] and increased prominence of Glisson’s capsule [females]). 

The NOAEL is 100 ppm (2.52 and 2.57 mg/kg-bw/day for males and females, respectively). 

STUDY RELIABILITY

[2] Reliable with restrictions. No information on statistical analyses was reported (although 

some effects were reported as “significant”) and a small number of animals (3 dogs/sex/group) 

was used. The full study, although not a guideline study, was well documented and adequate 

based on the information provided.

REFERENCES

Jolley, W.P., Stemmer, K.L., Grande, F., Richmond, J., and Pfitzer, E.A. 1969. The effects 

exerted upon beagle dogs during a period of two years by the introduction of 4,4'-methylenebis 

(2,6-ditertiary butylphenol) into their daily diets. Kettering Laboratory, University of Cincinnati, 

OH. Submitted to EPA in 1992 as part of a TSCA 8(e) submission (EPA Doc. No. 88-

920001353S, Fiche No. OTS0536073) entitled “Initial sub: the effect exerted upon beagle dogs 

during a period of 2-yrs by the introduction of 4,4'-methylenebis (2,6-ditertaryl butylphenol) into 

their daily diets (sanitized)”.
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Study 2: 2-Year repeated-dose oral toxicity study in dogs (Jolley et al., 1975)

Title: The effects upon beagle dogs following the ingestion of 4,4'-methylenebis (2,6-ditertiary 

butylphenol) for two years including the changes after an additional seven months on control diet

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Phenol, 4,4’-methylenebis[2,6-bis(1,1-dimethylethyl)-, CASRN 118-82-1

Remarks: 99% pure; Sample No. 1519-66; Appearance: fine, pale yellow-colored, crystalline 

solid.

METHOD

Method/guideline followed: Not specified

Test type: 2-Year Oral Repeated-Dose Toxicity 

GLP compliant?: Not specified

Year study performed: 1975

Test species: Beagle dogs; obtained from American Animal Industries Inc., Indiana; 10–

11 months old at study initiation. 

Number of animals/sex/dose: 6

Route of administration: Oral - diet

Vehicle: Corn oil (prior to incorporation into diet at a level of 5% corn oil by weight)

Exposure duration: Up to 27 months; at one dose level (35 mg/kg-bw/day), a 27-month dosing 

period was followed by 7 months on control diet 

Doses/concentrations tested: 0, 7, 35, and 75 mg/kg-bw/day; estimated by the investigators 

based on mean food consumption and body weight throughout the study.

Statistical methods used: None specified

Test method/conditions remarks: Beagle dogs (6/sex/dose) were fed diets containing the test 

substance in corn oil at 0, 7, 35, or 75 mg/kg-bw/day for periods up to 27 months. After 

6 months on the test diet, 3 animals/sex/dose were sacrificed. The group of animals (3/sex) that 

was dosed at 35 mg/kg-bw/day for 27 months was then shifted to the control diet for 7 months 

and sacrificed at 34 months. The remaining animals (3/sex/dose) were sacrificed after 27 months 
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at 0 and 7 mg/kg-bw/day and after 25 months at 75 mg/kg-bw/day. Dogs were observed daily 

for clinical signs of toxicity. Individual body weights and food consumption were recorded 

weekly. 

Analyses of hematological (packed cell volume, hemoglobin concentration, total leukocyte 

count, percentage of differentiated leukocytes, and prothrombin time) and clinical chemistry 

parameters (total serum cholesterol, alkaline phosphatase, SGPT, blood urea nitrogen, fasting 

blood sugar, and total protein) as well as urinalysis (color, appearance, specific gravity, pH, 

albumin, sugar and microscopic appearance) were conducted prior to treatment (baseline), after 

2 weeks, and after 1, 3, 6, 12, 18, 24, and/or 27 months of feeding on the test diets; the 

hematological parameters were also evaluated during recovery at 33 months and the above 

clinical chemistry parameters were evaluated during recovery at 27.5, 28, 28.75, 33, and/or 

34 months. Prior to the start of treatment and at 24 months, the following clinical chemistry 

parameters were assessed: total free serum lipids, serum sodium, serum potassium, thymol 

turbidity, and protein bound iodine. Additional clinical chemistry analyses (albumin, calcium, 

inorganic phosphate, SGOT, serum bilirubin, uric acid, and bromosulfalein retention) were 

conducted at 12, 18, 24, 27, 27.5, 28, 28.75, 33, and/or 34 months. At termination, all dogs were 

subjected to autopsy, the viscera were examined for gross pathological changes, and the major 

organs were weighed and processed for microscopic examination. The following organs were 

weighed individually: liver, heart, lungs, kidneys, spleen, brain, gonads, pituitary, adrenals, and 

thyroids. The following organs and tissues were examined microscopically: heart, lung, spleen, 

gall bladder, liver, kidney, stomach, two levels of small intestine, large intestine, pancreas, brain 

(cortex, brain stem, cerebellum, spinal cord), pituitary, thyroid gland, adrenal gland, gonads, 

uterus, mammary glands, aorta, bone, lens of the eyes, urinary bladder, lymph nodes, skin, 

muscle, and fat.

RESULTS

Endpoint value(s): LOAEL = 35 mg/kg-bw/day based on clinically significant increases in 

serum alkaline phosphatase activity and decreases in serum cholesterol levels, as well as 

histopathological changes in the liver (cirrhosis [with abundance of fibrous tissue, duplication of 

bile ducts, and cellular degeneration], marked lymphocytic cellular infiltration, dark brown 

pigmentation in Glisson’s capsule, and vacuolation of hepatocytes); NOAEL = 7 mg/kg-bw/day. 

Results remarks: Note that the study authors did not report group means or percent change 

from baseline (either for individual dogs or for treatment groups). Therefore, quantitative results 

reported as percent change from baseline or percent change from control were calculated by the 

EPA reviewers for this review. If baseline data were reported, the percent change relative to the 

baseline (mean of the two or three baseline values for each dog) for individual dogs was 

determined, and then group means for the percent change were calculated for each treatment 

group. If baseline data were not reported, group means for treatment groups were calculated and 

compared to control means for each time-point.

One female dog from the 75 mg/kg-bw/day group developed marked ascites, was moribund, and 

was sacrificed during the 14th month. Necropsy of this female revealed marked cirrhosis of the 

liver and congestion of the spleen. A male dog from the same group died during the 21st month 
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of treatment. The death of the male was considered to be due to intra-intestinal hemorrhages and

marked cirrhosis of the liver resulting from treatment with the test substance. No clinical signs 

of toxicity were observed in surviving dogs.

Weight loss was observed in the one male dog of the 75 mg/kg-bw/day group that died. No 

other effects on body weight were observed. No clear treatment-related effects were observed on 

growth rate (based on body weight gain) or food consumption. No treatment-related effects on 

hematology or urinalysis parameters were apparent.

Clinically significant increases (≥ 3-fold) in serum alkaline phosphatase levels, compared to 

baseline levels, were observed in male and female dogs in the 35 and 75 mg/kg-bw/day groups, 

with greater increases observed in females compared to males. In males in the 35 mg/kg-bw/day 

group, alkaline phosphatase levels were increased by approximately 3.3- and 3.4-fold after 6 and 

12 months, respectively, with subclinical increases of 2.5-, 2.6-, and 2.8-fold at 3, 18, and 

24 months, respectively. In males in the 75 mg/kg-bw/day group, alkaline phosphatase was 

increased by 3.6-, 4.6-, 3.8-, and 3.7-fold after 3, 6, 12, and 24 months, respectively, with 

subclinical increases of 2.7-, 2.6-, and 2.9-fold after 0.5, 1, and 18 months of treatment, 

respectively. In females in the 35 mg/kg-bw/day group, alkaline phosphatase was increased by 

4.8-, 4.3-, 7.3-, 9.0-, 5.9-, and 7.6-fold after 0.5, 3, 6, 12, 18, and 24 months of treatment, 

respectively; increases after 1 month of treatment were subclinical (2.6-fold increase). In 

females in the 75 mg/kg-bw/day group, alkaline phosphatase was increased by 3.0-, 4.4-, 5.9-, 

8.9-, 4.6-, and 4.6-fold after 0.5, 3, 6, 12, 18, and 24 months of treatment, respectively; increases 

after 1 month of treatment were subclinical (2.4-fold increase). Serum alkaline phosphatase in 

males and females in the 35 mg/kg-bw/day group returned to approximately baseline levels 

within 1.75 months into the recovery period. 

No consistent treatment-related, clinically significant increases (≥ 3-fold) in SGPT levels, 

relative to baseline, were observed. In the 75 mg/kg-bw/day group, SGPT levels were increased 

by 3.8-fold in both males and females at the 12-month time-point only. Measurements of SGOT 

at 12, 18, and 24 months showed no consistent treatment-related pattern of change, relative to 

controls (baseline data not reported). The two dogs that died at 75 mg/kg-bw/day showed 

increases in SGOT at 12 months of 3.6-fold (male) and 3.0-fold (female) relative to the control 

mean. Any increases in SGOT observed in other animals did not exceed 2-fold (relative to the 

control mean) at any time point.

Decreases (relative to baseline) in serum total cholesterol levels were observed in dogs in the 

35 mg/kg-bw/day group from 6 through 24 months and in the 75 mg/kg-bw/day group from 

3 through 24 months. During months 6–24 of treatment with 35 mg/kg-bw/day, decreases in 

serum total cholesterol ranged from 43 to 60% in male dogs and from 35 to 42% in female dogs; 

decreases did not exhibit duration-dependence in the 35 mg/kg-bw/day group. In male dogs in 

the 75 mg/kg-bw/day group, cholesterol was decreased by 52% after 3 months, with durationdependent decreases up to 79% of pre-treatment levels at 24 months. In female dogs in the 

75 mg/kg-bw/day group, cholesterol was decreased by 37% after 3 months, with decreases 

ranging from 61 to 75% during months 6–24, with no duration-dependence. Serum total 

cholesterol in dogs in the 35 mg/kg-bw/day group returned to pre-treatment levels within 

1.75 months into the recovery period. Serum total free lipids were decreased relative to baseline 
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in the 75 mg/kg-bw/day group at 6 and 24 months (not measured at other time points) by 33 and 

49%, respectively, in males and by 39 and 43%, respectively, in females. No consistent changes 

from baseline levels were observed for dogs in the 7 and 35 mg/kg-bw/day groups. 

Elevated bromsulfalein retention values (an indication of liver toxicity) were noted in all 

treatment groups; however, there was no dose response-relationship, the increases were less than 

2-fold, and there was “considerable variation” in measurements within groups. No treatmentrelated effects were observed on serum bilirubin, serum uric acid, or thymol turbidity. 

For serum total protein levels, study authors stated that levels were slightly decreased in dogs in 

the 75 mg/kg-day group, although they did not indicate if protein levels in treatment groups were 

compared to baseline levels or controls. Changes in serum total protein levels, relative to 

controls, were calculated for this review; decreases were found to be ≤ 13% in males and ≤ 19% 

in females. Comparison of treatment groups relative to baseline levels, conducted by the EPA 

reviewers, revealed a decrease in protein early in the study, followed by an increase; all groups 

displayed a similar pattern and, therefore, a treatment-related effect on protein was not apparent. 

Serum albumin levels at 12, 18, and 24 months were decreased in a dose-related manner, 

compared to controls (baseline levels not reported); no pattern of duration-dependence was 

observed. In male dogs during months 12 to 24, decreases ranged from 7 to 11%, 16 to 36%, 

and 22 to 49% in the 7, 35, and 75 mg/kg-bw/day groups, respectively. In females, decreases 

ranged from 21 to 27%, 24 to 35%, and 43 to 50% in the 7, 35, and 75 mg/kg-bw/day groups, 

respectively. Changes at 75 mg/kg-bw/day were of likely toxicological significance, while the 

toxicological significance of changes at 7 and 35 mg/kg-bw/day is unclear. 

For serum urea nitrogen, study authors stated that levels in the 35 and 75 mg/kg-bw/day groups 

“tended to be slightly higher than controls” at 1, 3, and 6 months. However, review of the data 

for this report showed that levels in treatment groups were not consistently higher or lower than 

controls throughout the study. The largest increases in serum urea nitrogen at 35 and 75 mg/kgbw/day, relative to controls, were observed at 3 months and were 14 and 43%, respectively, in 

males and 33 and 57%, respectively, in females. Decreased serum urea nitrogen was observed 

later in the study period; at 24 months, decreases at 35 and 75 mg/kg-bw/day, relative to 

controls, were 24 and 35% in males, respectively, and 19 and 32%, respectively, in females. 

Changes relative to baseline values showed a similar pattern. In the absence of consistent effects 

on serum urea nitrogen, the toxicological significance of these changes is not clear.

Serum sugar levels were slightly and sporadically increased in the 35 and 75 mg/kg-bw/day 

groups, compared to controls as well as baseline values, at 1, 3, and/or 6 months. Study authors 

stated that values were within the normal range, although the normal range was not reported. 

Increases in serum sugar levels were not observed after 12 months, leading study authors to 

conclude that changes in serum sugar levels were not clinically significant. 

Serum protein-bound iodine levels after 24 months of treatment (only time point tested) were 

decreased, based on comparison with baseline values, by 19, 27, 39, and 47% in males and by 0,

21, 34, and 53% in females at 0, 7, 35, and 75 mg/kg-bw/day, respectively. The clinical 

significance of this finding is uncertain. 
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No treatment-related effects were observed for serum sodium, potassium, calcium, or inorganic 

phosphorus levels at 12, 18, or 24 months, relative to baseline levels. Study authors stated that 

slight decreases in calcium were observed in dogs in the 75 mg/kg-bw/day group; however, 

study authors do not indicate how comparisons between treatment groups were made. 

Comparison of the 75 mg/kg-bw/day group relative to control values, conducted for this review, 

showed decreases that were ≤ 13% and were not considered clinically significant by the EPA 

reviewers. Comparison of treatment groups relative to baseline levels, conducted by the EPA 

reviewers, revealed that calcium increased in all groups during the first 12 months, and all 

groups showed a similar pattern of change compared to baseline. 

Absolute and relative liver weights were increased, relative to controls, in male and female dogs 

in the 75 mg/kg-bw/day group after 25 months. In high-dose males, absolute and relative liver 

weights were increased by 86 and 91%, respectively, and in females by 42 and 49%, 

respectively. At 35 mg/kg-bw/day, small increases in absolute (7 and 14% in males and females, 

respectively) and relative (13 and 20% in males and females, respectively) liver weights were 

observed after 6 months; the toxicological significance of these changes was unclear. The 

remaining dogs in the 35 mg/kg-bw/day group were sacrificed after the 7-month recovery period 

and had liver weights similar to control values. Liver weights at 7 mg/kg-bw/day did not appear 

to be affected by treatment. No consistent dose-related changes were observed for weights of 

other organs. 

Histopathological examination revealed morphological changes in the livers of dogs from the 

35 and 75 mg/kg-bw/day groups. After 25 months at 35 mg/kg-bw/day, three dogs (one male 

and two females) showed moderately increased prominence of Glisson’s capsule (also observed 

in some control animals); two other dogs (males) showed liver cirrhosis (with abundance of 

fibrous tissue, duplication of bile ducts, and cellular degeneration) accompanied by marked 

lymphocytic cellular infiltration and dark brown pigment in Glisson’s capsule. The sixth animal 

in this dose group (a female) showed no remarkable histological findings in the liver at 

25 months. Vacuolation of hepatocytes was observed in one male and one female at 35 mg/kgbw/day. The majority of dogs fed 75 mg/kg-bw/day suffered from moderate to severe liver 

cirrhosis by 2 years, accompanied by mild-to-moderate lymphocytic cellular infiltration, dark 

brown pigmentation in Glisson’s capsule, and vacuolation of hepatocytes. Mild to moderate 

prominence of Glisson’s capsule and mild lymphocytic cellular infiltration were observed in the 

controls. Changes similar to those in the controls were observed in livers of dogs in the 7 mg/kgbw/day group. 

After being fed the control diet for 7 months, the severity of the liver lesions was markedly 

decreased in all affected animals of the 35-mg/kg-bw/day group. Additionally, the 

characteristics of the lesions changed; the fibrous tissue became denser and was in most 

instances a fine band. Therefore, the hepatic lesions in dogs receiving 35 mg/kg-bw/day appear 

to be partially reversible. The residual lesions in the liver were not considered by study authors 

to be cirrhotic. Complete recovery of the liver did not occur following a 7-month period on the 

control diet. 

Adverse, treatment-related histopathological changes were not seen in the other organs.
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CONCLUSIONS

The LOAEL is 35 mg/kg-bw/day based on clinically significant increases in serum alkaline 

phosphatase activity and decreases in serum cholesterol levels, as well as histopathological 

changes of the liver (cirrhosis [with abundance of fibrous tissue, duplication of bile ducts, and 

cellular degeneration], marked lymphocytic cellular infiltration, dark brown pigmentation in 

Glisson’s capsule, and vacuolation of hepatocytes); the NOAEL is 7 mg/kg-bw/day. 

STUDY RELIABILITY

[2] Reliable with restrictions. No information on statistical analyses was reported. The full 

study, although not a guideline study, was well documented and adequate based on the 

information provided.

REFERENCES

Jolley, W.P., Stemmer, K.L., Pfitzer, E.A., Grande, F., and Richmond, J. 1975. The effects 

upon beagle dogs following the ingestion of 4,4'-methylenebis (2,6-ditertiary butylphenol) for 

two years including the changes after an additional seven months on control diet. Kettering 

Laboratory, University of Cincinnati, OH. Submitted to EPA in 1992 as part of a TSCA 8(e) 

submission (EPA Doc. No. 88-920001353S, Fiche No. OTS0536073) entitled “Initial sub: the 

effect exerted upon beagle dogs during a period of 2-yrs by the introduction of 4,4'-methylenebis 

(2,6-ditertaryl butylphenol) into their daily diets (sanitized)”. 
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Study 3: Combined repeated-dose and carcinogenicity toxicity study (ECHA website, Exp 

WoE Repeated dose toxicity: oral.003)

Title: Not provided

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Phenol, 4,4’-methylenebis[2,6-bis(1,1-dimethylethyl)-, CASRN 118-82-1; referred to 

as 4,4'-Methylenebis(2,6-di-tert-butylphenol) in the study report (other synonyms: BHD, AN2, 

Ethyl Antioxidant 702).

Remarks: Purity not specified; crystalline solid 

METHOD

Method/guideline followed: Similar to OECD TG 453 “Combined Chronic 

Toxicity/Carcinogenicity Studies”

Test type: Combined Repeated-Dose Toxicity/Carcinogenicity Study

GLP compliant?: N/A (Pre-GLP)

Year study performed: 1969

Test species: Crj:CD(SD) rat; source: Charles River Breeding Laboratories Inc., Brookline, 

MA; age: weanlings at initiation; weight (average during week 1): 394–436 g (males) and 248–

269 g (females).

Number of animals/sex/dose: 25 in the test groups; 50 in the control group

Route of administration: Oral - Diet

Vehicle: Mazola corn oil (reported to be free of any antioxidants)

Exposure duration: 2 years

Doses/concentrations tested: 0, 15, 60, 100, 500, and 3000 ppm (corresponding approximate 

dose levels were 0, 0.55, 2.14, 3.56, 18.1, and 107 mg/kg-bw/day, respectively, for males and 0, 

0.67, 2.63, 4.35, 22.2, and 138 mg/kg-bw/day, respectively, for females)

Statistical methods used: Not specified
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Test method/conditions remarks: 

Dosing and Environmental Conditions: Rats (25/sex/dose) were fed diets containing the test 

substance at various concentrations for up to 2 years. A control group of 50 rats was fed a diet 

containing only the corn oil vehicle. Four rats per sex were assigned to subgroups of each dose 

group for interim kills at 13, 26, 52, and 78 weeks (3, 6, 12, and 18 months). Terminal sacrifice 

was performed at 104 weeks (24 months). 

The animals were acclimated for 2 weeks and were not fasted prior to the start of the study. The 

test animals were housed three males and four females per cage prior to the start of the study. 

Diet and water were provided ad libitum throughout the study. During the study, the test 

temperature was maintained at 76 ± 2°F and the humidity was not controlled.

Preparation of Test Diets: For preparation of the test diets, “1 kg of corn oil was added to 19 kg 

of the base diet with or without the test substance. The mixture was stirred for 20 min in a 

Hobart mixer (stainless steel). [Five] diets containing the test item were prepared... The diet 

was freshly prepared every week.” [ECHA]

The test diets were prepared by “mixing appropriate amounts with [a] special preparation of 

Rockland Mouse and Rat diet containing no more than 1% of fat.” [ECHA] The rodent diet food 

was obtained fresh once each month.

Clinical Observations, Body Weights, and Food Consumption: Cage-side observations were 

made on a daily basis for general appearance and behavior. Animals were observed weekly for a 

detailed assessment of clinical signs. Body weight and food consumption were recorded weekly. 

Mean test substance intake for males and females in each group was calculated by an unspecified 

method. 

Ophthalmological examinations were performed weekly, and all groups were examined for the 

presence of cataracts. 

Hematology and Clinical Chemistry Measurements: Hematological examinations were 

performed on unfasted treated and control animals (10 rats/sex/dose) at 1 week before the start of 

dosing, and again at 13, 26, 52, 78, and 104 weeks. Hematological parameters examined 

included the following: prothrombin time, hemoglobin, packed cell volume of erythrocytes, and 

total and differential leukocytes. Clinical chemistry investigations were performed on blood 

samples collected at scheduled sacrifice of all test animals. Clinical chemistry parameters 

examined included the following: total lipids, cholesterol, and alkaline phosphatase. 

No urinalysis assessments were performed.

Gross Necropsy and Histopathology: It appears that organ weights were recorded, but, with the 

exception of the liver, the organs weighed were not specified in the ECHA study summary (the 

full study report was not available for review). Pathological examinations were performed on 

the following organs at gross necropsy: liver, heart, lung, kidneys, spleen, brain, gonads, 

pituitary, adrenals, and thyroid. Histological examinations were performed on selected tissues, 

including the heart, aorta, lung, liver, spleen, kidney, stomach, duodenum, pylorus, small 
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intestine (ileum), large intestine (colon), mesenteric lymph nodes, urinary bladder, prostate, 

testes, ovaries, uterus, pancreas, thyroid, thymus, adrenals, pituitary, brain, spinal cord, eye, bone 

marrow, bone, skeletal muscle, and skin.

RESULTS

Endpoint value(s): 

LOAEL (males) = 500 ppm (18.1 mg/kg-bw/day) based on increased incidence and severity of 

fatty degeneration of the liver and increased incidence of bile duct multiplication; NOAEL 

(males) = 100 ppm (3.56 mg/kg-bw/day). 

LOAEL (females) = 100 ppm (4.35 mg/kg-bw/day) based on increased incidence and severity of 

bile duct multiplication; NOAEL (females) = 60 ppm (2.63 mg/kg-bw/day).

Results remarks: 

Mortality: Mortalities occurred at all dose levels at incidences similar to controls. The mortality 

percentages at dietary concentrations of 0, 15, 60, 100, 500, and 3000 ppm were 54.9, 46.2, 57.1, 

44.0, 61.5, and 46.2%, respectively, for males and 52.9, 40.0, 44.0, 68.0, 38.5, and 52.0%, 

respectively, for females. Several rats in all groups had various infectious diseases (not specified 

on the ECHA website), which resulted in death. These illnesses were not considered to be 

related to treatment. 

Clinical Observations, Body Weight, and Food Consumption: Greenish-yellow discoloration of 

the fur was observed at 500 and 3000 ppm beginning at 1 year and 6 months of treatment, 

respectively. Occasional discoloration was also observed in some rats at 100 ppm after 

15 months of treatment. No treatment-related effects were found on mean body weight, mean 

body weight gain, or food consumption in any group. Although variations were observed in 

individual body weight, body weight gain, and food consumption for some test animals, these 

effects were considered to be related to illness and were not considered to be treatment-related. 

Food efficiencies in all treated groups were comparable to controls. No treatment-related 

findings were observed during ophthalmological examinations. 

Hematology and Clinical Chemistry Measurements: Mild to moderate leukopenia was observed 

in some females after up to 78 weeks of feeding, but this finding was comparable to controls. 

Anaemia or polychromasia, which was not considered to be treatment-related, was noted in 

individual males and females in all groups, including controls, at weeks 78 and 104. One week 

prior to treatment, a statistically significant difference (p-value was not provided) in prothrombin 

time was noted in males at 15 and 500 ppm, and in females at 60, 100, 500, and 3000 ppm, 

relative to controls (the direction and magnitude of these changes were not provided on the 

ECHA website). Additionally, this same effect was noted in males at 15, 100, and 3000 ppm at 

13 weeks and in females at 500 ppm at 26 weeks. No other differences in prothrombin time 

relative to controls were noted at any other observation times (weeks 52, 78, and 104). “As this 

finding was not dose related and did not persist throughout the course of the study, it was not 

considered treatment-related.” [ECHA] No other effects were observed for any other 

hematological parameters that were analyzed.
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A statistically significant increase (p-value was not provided) in alkaline phosphatase levels was 

observed in males at 3000 ppm at week 104. “Due to major fluctuations in the measurements 

this finding was not considered treatment related by the [study] authors.” [ECHA] However, 

due to the histopathological effects observed in the liver of rats in this group, the EPA reviewers 

consider the increase in alkaline phosphatase to be toxicologically significant. 

“No consistent treatment related effect was observed on the serum cholesterol and total lipid 

levels.” [ECHA]

Gross Necropsy and Histopathology: “All relative organ weights and most absolute organ 

weights in the treated groups were comparable to the respective control values. Single rats 

receiving 3000 ppm of the test substance were [reported] to have enlarged livers, but the group 

averages did not reach statistical significance, with the exception of females killed after week 13 

and males killed after week 78.” [ECHA] A further description of the incidence and statistical 

significance of enlarged livers after these time points was not provided on the ECHA website. 

Mean relative liver weight was statistically significantly reduced (p-value was not provided) 

compared to controls in females fed 15 ppm of the test diet for 104 weeks. Since a dose-related 

trend was not observed and the finding appeared to be an isolated observation, the effect was not

considered to be treatment-related. 

Necropsy showed no treatment-related gross organ changes in male or females.

Histopathological examination revealed that all of the animals (treatment and control) killed at 

24 months had pneumonia or interstitial nephritis or both. Throughout the study, 183 rats died. 

“[For] 52 [rats] no postmortem pathology could be done due to autolysis. For 131 [rats] 

postmortem pathology revealed in most instances massive pulmonary infection as the cause of 

death. Liver lesions were the most prominent findings and consisted of fatty degeneration of the 

hepatic [cells] and a proliferation of the bile ducts. Fatty degeneration [ranged] from small fat 

vacuoles to a fatty liver. There was no special localization of the [degenerated] cells in the liver 

lobe... Two types of proliferation of the bile ducts could be distinguished morphologically: 

1. multiplication of the bile ducts with retainment of their normal structure occurred in small 

clusters. 2. A dilatation and multiplication of the bile ducts with fibrotic walls without 

epithelium. In some of the dilated bile ducts mucous plaques were observed. No inflammatory 

reaction was present. [With] [u]p to 3 months of feeding no alterations in the livers were 

observed. Both lesions [increased] in control and treated rats from 18 months of treatment. 

After 24 [months] more severe lesions were observed in the 500 and 3000 ppm dose groups.” 

[ECHA]

At 12 months in males, the incidences of fatty degeneration of the liver at dietary concentrations 

of 0, 15, 60, 100, 500, and 3000 ppm were 0/4, 1/4, 0/4, 0/4, 4/4, and 4/4 (0, 25, 0, 0, 100, and 

100%), respectively. At 18 months in males, the incidences at 0, 15, 60, 100, 500, and 3000 ppm 

were 0/4, 0/4, 0/11, 0/4, 3/3, and 2/3 (0, 0, 0, 0, 100, and 67%), respectively. At 24 months in 

males, the incidences at 0, 15, 60, 100, 500, and 3000 ppm were 8/22, 8/14, 5/12, 8/14, 6/11, and 

 

1 Only one male was examined histologically in the 60 ppm group at 18 months, according to the data tables for 

fatty degeneration and bile duct multiplication provided on the ECHA website. The reason(s) for the small number 

of males in this subgroup was not explained.
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14/14 (36, 57, 42, 57, 55, and 100%), respectively. Statistical analyses1by Fisher’s exact test 

revealed significant increases in incidence of hepatic fatty degeneration in males at 500 ppm at 

12 (p = 0.03) and 18 months (p = 0.03) and at 3000 ppm at 12 (p = 0.03) and 24 months 

(p < 0.0001). In addition, there appeared to be a dose-related increase in severity of hepatic fatty 

degeneration in males at ≥ 500 ppm at 24 months. 

At 12 months in males, the incidences of bile duct multiplication at 0, 15, 60, 100, 500, and 

3000 ppm were 0/4, 1/4, 1/4, 2/4, 3/4, and 4/4 (0, 25, 25, 50, 75, and 100%), respectively. At 

18 months in males, the incidences at 0, 15, 60, 100, 500, and 3000 ppm were 0/4, 0/4, 0/1, 0/4, 

3/3, and 3/3 (0, 0, 0, 0, 100, and 100%), respectively. At 24 months in males, the incidences at 0, 

15, 60, 100, 500, and 3000 ppm were 5/23, 3/14, 2/12, 5/14, 5/11, and 10/14 (22, 21, 17, 36, 45, 

and 71%), respectively. Statistical analyses by Fisher’s exact test revealed significant increases 

in incidence of bile duct multiplication in males at 500 ppm at 18 months (p = 0.03) and at 

3000 ppm at 12 (p = 0.03), 18 (p = 0.03), and 24 months (p = 0.005). No dose-related increase in 

severity of bile duct multiplication in males was apparent.

At 12 months in females, the incidences of fatty degeneration of the liver at 0, 15, 60, 100, 500, 

and 3000 ppm were 0/4, 0/4, 0/4, 0/4, 1/4, and 3/4 (0, 0, 0, 0, 25, and 75%), respectively. At 

18 months in females, the incidences at 0, 15, 60, 100, 500, and 3000 ppm were 0/4, 0/4, 1/4, 

0/4, 3/4, and 3/3 (0, 0, 25, 0, 75, and 100%), respectively. At 24 months in females, the 

incidences at 0, 15, 60, 100, 500, and 3000 ppm were 4/24, 3/14, 4/14, 2/8, 6/16, and 8/12 (17, 

21, 29, 25, 38, and 67%), respectively. Statistical analyses by Fisher’s exact test revealed 

significant increases in incidence of hepatic fatty degeneration in females at 3000 ppm at 

18 (p = 0.03) and 24 months (p = 0.007). No dose-related increase in severity of hepatic fatty 

degeneration in females was apparent.

At 12 months in females, the incidences of bile duct multiplication at 0, 15, 60, 100, 500, and 

3000 ppm were 0/4, 0/4, 1/4, 1/4, 1/4, and 3/4 (0, 0, 25, 25, 25, and 75%), respectively. At 

18 months in females, the incidences at 0, 15, 60, 100, 500, and 3000 ppm were 1/4, 0/4, 3/4, 

0/4, 3/4, and 4/4 (25, 0, 75, 0, 75, and 100%), respectively. The incidences in females at 

24 months at 0, 15, 60, 100, 500, and 3000 ppm were 2/24, 3/15, 4/14, 4/8, 11/16, and 8/12 (8, 

20, 29, 50, 69, and 67%), respectively. Statistical analyses by Fisher’s exact test revealed 

significant increases in incidence of bile duct multiplication in females at 100 ppm at 24 months 

(p = 0.02), at 500 ppm at 24 months (p = 0.0001), and at 3000 ppm at 18 (p = 0.05) and 24 

months (p = 0.0006). In addition, there appeared to be a dose-related increase in severity of bile 

duct multiplication in females at ≥ 100 ppm at 24 months.

CONCLUSIONS

The LOAEL was 500 ppm (18.1 mg/kg-bw/day) in males based on increased incidence and 

severity of fatty degeneration of the liver and increased incidence of bile duct multiplication. 

The NOAEL was 100 ppm (3.56 mg/kg-bw/day) in males. 

 

1 All statistical analyses of histopathological results were conducted by EPA reviewers using GraphPad Software 

(Scientific Software), available at http://graphpad.com/quickcalcs/index.cfm.
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The LOAEL was 100 ppm (4.35 mg/kg-bw/day) in females based on increased incidence and 

severity of bile duct multiplication. The NOAEL was 60 ppm (2.63 mg/kg-bw/day) in females.

STUDY RELIABILITY

[2] Reliable with restrictions. The study is considered to be reliable with restrictions based on 

weight of evidence. When compared to the TSCA 799.9430 guideline, the following deviations 

were noted: (1) the total number of animals at test initiation was not specified; (2) the lot or 

batch number of the test substance was not provided; (3) the environmental test temperature 

(76 ± 2°F) exceeded the recommended temperature of 22 ± 3°C (71.6°F); (4) humidity was not 

controlled; (5) limited hematology and clinical chemistry parameters were examined;

(6) urinalysis was not conducted; (7) organs weighed at necropsy were not specified; and 

(8) group mean data for endpoints other than histopathological lesions were not reported. 

Inconsistencies in some of the data tables on the ECHA website for severity grades of fatty 

degeneration of the liver and bile duct multiplication (incidences and percentages) render some 

of this information of questionable value for fully describing the potential toxicity of CASRN 

118-82-1. However, these inconsistencies did not impact the assignment of the endpoint values. 

REFERENCE

Study summary available from the ECHA website at

http://apps.echa.europa.eu/registered/data/dossiers/DISS-dcee47d2-62ad-0171-e044-

00144f67d031/AGGR-bf547f31-7fe6-4a5f-a06a-e41a1a9a2ab0_DISS-dcee47d2-62ad-0171-

e044-00144f67d031.html#AGGR-bf547f31-7fe6-4a5f-a06a-e41a1a9a2ab0 (study indicated as 

Exp WoE Repeated dose toxicity: oral.003 in online entry for CASRN 118-82-1) as of December 

2, 2013. The data source is referred to as a “study report”, with a report date of 1969-07-08. No 

other reference information is provided. 

Text in quotes is taken directly from the ECHA website.

Copyright Notice: © European Chemicals Agency, 2007-2011. Reproduction is authorised 

provided the source is fully acknowledged in the form “Source: European Chemicals Agency, 

http://echa.europa.eu/”, and provided written notification is given to the ECHA Communication 

Unit (info (at) echa.europa.eu).
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C-7. Genetic Toxicity – Gene Mutation

Summary of endpoint: Adequate 

Study 1: Mutagenicity studies in Salmonella typhimurium and Escherichia coli (Brooks and 

Wiggins, 1987)

Title: Microbial mutagenicity studies with IONOX 220

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Phenol, 4,4’-methylenebis[2,6-bis(1,1-dimethylethyl)-, CASRN 118-82-1; referred to 

as IONOX 220 in study report.

Remarks: 99.04% purity; Appearance: pale yellow powder; obtained from San Celoni, 

Barcelona, Spain.

METHOD

Method/guideline followed: Other (based on Ames et al., 1975); similar to guideline study 

OECD 471

Test type: Ames reverse gene mutation assay

GLP compliant?: Yes

Year study performed: 1987

Test species: Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100

and Escherichia coli strain WP2uvrA pKM 101

Metabolic activation: Without and with (S-9 metabolic activation from rat livers)

Concentrations tested: 0 (solvent control), 31.25, 62.5, 125, 250, 500, 1000, 2000, or 4000 

μg/plate (nominal); precipitate was observed at concentrations ≥ 500 μg/plate. 

Statistical methods used: Not specified

Test method/conditions remarks: S. typhimurium and E. coli strains were exposed to the test 

substance in acetone both in the presence and absence of rat liver S-9 mix and incubated at 37°C 

for 48 to 72 hours in a plate-incorporation assay. The test was conducted twice. Three plates per 

test concentration were used for each test. The initial number of cells per plate was not reported. 

The number of revertants per plate and ratio of the number of revertants for the test substance to 

the number of revertants for control (including means and standard deviations) were reported. 
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Increases in the ratio of revertants per treated plate to revertants per control plate of ≥ 2.5-fold 

were considered a positive mutagenic response. 

Positive Controls:

TA98, TA100, and TA1538: Benzo(a)pyrene (20 μg)

TA1537: Neutral red (20 μg)

TA1535: Sodium azide (5 μg)

WP2uvrA pKM 101: Potassium dichromate (20 μg)

RESULTS 

Genotoxicity result: Negative in the presence and absence of metabolic activation

Cytotoxic concentration (if applicable): > 4000 μg/plate (nominal), in the absence and 

presence of metabolic activation

Results remarks: No effects of the test substance on mutation frequency were observed with or 

without activation. Precipitation of the test substance was observed at nominal concentrations 

≥ 500 μg/plate. 

The addition of 2000 μg/mL test article (equivalent to 4000 μg/plate) changed media pH from 

7.49 to 7.45. No information was reported on the pH at other concentrations.

Solvent controls responded appropriately.

Positive control substances responded appropriately for the following strains and activation 

combinations: WP2uvrA pKM 101 without activation; TA1535 with and without activation; 

TA98, TA100, TA1537, and TA1538 with activation. Positive control substances did not 

produce positive responses for WP2uvrA pKM 101 with activation and TA98, TA100, TA1537, 

and TA1538 without activation.

CONCLUSIONS

The test article did not induce gene mutations in any strain in the presence or absence of 

metabolic activation.

STUDY RELIABILITY

[2] Reliable with restrictions. The study meets the current guidelines for the assay with the 

exception that the investigators did not achieve appropriate positive control responses for all 

strains with and without activation, as indicated by the lack of a positive response for some 

combinations of positive control substance, strain, and presence/absence of activation. 

Therefore, the results are only valid for the combinations of strain and activation for which 

positive controls responded appropriately.
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REFERENCE

Brooks, T.M. and D.E. Wiggins. 1987. Microbial Mutagenicity Studies with IONOX 220. 

Shell Research Limited, Sittingbourne Research Center. Submitted to EPA on May 17, 2007. 

Available online at http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2005-

0033-0250 as of December 4, 2013.
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Study 2: Mutagenicity study in Saccharomyces cerevisiae (Brooks and Wiggins, 1987)

Title: Microbial mutagenicity studies with IONOX 220

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Phenol, 4,4’-methylenebis[2,6-bis(1,1-dimethylethyl)-, CASRN 118-82-1; referred to 

as IONOX 220 in study report.

Remarks: 99.04% purity; Appearance: pale yellow powder; obtained from San Celoni, 

Barcelona, Spain.

METHOD

Method/guideline followed: Other (based on Zimmermann, 1977 and Davies, P.J. et al., 1975)

Test type: Saccharomyces mitotic gene conversion assay

GLP compliant?: Yes

Year study performed: 1987

Test species: Saccharomyces cerevisiae strain JD1

Metabolic activation: Without and with (S-9 metabolic activation from rat livers)

Concentrations tested: 0 (solvent control), 0.01, 0.1, 0.5, 1.0, and 2.5 mg/mL. The study 

report does not provide any information regarding precipitation of the test substance.

Statistical methods used: Not specified

Test method/conditions remarks: Liquid suspension cultures of log-phase cells of 

Saccharomyces cerevisiae JD1 were exposed to the test substance in acetone in the presence and 

absence of rat liver S9 fraction. Cells were incubated for 18 hours at 30°C in the absence of S9 

or for 2 hours at 37°C followed by 16 hours at 30°C in the presence of S9. Cultures were then 

seeded onto the appropriate culture media and incubated for 3 days at 30°C prior to counting 

prototrophic colonies. The test was conducted twice. Three plates per concentration were used 

for each test. The number of prototrophs per plate at the histidine locus and at the tryptophan 

locus and the ratios of number of prototrophs for the test substance to the number of prototrophs 

for control for each locus were reported. A 2-fold increase above control was considered a 

positive mutagenic response. 
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Positive controls:

4-nitroquinoline-N-oxide (0.00025 mg/mL) without metabolic activation

Cyclophosphamide (1.25 mg/mL) with metabolic activation

RESULTS 

Genotoxicity result: Negative in the presence and absence of metabolic activation

Cytotoxic concentration (if applicable): > 2.5 mg/mL

Results remarks: There was no increase in the rate of mitotic gene conversion either in the 

presence or absence of S9. No effect on cell viability was observed. Positive and solvent 

controls responded appropriately.

CONCLUSIONS

The test substance did not induce mitotic gene conversions in Saccharomyces either in the 

presence or absence of metabolic activation.

STUDY RELIABILITY

[2] Reliable with restrictions. Similar to OECD guideline study 481. This study is rated as 

“reliable with restrictions” because the established limit concentration for this assay was not 

identified and no justification was provided for choice of the highest concentration tested. 

(OECD TG 481 does not specify a limit concentration.)

REFERENCE

Brooks, T.M. and D.E. Wiggins. 1987. Microbial Mutagenicity Studies with IONOX 220. 

Shell Research Limited, Sittingbourne Research Center. Submitted to EPA on May 17, 2007. 

Available online at http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2005-

0033-0250 as of December 4, 2013.
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Study 3: Mammalian gene mutation assay (Sandoz Chemical Ltd, 1991, as summarized in the 

online ECHA entry for 2,6-di-tert-butylphenol, Exp Key Genetic toxicity in vitro.001)

Title: Mammalian gene mutation assay

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Analog: 2,6-Di-tert-butylphenol, CASRN 128-39-2

Remarks: 98.7% purity; Lot/batch No.: Charge 10/6; solid at 20°C; stability in solvent: at least 

48 hours in water and DMSO

METHOD

Method/guideline followed: OECD 476; 40 CFR, Ch. I part 798; EEC Directive 87/302, L 133

Test type: Mammalian cell gene mutation assay

GLP compliant?: Yes

Year study performed: 1991

Test species: Chinese hamster lung fibroblasts (V79)

Metabolic activation: With and without S9 mix (liver microsome preparations)

Concentrations tested: 

Pre-experiment for toxicity (colony forming ability): 0 (negative [untreated] control), 0 (solvent 

control), 0.1, 1.0, 3.0, 6.0, 10.0, 30.0, 50.0, and 100 μg/mL without and with S9 mix

Main experiment: 0 (negative [untreated] control), 0 (solvent control), 0.3, 1.0, 2.0, 4.0, 6.0, and 

8.0 μg/mL without S9 mix; 0 (negative [untreated] control), 0 (solvent control), 3.0, 10.0, 20.0, 

30.0, 40.0, and 50.0 μg/mL with S9 mix

Statistical methods used: “Since the distribution of mutant cells does not follow known 

statistical models, an adequate statistical method is not available.” [ECHA]

Test method/conditions remarks: A pre-experiment study of cytotoxicity (plating efficiency 

assay) was conducted to determine the toxicity of the test substance both with and without 

metabolic activation. The main study consisted of two independent experiments, using identical 

procedures, each conducted with and without the presence of metabolic activation. In the main 

study, Chinese hamster V79 cells were exposed to the test substance for 4 hours at 

concentrations of 0.3, 1.0, 2.0, 4.0, 6.0, and 8.0 μg/mL without S9 mix and at 3.0, 10.0, 20.0, 

30.0, 40.0, and 50.0 μg/mL with S9 mix.
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Cells: V79 cells were stored as frozen stock cultures, and subcultures were made from these 

stocks. Cultures were periodically checked for mycoplasma contamination and karyotype 

stability. The medium for culturing the V79 cells was MEM (minimal essential medium), 

supplemented with 10% fetal calf serum (FCS). The cells were subcultured twice per week and 

were incubated at 37°C with 4.5% carbon dioxide atmosphere. For the selection of mutants at 

the HGPRT locus, the medium was supplemented with 11 μg thioguanine/mL.

Preparation of Dose Levels: Dimethyl sulfoxide (DMSO) was chosen for the vehicle based on 

its solubility properties and its non-toxicity to cells.

Solvent (Vehicle) Control Substance: DMSO

Positive Control Substances: Without metabolic activation: EMS (indicated to be used at a 

concentration of 0.6 μg/mL in Tables I through V but 0.6 mg/mL in the “Controls” section, 

which also named this positive control “1890” rather than EMS); With metabolic activation: 

DMBA (3.85 μg/mL; the “Controls” section named this positive control “26” rather than 

DMBA).

Methods of Application: “2 or 3 days old logarithmically growing stock cultures more than 50% 

confluent were trypsinized at 37°C for 5 min. Then digestion [was] stopped by adding complete 

medium and a single cell suspension prepared. Prior to trypsin treatment cells were rinsed with 

Ca-Mg-free salt solution containing 200 mg/l EDTA. The cell suspension was seeded into 

plastic culture flasks (Greines, D-7440 Nürtingen, Germany). Approx. 8×100000 [in 30 mL 

medium for the mutagenicity tests] and 5×100 cells [in 5 mL medium for the determination of 

cytotoxicity], respectively, were seeded in each flask. The medium was MEM with 10% FCS 

(complete medium).” [ECHA]

After a 24-hour preincubation period, the medium was replaced with serum-free medium 

containing the test substance, either with or without 50 μL/mL S9 mix. After a 4-hour exposure 

period, the treatment medium was replaced with normal medium after the cells had been rinsed 

twice with “saline G”. 

Evaluation Criteria: “A test article is classified positive if it induces either a significant 

concentration-related increase in the mutant frequency or a reproducible and significant positive 

response for at least one of the test points.” [ECHA]

“A test article producing neither a significant concentration related increase in the mutant 

frequency nor a significant and reproducible positive response at any one of the test points is 

considered non-mutagenic in this system.” [ECHA]

“A significant response is described as follows: The test article is classified as mutagenic if it 

induces reproducibly with at least one of the concentrations a mutation frequency that is three 

times higher than the spontaneous mutation frequency in the experiment.” [ECHA]
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“The test article is classified as mutagenic if there is a reproducible concentration-related 

increase in the mutation frequency. Such evaluation may be considered independently of an 

enhancement factor for induced mutants.” [ECHA]

“However, in a case by case evaluation this decision depends on the level of the corresponding 

negative control data. If there is by chance a low spontaneous mutation rate as compared to the 

range normally found (5–45 mutants per 1000000 cells) a seemingly concentration-related 

increase of the mutations within this range will be regarded as to be irrelevant.” [ECHA]

RESULTS 

Genotoxicity result: Negative in the presence and absence of metabolic activation.

Cytotoxic concentration (if applicable): 6.0 μg/mL without metabolic activation and 

50.0 μg/mL with metabolic activation based on relative plating efficiency in the pre-experiment 

for toxicity.

Results remarks: “Taking into account the mutation rates found in the groups treated with the 

test article compared to the negative and solvent controls it can be concluded that no biologically 

relevant increase of point mutations was observed. The test article did not induce reproducible 

concentration-related increase in the mutant colony numbers. The mutant values of the groups 

treated with the test article were in the range of the negative controls.” [ECHA]

“In this study in both experiment[s] (with and without S9 mix) the range of the negative controls 

was from 0.7 up to 49.1 mutants per 1000000 cells, the range of the groups treated with the test 

article was from 0.5 up to 36.8 mutants per 1000000 cells.” [ECHA]

The positive control substances showed a distinct increase in induced mutant colonies. Negative 

and solvent controls responded appropriately.

CONCLUSIONS

Under the experimental conditions reported, the test substance did not induce point mutations at 

the HGPRT locus in V79 cells either in the presence or absence of metabolic activation.

STUDY RELIABILITY

[2] Reliable with restrictions. The test concentrations used were below the maximum 

recommended in the test guidelines; however, the cytotoxicity findings from the pre-experiment 

justify the concentrations tested. The ECHA summary was inconsistent about the concentration 

of the substance (EMS) that was used as a positive control in absence of metabolic activation. 

The study summary did not indicate whether or not precipitation of test substance was observed

at the concentrations tested. In the ECHA summary, the final table, which should have presented 

mutation rates found in experiment II, was missing. ECHA did not provide a full citation for this 

study (the ECHA Reference section indicates only “study report” and “1991”), but it appears to 

be the same study that was attributed to Sandoz Chemical Ltd. (1991) by the OECD/SIDS 
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documents for CASRN 128-39-2 [2,6-di-tert-butylphenol]. These factors/deviations do not 

appear to affect the validity of the conclusion of this study. 

REFERENCE

Sandoz Chemical Ltd. 1991. Unpublished CCR Report [on a mammalian cell gene mutation 

assay] - Sandoz, 243617. Muttenz, Switzerland. As summarized in the online ECHA 

(http://apps.echa.europa.eu/registered/data/dossiers/DISS-97d6a15b-c218-5de0-e044-

00144f67d031/AGGR-0d490876-6ff9-4b92-be19-3c34473107ba_DISS-97d6a15b-c218-5de0-

e044-00144f67d031.html#AGGR-0d490876-6ff9-4b92-be19-3c34473107ba) entry for CASRN 

128-39-2 (study indicated as Exp Key Genetic toxicity in vitro.001, available online as of 

December 2, 2013). Secondary reference: OECD. 2002. SIDS/IRPTC Data Profile for 2,6-Ditert-butylphenol (CASRN 128-39-2). UNEP Publications. 2002. Available online at 

http://www.chem.unep.ch/irptc/sids/OECDSIDS/128392.pdf as of December 4, 2013.

Text in quotes is taken directly from ECHA.

Copyright Notice: © European Chemicals Agency, 2007-2011. Reproduction is authorized 

provided the source is fully acknowledged in the form “Source: European Chemicals Agency, 

http://echa.europa.eu/”, and provided written notification is given to the ECHA Communication 

Unit (info (at) echa.europa.eu).
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Study 4: Mutagenicity study in Salmonella typhimurium (Johnson, 1984)

Title: Genetic Toxicology: Salmonella/Microsomal Assay

This study is: Adequate Not Adequate

Numerous experimental details were not included in the study report, including test substance 

purity, test method/guideline followed, statistical methods, criteria to define a positive response, 

identity of positive control(s), quantitative results for untreated control, detailed results for 

triplicate plating, and quantitative data on cytotoxicity at 25 μg/plate in strains TA1535, 

TA1537, and TA100. 

TEST SUBSTANCE 

Identity: Analog: 2,6-Di-tert-butylphenol, CASRN 128-39-2; referred to as AN 701 in the study 

report.

Remarks: Purity not provided; received as a yellow-brown liquid

METHOD

Method/guideline followed: Other (plate incorporation Ames Assay was carried out according 

to S.O.P. ETTOX-027)

Test type: Salmonella Gene Mutation Assay

GLP compliant?: Yes

Year study performed: 1984

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

Metabolic activation: Without and with (Aroclor 1254 induced rat liver S-9 fraction)

Concentrations tested: 0.25, 0.5, 2.5, 5, and 25 μg/plate; test substance was soluble at all 

concentrations tested.

Statistical methods used: Not specified

Test method/conditions remarks: The compound was tested using the Ames plate 

incorporation assay. Salmonella strains were exposed to the test substance in acetone in the 

presence and absence of metabolic activation. Each concentration was tested in triplicate. An 

untreated control, solvent control, and positive control were tested concurrently. The positive 

control was not identified.
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RESULTS 

Genotoxicity result: Negative in the presence and absence of metabolic activation

Cytotoxic concentration (if applicable): 25 μg/plate in strains TA1535, TA1537, and TA100 

with and without metabolic activation; cytotoxicity not reported for TA98

Results remarks: The test substance did not induce a dose-related increase in mutant colonies 

in any strains tested with or without metabolic activation.

Numerous experimental details were not included in the study report, including test substance 

purity, test method/guideline followed, statistical methods, a clear statement of criteria used to 

define a positive response, identity of positive control(s), quantitative results for untreated 

control, detailed results for triplicate plating, and quantitative data on cytotoxicity at 25 μg/plate 

in strains TA1535, TA1537, and TA100. 

CONCLUSIONS

The test substance did not induce gene mutations in Salmonella typhimurium strains TA1535, 

TA1537, TA98, or TA100 in the absence or presence of metabolic activation.

STUDY RELIABILITY

[4] Not assignable. Numerous experimental details were not included in the study report, 

including test substance purity, test method/guideline followed, statistical methods, a clear 

statement of criteria to define a positive response, identity of positive control(s), quantitative 

results for untreated control, detailed results for triplicate plating, and quantitative data on 

cytotoxicity at 25 μg/plate in strains TA1535, TA1537, and TA100. 

REFERENCE

Johnson, D.E. 1984. Genetic Toxicology Salmonella/Microsomal Assay. Performing 

Laboratory: Genetic Toxicology Laboratory, Baton Rouge, LA. Study No.: Ames 086-068. 

Submitted by Ethyl Corporation to EPA in 1986 as part of a TSCA 8(e) submission (EPA Doc. 

No. 40-8627016, Fiche No. OTS0526562) entitled “Letter from Ethyl Corp to US EPA regarding 

submission of 2,6-di-tert-butylphenol toxicological studies with attachments”.
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Study 5: Mutagenicity study in S. typhimurium and E. coli (Hachiya and Takizawa, 1994)

Title: Mutagenicity of plastic additives

This study is: Adequate Not Adequate

Information for this study was reviewed in HPVIS (4/8/2005) based on a partial English 

translation of the original study; a complete translation of this study was not available. The 

HPVIS (4/8/2005) summary indicated that positive controls were not included in this assay. 

Positive controls, with and without metabolic activation, are required to validate assay 

performance. Since none of the 20 chemicals tested yielded a positive response, performance of 

this assay cannot be validated. Additional reporting inadequacies include lack of information on 

replicate plating, purity of the test substance, cytotoxic concentrations, quantitative results, and 

justification for testing up to a concentration that was only 40% of the recommended limit 

concentration of 5000 μg/plate.

TEST SUBSTANCE 

Identity: Phenol, 4,4’-methylenebis[2,6-bis(1,1-dimethylethyl)-, CASRN 118-82-1

Remarks: Purity not provided

METHOD

Method/guideline followed: Not specified

Test type: S. typhimurium and E. coli gene conversion assay

GLP compliant?: Not specified

Year study performed: 1994

Test species: S. typhimurium strains TA97, TA98, TA100, and TA102 and E. coli strain WP2 

uvrA (pKM101)

Metabolic activation: With and without 

Concentrations tested: 0, 50, 100, 200, 500, 1000, and 2000 μg/plate

Statistical methods used: Not specified

Test method/conditions remarks: No details on test method/conditions were reported. Solvent 

was acetone and Tween 80.

“There was no indication of either cytotoxicity or solubility problems beyond the statement that 

the test substance was insoluble but homogeneously dispersed in phosphate buffer in the assay 
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with metabolic activation. It thus appears that the chemical should have been tested up to the 

limit concentration of 5000 μg/plate.” (HPVIS, 4/8/2005)

“There was no repeat experiment to confirm the negative result. Especially since the original

experiment only tested up to 40% of the limit concentration, a justification for having no repeat 

experiment should have been provided. It will be important to know if they used more than one 

plate per concentration; they should have used triplicate plating with the reported values being 

the mean and the standard error of the mean for those three values. They provided only one 

number per plate, with no measure of the variability. It will be important to know how they 

prepared their S-9 mix to provide endogenous metabolic activation. The purity of the test 

chemical is unknown.” (HPVIS, 4/8/2005)

Note that 20 different chemicals were evaluated for mutagenicity in this study. None of the 

chemicals had a positive response.

RESULTS 

Genotoxicity result: Negative

Cytotoxic concentration (if applicable): Not specified

Results remarks: The authors reported negative results for the subject chemical in all strains of 

S. typhimurium and E. coli.

CONCLUSIONS

This test was not adequate to rule out induction of gene mutations in bacteria with or without 

metabolic activation due to the lack of positive controls. However, results that were reported for 

the test substance were negative for induction of gene mutations in bacteria up to a concentration 

that was 40% of the guideline-recommended limit concentration of 5000 μg/plate.

STUDY RELIABILITY

[4] Not assignable. Information for this study was reviewed in HPVIS (4/8/2005) based on a 

partial English translation of the original study; a complete translation of this study was not 

available. The HPVIS (4/8/2005) summary indicated that positive controls were not included in 

this assay. Positive controls, with and without metabolic activation, are required to validate 

assay performance. Since none of the 20 chemicals tested yielded a positive response, 

performance of this assay cannot be validated. Additional reporting inadequacies include lack of 

information on replicate plating, purity of the test substance, cytotoxic concentrations, 

quantitative results, and justification for testing up to a concentration that was only 40% of the 

recommended limit concentration of 5000 μg/plate.
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REFERENCES

Hachiya, N. and Y. Takizawa. 1994. Mutagenicity of Plastic Additives (translation). 

Hen'lgensei Shiken 3(3):147-154. Study summary available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2005-0033-0181 (summarized 

in “End_Point_E1_-_Mammalian_Toxicity_-_Genotoxicity.pdf”) as of December 4, 2013. 

US EPA. OPPT. High Production Volume Information System (HPVIS). Submission ID: 

24929673. Submission Name: Phenol, 4,4’-methylenebis[2.6-bis(1,1-dimethylethyl)-. Sponsor: 

Orphan Chemical. Submission created on 04/08/2005.

Text in quotes is taken directly from HPVIS (4/8/2005).
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C-8. Genetic Toxicity – Chromosomal Aberrations

Summary of endpoint: Adequate

Study 1: Chromosomal aberrations study in CHO cells (de Vogel and Usta, 2007)

Title: Chromosomal Aberration test with AN-2 in Cultured Chinese Hamster Ovary (CHO) 

Cells

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 4,4'-methylenebis(2,6-di-tert-butylphenol) (AN-2; Phenol, 4,4’-methylenebis[2,6-

bis(1,1-dimethylethyl)-), CASRN 118-82-1

Remarks: 99.45% purity; white powder; production lot: 5127; MW = 424.7; supplier: 

Albemarle Corporation; TNO Dispense No. 060123; vehicle: DMSO

METHOD

Method/guideline followed: OECD TG 473, 40 CFR 799.9537

Test type: Chromosomal Aberration Test in Cultured Chinese Hamster Ovary Cells

GLP compliant?: Yes

Year study performed: 2006

Test species: Chinese hamster ovary (CHO) cells

Metabolic activation: With and without

Concentrations tested: First test (pulse exposure; with and without activation): 0, 0.1, 0.2, 

0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, 100, and 200 μg/mL; second test (pulse exposure; with 

activation): 0, 5, 10, 30, 50, 75, 100, 150, 200, and 300 μg/mL; second test (continuous 

exposure; without activation): 0, 1.25, 2.5, 5, 10, 30, 50, 75, 100, 150, 200, and 300 μg/mL.

Statistical methods used: Fisher’s exact probability test (two-sided) was conducted to 

determine significant differences between treated and control cultures.
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Test method/conditions remarks: 

Cells: Chinese Hamster Ovary cells (CHO K-1 line) were stored as frozen stock cultures, and 

subcultures were made from these stocks. Each passage was tested for mycoplasma 

contamination and karyotype stability. The medium for culturing the CHO cells was Ham's F-12 

medium (with Glutamax-I) supplemented with heat-inactivated fetal calf serum (10%), 

penicillin, and streptomycin. 

Metabolic Activation System: The S-9 mix consisted of a liver homogenate fraction and 

cofactors, derived from Aroclor 1254-induced Wistar rats. The final concentration of S-9 mix in 

culture medium used in the tests requiring activation was 4%. 

Preparation of Dose Levels: Dimethyl sulfoxide (DMSO) was chosen for the vehicle. Based on 

the solubility pre-test, 200 μg/mL final concentration in the culture medium was chosen as the 

highest dose level for the first test.

Methods for 1st Chromosomal Aberration Test: Exponentially growing cells were seeded in 

sterile tissue culture flasks containing culture medium, and incubated at 37°C in humidified air 

containing 5% CO2. Approximately 24 hours after seeding, the cells were exposed to the test 

substance or control substance, with or without S-9 mix. In the presence of S-9, the culture 

medium with fetal calf serum was replaced by culture medium with penicillin and streptomycin, 

but without calf serum. In the first test (pulse exposure), the cells were exposed (in duplicate 

cultures) to the test substance for 4 hours at the following concentrations, either with or without 

activation: 0 (vehicle control), 0.1, 0.2, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, 100, and 200 

μg/mL. At the end of the exposure, the cultures were checked, the medium with test substance 

was removed, and the cells were washed twice and supplied with fresh culture medium. The 

cells were incubated for another 14 hours at 37°C in humidified air with 5% CO2. Two hours 

before the end of the culture period, the cells and culture media were checked visually and 

selected cultures were arrested in metaphase by the addition of colcemid. The cells were 

harvested at 18 hours by trypsinization. The following concentrations were selected for cell 

sampling: 0, 6.25, 12.5, 25, 50, 100, and 200 μg/mL.

Methods for 2nd Chromosomal Aberration Test: In the second test, the 4-hour pulse exposure 

with activation was repeated at the following concentrations: 0, 5, 10, 30, 50, 75, 100, 150, 200, 

and 300 μg/mL. The following concentrations were selected for cell sampling: 0, 50, 75, 100, 

150, 200, and 300 μg/mL. In the absence of activation, the cells were exposed continuously for 

18 hours and harvested immediately after the exposure period. The concentrations used for 

continuous exposure were: 0, 1.25, 2.5, 5, 10, 30, 50, 75, 100, 150, 200, and 300 μg/mL. The 

concentrations selected for cell sampling were: 0, 1.25, 2.5, 5, 10, and 30 μg/mL.

Positive Control Substance: The positive control substances were: cyclophosphamide 

(5 μg/mL) with activation and mitomycin C (0.1 μg/mL) without activation.

Methods for Determining Mitotic Index: Two slides were prepared from each culture that had 

been selected for cell sampling. For each culture, at least 1000 nuclei were examined (500 per 

slide) to determine the mitotic index (percentage of cells in mitosis). On the basis of the mitotic 

index observed, three concentrations of the test substance were selected for the scoring of 
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chromosomal aberrations. If possible, the highest concentration chosen reduced the mitotic 

index by at least 50% (but not more than 70%) when compared to the negative control, or 

exhibited some other clear indication of cytotoxicity. For each chosen concentration, 200 wellspread metaphases (100 metaphases/culture, each containing 20–22 centromeres) were examined 

for chromatid-type aberrations (gaps, breaks, fragments, and interchanges) and chromosome-type 

aberrations (gaps, breaks, minutes, rings, and dicentrics). Heavily damaged cells were not 

included in the 200 analyzed cells.

Evaluation of Test Results: The criteria for a clastogenic response are: a concentration-related 

increase in the percentage of cells with chromosomal aberrations over the concurrent control 

frequencies or a single positive test point in both tests at approximately the same dose level. 

Cells with only gaps (achromatic lesions) were not included in the final assessment of 

clastogenic activity. If the increase in cells with chromosomal aberrations is marginally 

significant (0.05 < p < 0.1), then the response is considered equivocal.

RESULTS

Genotoxicity result: Negative

Cytotoxic concentration (if applicable):1 ≥ 100 μg/mL in pulse exposures with or without 

activation; ≥ 10 μg/mL in continuous exposure without activation

Results remarks: 

1

st Chromosomal Aberration Test: In the first test, in the pulse treatment group with metabolic 

activation, the three concentrations of test material chosen for chromosomal aberration analysis 

were 50, 100, and 200 μg/mL. The mitotic index of the highest concentration analyzed 

(200 μg/mL) was reduced to 71% of the concurrent vehicle control (DMSO). The test substance 

did not induce a statistically significant increase in the number of aberrant cells at any of the 

dose levels analyzed when compared to the number of aberrant cells observed in the concurrent 

control (DMSO).

In the first test, in the pulse treatment group without activation, the three concentrations of test 

material chosen for chromosomal aberration analysis were 50, 100, and 200 μg/mL. The mitotic 

index of the lowest concentration analyzed (50 μg/mL) was not reduced; the mitotic indices of 

the middle concentration (100 μg/mL) and the highest concentration (200 μg/mL) were reduced 

to 59 and 54%, respectively, of that of the concurrent vehicle control. The test substance did not 

induce a statistically significant increase in the number of aberrant cells at any of the dose levels 

analyzed when compared to the number of aberrant cells in the vehicle control group.

2

nd Chromosomal Aberration Test: In the second test, in the pulse treatment group with 

metabolic activation, dose-related toxicity could be demonstrated. The test concentrations 

chosen for chromosomal aberration analysis were 100, 200, and 300 μg/mL. The mitotic index 

of the lowest concentration analyzed (100 μg/mL) was reduced to 80%; the mitotic index of the 

mid-concentration analyzed (200 μg/mL) was reduced to 49%; and the mitotic index of the 

highest concentration analyzed was reduced to 40% of that of the concurrent vehicle control. 

 

1 Mitotic index was ≤ 80% of control value at cytotoxic concentrations.
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The test substance did not induce a statistically significant increase in the number of aberrant 

cells at any of the dose levels analyzed, when compared to the aberrant cells found in the 

concurrent (DMSO) control cultures.

In the second test, in the continuous treatment group of 18 hours without metabolic activation, 

dose-related toxicity could be demonstrated. Dose levels chosen for chromosomal aberration 

analysis were 2.5, 5, and 10 μg/mL. The mitotic index of the lowest concentration analyzed 

(2.5 μg/mL) was 91%, the mitotic index of the mid-concentration analyzed (5 μg/mL) was 

reduced to 87%, and the mitotic index of the highest concentration analyzed (10 μg/mL) was 

reduced to 47% of that of the concurrent vehicle control. At the next higher concentration 

selected for cell sampling (30 μg/mL), the mitotic index was reduced to 11%. This strong

reduction was above the acceptable toxicity level and thus this dose was not selected for 

chromosomal aberration analysis. The test substance did not induce a statistically significant 

increase in the number of aberrant cells at any of the dose levels analyzed, when compared to the 

number of aberrant cells found in the concurrent control cultures.

Precipitation of the test substance was observed at 100 and 200 μg/mL in the first test and at 

300 μg/mL in the second test.

Criteria for validity of the study were met by the positive controls (mitomycin C without S-9, 

and cyclophosphamide with S-9) giving the expected statistically increased numbers of aberrant 

cells and the negative (vehicle) control results falling within the historical range.

CONCLUSIONS

Under the conditions of the study, the test substance was not clastogenic to CHO cells.

STUDY RELIABILITY

[1] Reliable without restrictions. 

REFERENCE

de Vogel, N. and B. Usta. 2007. Chromosomal Aberration test with AN-2 in Cultured Chinese 

Hamster Ovary (CHO) Cells. TNO Laboratories, Zeist, Netherlands, Project No. 

031.11113/01.42, Study No. 6802/24, May 9, 2007. Submission documented online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2005-0033-0257 as of 

December 4, 2013. 
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Study 2: Chromosomal aberrations study in Chinese hamster V79 cells (Sandoz Chemical 

Ltd, 1992)

Title: In vitro mammalian chromosome aberration test

This study is: Adequate Not Adequate

Important study details missing from the study summary included description of the metabolic 

activation system, solvent and positive control information (named by strain and metabolic 

activation), statistical methods used, and additional quantitative data on the results and 

information on cytotoxicity. Other deficiencies include: (1) The shortest exposure period was 

18 hours rather than the OECD-recommended 3–6 hour exposure period. (2) The SIDS/IRPTC 

Data Profile stated that the test substance negatively affected the plating efficiency of the cells at 

concentrations greater than 3 μg/mL in the absence of S-9 mix and at concentrations greater than 

30 μg/mL in the presence of S-9 mix. It is uncertain whether or not this is indicative of 

cytotoxicity. (3) A limit concentration of 5 mg/mL is recommended by OECD TG 473; 

however, no rationale was provided for testing only up to 10.0 μg/mL without activation and 

only up to 50.0 μg/mL with activation.

TEST SUBSTANCE 

Identity: Analog: 2,6-Di-tert-butylphenol, CASRN 128-39-2

Remarks: 98.7% purity

METHOD

Method/guideline followed: OECD TG 473 (EEC Directive 84/449, B 10)

Test type: In vitro mammalian chromosomal aberration test

GLP compliant?: Yes

Year study performed: 1992

Test species: Chinese hamster V79 cells

Metabolic activation: With and without S-9 mix

Concentrations tested: 0.3, 3.0, 6.0, 10.0 μg/mL for 18 hrs and 6.0 μg/mL for 28 hrs without 

S9 mix; 3.0, 30.0, 50.0 μg/mL for 18 hrs and 50.0 μg/mL for 28 hrs with S9 mix. 

Statistical methods used: Not specified

Test method/conditions remarks: The exposure period was 18 to 28 hours.
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RESULTS

Genotoxicity result: Negative with and without metabolic activation

Cytotoxic concentration (if applicable): > 3.0 μg/mL without S-9 mix and > 30.0 μg/mL with 

S-9 mix.

Results remarks: No mutagenic effects were obtained with or without metabolic activation. 

CONCLUSIONS

The test substance did not induce chromosomal aberrations in Chinese hamster V79 cells either 

in the absence or presence of metabolic activation. 

STUDY RELIABILITY

[3] Not reliable. Important study details missing from the study summary included description 

of the metabolic activation system, solvent and positive control information (named by strain and 

metabolic activation), statistical methods used, and additional quantitative data on the results and 

information on cytotoxicity.

Other deficiencies include: (1) The shortest exposure period was 18 hours rather than the

OECD-recommended 3–6 hour exposure period. (2) The SIDS/IRPTC Data Profile stated that 

the test substance negatively affected the plating efficiency of the cells at concentrations greater 

than 3 μg/mL in the absence of S-9 mix and at concentrations greater than 30 μg/mL in the 

presence of S-9 mix. It is uncertain whether or not this is indicative of cytotoxicity. (3) A limit 

concentration of 5 mg/mL is recommended by OECD TG 473; however, no rationale was 

provided for testing only up to 10.0 μg/mL without activation and only up to 50.0 μg/mL with 

activation.

REFERENCE

Sandoz Chemical Ltd. 1992. Unpublished CCR Report [on a chromosomal aberration assay] -

Sandoz, 243628 (full study report not available). Muttenz, Switzerland. As summarized in: 

OECD. 2002. SIDS/IRPTC Data Profile for 2,6-Di-tert-butylphenol (CASRN 128-39-2). 

UNEP Publications. 2002. Available online at 

http://www.chem.unep.ch/irptc/sids/OECDSIDS/128392.pdf as of December 4, 2013.
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C-9. Reproductive/Developmental Toxicity

Summary of endpoint: Adequate

Study 1: Preliminary reproduction/developmental toxicity screening test in rats (Sandoz 

Chemical Ltd., 1992, as summarized in the online ECHA entry for 2,6-di-tert-butylphenol, 

Exp Key Toxicity to reproduction.001)

Title: Not provided

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Analog: 2,6-Di-tert-butylphenol, CASRN 128-39-2

Remarks: 98.7% purity; Lot/batch No. 10/6; solid at 20°C (colorless solid)

METHOD

Method/guideline followed: OECD Preliminary Reproduction/Developmental Toxicity 

Screening Test (Precursor Protocol of OECD TG 421 [1995])

Test type: One-Generation Reproduction/Developmental Toxicity

GLP compliant?: Yes

Year study performed: 1992

Test species: Wistar rats; source: Biological Research Laboratories Ltd., Switzerland;

age: at least 11 weeks old; weight: 304–339 g (males) or 182–229 g (females) at study initiation.

Number of animals/sex/dose: 10

Route of administration: Oral gavage

Vehicle: Bi-distilled water with 4% carboxymethyl cellulose (CMC)

Exposure duration: Parental females were exposed for 2 weeks prior to mating, throughout the 

mating period (up to 13 days), and through gestation and lactation up to postpartum day 4. 

Parental males were exposed for 2 weeks prior to mating, throughout the mating period, and 

following mating for a total exposure period of 43 days.

Doses/concentrations tested: 0 (vehicle control), 30, 150, and 750 mg/kg-bw/day
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Statistical methods used: Statistical analyses were performed on data for body weight, food 

consumption, organ weights, reproduction, and breeding. If the variables followed a normal 

distribution, the Dunnett’s many-to-one t-test, based on a pooled variance estimate, was used for 

intergroup comparisons (i.e., single treatment groups against the control group). The Steel-test 

(many-to-one rank test) was applied when the data did not follow a normal distribution. For the 

spontaneous mortality of pups data, Fisher's Exact test (2×2 tables) was applied.

Test method/conditions remarks: Dose selection was based on the results of a subacute 28-day 

toxicity study (RCC NOTOX Project 057454). Mixtures of test substance and vehicle were 

prepared daily before administration. 

Male and female Wistar rats (10/sex/dose) were administered the test substance via gavage at 

doses of 0, 30, 150, and 750 mg/kg-bw/day daily. Females received the test substance for 

2 weeks prior to mating, throughout the mating period (up to 13 days, except for one female 

which was paired for 17 days), and through gestation and lactation up to postpartum day 4. 

Males received the test substance for 2 weeks prior to mating, throughout the mating period, and 

following mating for a total exposure period of 43 days. The day on which spermatozoa were 

found in the vaginal smear or a vaginal plug was observed was designated as day 0 post coitum. 

After successful mating, each pregnant female was separated and housed individually. 

Animals were observed at least twice daily for mortality and clinical signs; body weights were 

recorded daily. Females were observed for signs of difficult or prolonged parturition. Food

consumption in males was recorded weekly during the pre-mating and postmating periods. Food 

consumption in females was recorded for days 1–8 and 8–14 of the pre-mating period, days 0–7, 

7–14, and 14–21 post coitum, and days 0–4 postpartum. No food consumption was recorded 

during the mating period. Males were sacrificed when they were no longer necessary for the 

assessment of reproductive effects, and dams were sacrificed on day 4 postpartum.

All parental animals were necropsied and examined macroscopically for any structural 

abnormalities or pathological changes. Uteri of non-pregnant females were placed in a solution 

of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites. The testes

and epididymides of all parental males were weighed. Histopathological examination of the 

testes and ovaries was performed on animals from the control and high-dose group.

The following reproductive and developmental parameters were recorded: number of corpora 

lutea, number of implantations, litter size, number of live births, number of stillbirths, sex ratio,

number of pups living on day 4 postpartum, and pup body weights on days 0, 1, and 4 

postpartum. Dead pups (except if excessively cannibalized) and pups killed at day 4 of lactation 

were examined macroscopically for gross abnormalities. 

RESULTS

Endpoint value(s): The LOAEL for parental toxicity was 750 mg/kg-bw/day based on severe 

clinical signs of toxicity (sedation, ataxia, lateral or ventral recumbency, tremor, clonic spasms 

of the forepaws, hunched posture, and salivation) in both sexes, reduced food consumption in 

females during the pre-mating period, and reduced body weight gain in both sexes (statistical 
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significance not reported). The NOAEL for parental toxicity was 150 mg/kg-bw/day. The 

LOAEL for reproductive and developmental toxicity was 750 mg/kg-bw/day based on reduced 

viability index and a reduction in pup body weight gain. The NOAEL for reproductive and 

developmental toxicity was 150 mg/kg-bw/day.

Results remarks: No deaths of parental animals occurred during the course of the study. 

Severe clinical signs of toxicity were observed in high-dose males and females. In males, the 

signs were observed from day 2 of the pre-mating period until day 5 of the mating period. Signs 

included ruffled fur, sedation, ataxia, lateral or ventral recumbency, tremor, clonic spasms of the 

forepaws, hunched posture, and salivation. After day 5 of the mating period, until termination of 

the study, the number and intensity of the signs diminished continuously and/or disappeared. In 

parental females, the identical signs were noted during the pre-mating, mating, and gestation 

periods. During the lactation period, only ruffled fur was observed.

There was a marginal reduction in body weight gain for males of the high-dose group during the 

pre-mating and mating periods. During the gestation period (days 0–21 post coitum), the mean 

body weight gain in females from the vehicle control, and low-, mid-, and high-dose groups was 

51.1, 52.8, 55.2, and 47.3%, respectively. During days 1–4 of lactation, mean body weight gains 

of 8.3, 4.5, 6.9, and 2.8% were observed in control, and low-, mid-, and high-dose females, 

respectively. Neither secondary source (OECD/SIDS or ECHA) reported statistical significance 

for these findings. 

The mean food consumption of high-dose males was statistically significantly increased from 

days 8 to 14 of the pre-mating period and from days 1 to 10 after the mating period; secondary 

sources did not report the magnitude of changes. No differences were observed among mean 

food consumption rates during both periods in males from the vehicle control, low-dose, and 

mid-dose groups. The mean food consumption of high-dose females was statistically 

significantly reduced between days 1 and 8 of the pre-mating period (magnitude of change not 

reported in secondary sources). During days 8 to 14 of the pre-mating period, the mean food 

consumption in high-dose females was only slightly increased (compensatory reaction; 

magnitude not reported). No test article-related differences in the mean food consumption of the 

females were noted during the gestation and lactation periods. Statistically significantly 

increased mean food consumption was observed in females from the mid-dose group between 

days 14 and 21 post coitum; however, this increase “is considered to be incidental because from 

the beginning of the study in this group, the highest food consumption was recorded.” [ECHA]

The mean organ weights and organ/body weight ratios for testes and epididymides of males were 

similar in all groups. Macroscopic examination of parental animals revealed no test substancerelated effects. The microscopic examination of testes and ovaries of parental animals from the 

control and high-dose groups showed no indication of test substance-related effects. 

A significant (p ≤ 0.01) decrease in viability index (survival from postnatal day 0 to 4) was 

observed in pups from the high-dose group (90.3%) compared to controls (99.1%). No test 

substance-related effects were observed for pre-coital time, percentage mating, fertility index,

and conception rate in any dose group. 
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Pup body weight at birth was similar among all groups. However, body weight gain of pups 

from days 1 to 4 postpartum was reduced in the high-dose group (23.3% weight gain) compared 

to controls (38.3% weight gain). No effects were observed upon external examination or

necropsy of the pups in any group.

CONCLUSIONS

The LOAEL for parental toxicity was 750 mg/kg-bw/day based on severe clinical signs of 

toxicity (sedation, ataxia, lateral or ventral recumbency, tremor, clonic spasms of the forepaws, 

hunched posture, and salivation) in both sexes, reduced food consumption in females during the 

pre-mating period, and reduced body weight gains in both sexes (statistical significance not 

reported). The NOAEL for parental toxicity was 150 mg/kg-bw/day. The LOAEL for 

reproductive and developmental toxicity was 750 mg/kg-bw/day based on reduced viability 

index and a reduction in pup body weight gain. The NOAEL for reproductive and 

developmental toxicity was 150 mg/kg-bw/day.

STUDY RELIABILITY

[2] Reliable with restrictions. Histological examination of the epididymides, which is 

recommended by OECD TG 421, was not conducted. Also, reviews of this study reported by 

secondary sources did not include results of statistical analyses for all variables examined, 

including maternal weight gain, postnatal loss during lactation, and fetal body weight. 

REFERENCE

Sandoz Chemical Ltd. 1992. Unpublished CCR Report [on a reproduction/developmental 

toxicity test] - Sandoz, 321794. Muttenz, Switzerland. As summarized in the online ECHA 

(http://apps.echa.europa.eu/registered/data/dossiers/DISS-97d6a15b-c218-5de0-e044-

00144f67d031/AGGR-e91a95e9-69e4-4f56-a8ac-562a89c8c06f_DISS-97d6a15b-c218-5de0-

e044-00144f67d031.html#AGGR-e91a95e9-69e4-4f56-a8ac-562a89c8c06f) entry for CASRN 

128-39-2 (study indicated as Exp Key Toxicity to reproduction.001, available online as of 

December 2, 2013). Secondary Reference: OECD. 2002. SIDS/IRPTC Data Profile for 2,6-Ditert-butylphenol (CASRN 128-39-2). UNEP Publications. 2002. Available online at 

http://www.chem.unep.ch/irptc/sids/OECDSIDS/128392.pdf as of December 4, 2013.

Text in quotes is taken directly from ECHA.

Copyright Notice: © European Chemicals Agency, 2007-2011. Reproduction is authorized 

provided the source is fully acknowledged in the form “Source: European Chemicals Agency, 

http://echa.europa.eu/”, and provided written notification is given to the ECHA Communication 

Unit (info (at) echa.europa.eu).
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Study 2: Fertility screening study in rats (Davis and Jolley, 1961)

Title: A comparison of the effects of two antioxidants on gestation and parturition in the rat 

when fed several concentrations in the diet

This study is: Adequate Not Adequate

The study was not conducted according to standard guidelines. The highest dose tested did not 

induce toxic effects as recommended in guideline 40 CFR 799.9355 and was below the 

recommended limit dose of 1000 mg/kg-bw/day. Male and female parental animals were not 

dosed for a minimum of 2 weeks prior to mating as recommended in the guideline (females were 

exposed for only 1 week before mating and males were exposed only during mating). The 

number of pregnant females per group was less than the minimum number of eight that is

recommended in guideline 40 CFR 799.9355. Only two males were used per group, whereas the 

guideline recommends ten. Guideline-required parameters that were not evaluated included 

body weight, food consumption, clinical signs of parental rats, and weights and histology of

reproductive organs. Pups were not examined for gross abnormalities. Important study details 

that were missing from the study summary included test substance purity and statistical methods 

used.

TEST SUBSTANCE 

Identity: Phenol, 4,4’-methylenebis[2,6-bis(1,1-dimethylethyl)-, CASRN 118-82-1; Referred to 

as Antioxidant 702 and 4,4’-methylenebis(2,6-di-tert-butylphenol) in study report

Remarks: Purity not provided; Light yellow crystalline solid

METHOD

Method/guideline followed: Not specified

Test type: Reproductive/Developmental Toxicity Study - Rats

GLP compliant?: N/A (pre-GLP)

Year study performed: 1961

Test species: CFN rats

Number of animals/sex/dose: 2 males and 10 females/group

Route of administration: Oral - Diet

Vehicle: Alphacel (non-nutritive cellulose flour)
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Exposure duration: Females were exposed during a pre-mating period for 7 days and

throughout mating (12 days), gestation, and parturition to day 4 of lactation. Males were 

exposed only during mating. 

Doses/concentrations tested: 0 (control), 0.125, 0.5, and 1.25% in feed (approximately 62.5, 

250, and 625 mg/kg-bw/day, calculated for this review, using a food factor of 0.05)1

Statistical methods used: None specified

Test method/conditions remarks: Females were exposed during a pre-mating period for 7 days

and throughout mating (12 days), gestation, and parturition to day 4 of lactation. Males were 

exposed only during the mating period. Concentrations in feed were 0.125, 0.5, and 1.25%. A 

negative control group was maintained on a basal diet and a positive control group was 

maintained on the basal diet plus 1.25% N,N'-diphenyl-p-phenylenediamine (DPPD). 

Females were housed in groups of five per cage for the duration of the experiment. During the 

mating period, one male was housed with each group of five females.

RESULTS

Endpoint value(s): NOAEL (reproductive/developmental toxicity) = 1.25% (equivalent to 

625 mg/kg-bw/day; based on no effects at the highest dose tested).

Results remarks: The test substance had no effect on fertility, length of gestation, number of 

live or dead pups per litter at birth, pup weight, or the viability of offspring during the initial 

4 days after delivery.

Results of reproductive performance are provided in the table below.

% 

compound 

in diet

No. of females

Avg. length 

of gestation 

(days)

No. of 

litters

Avg. 

weight of 

pups (g)

Avg. nos. per litter Avg. no. of 

surviving 

pups at 

postpartum 

Bred Pregnant Live births Still births day 4

0 10 7 22 7 5.8 10.9 0.7 9.3

1.25 10 6 22 6 5.9 11.8 0.2 11.3

0.5 10 10 22 10 6.2 9.9 0.3 9.1

0.125 10 9 22 9 6.2 9.6 1.3 8.7

Rats fed DPPD (positive control) had an increased incidence of stillborn pups (~ 50%), but there 

was no effect on the number of young or the weight of offspring. The period of gestation of 

3/10 rats fed on the diet containing DPPD was prolonged (24–25 days). Two of these rats died 

during the 25th day of pregnancy and prior to parturition. At autopsy, their unborn litters were 

found to be normal in number and size. 

 

1 1 mg/kg in feed = 0.05 mg/kg-bw/day.
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CONCLUSIONS

The NOAEL for reproductive and developmental toxicity was 1.25% (equivalent to 625 mg/kgbw/day; based on no effects at the highest dose tested).

STUDY RELIABILITY

[3] Not reliable. The study was not conducted according to standard guidelines. The highest 

dose tested did not induce toxic effects as recommended in guideline 40 CFR 799.9355 and was 

below the recommended limit dose of 1000 mg/kg-bw/day. Male and female parental animals 

were not dosed for a minimum of 2 weeks prior to mating as recommended in the guideline 

(females were exposed for only 1 week before mating and males were exposed only during 

mating). The number of pregnant females per group was less than the minimum number of eight

that is recommended in guideline 40 CFR 799.9355. Only two males were used per group, 

whereas the guideline recommends ten. Guideline-required parameters that were not evaluated 

included body weight, food consumption, clinical signs of parental rats, and weights and 

histology of reproductive organs. Pups were not examined for gross abnormalities. Important 

study details that were missing from the study summary included test substance purity and 

statistical methods used.

REFERENCE

Davis, R.K. and W. Jolley. 1961. “A Comparison of the Effects of Two Antioxidants on 

Gestation and Parturition in the Rat When Fed Several Concentrations in the Diet,” The 

Kettering Laboratory in the Department of Preventive Medicine and Industrial Health, College of 

Medicine, University of Cincinnati, Cincinnati, Ohio, for Ethyl Corporation. (Submitted as part 

of Food Additive Petition No. 4B1469 and Food Additive Petition 1397, Food and Drug 

Administration). Available online at http://www.regulations.gov/#!documentDetail;D=EPAHQ-OPPT-2005-0033-0181 (on pp. 10-21 of “End_Point_F2 –Mammalian_Toxicity_-

_Repeated_Dose_Reproduction_Developmental.pdf”) as of December 4, 2013. 
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Study 3: Three-generation reproductive toxicity study in rats (Witherup et al., 1969)

Title: The effects exerted upon the fertility of rats and upon the viability of their offspring by 

the introduction of 4,4'-methylenebis (2,6-ditertiary butylphenol) into their diets

This study is: Adequate Not Adequate

The highest dose tested did not induce toxic effects as recommended in guideline 40 CFR 

799.9355 and was below the recommended limit dose of 1000 mg/kg-bw/day. In addition, 

results of this study are considered equivocal due to a potential treatment-related decrease in the 

lactation index in F2b pups; therefore, NOAEL and LOAEL values for developmental toxicity 

cannot be determined. Important study details that were missing from the study summary 

included test substance purity, test method/guideline followed, and statistical methods used. 

Measurements of body weights and food consumption were not conducted for the parental 

animals. Additionally, extraneous disease and underlying environmental factors complicate the 

interpretation of the results.

TEST SUBSTANCE 

Identity: Phenol, 4,4’-methylenebis[2,6-bis(1,1-dimethylethyl)-, CASRN 118-82-1; Referred to 

as BHD in study report

Remarks: Purity not provided; obtained from the Ethyl Corporation; finely divided crystalline 

cream-colored solid

METHOD

Method/guideline followed: Not specified

Test type: Three-Generation Reproduction Toxicity Study - Rats

GLP compliant?: N/A (pre-GLP)

Year study performed: 1969

Test species: CD Rats; obtained from the Charles River Breeding Laboratories, Inc., Brookline, 

Massachusetts.

Number of animals/sex/dose: 20 females and 10 males/group

Route of administration: Oral - Diet

Vehicle: Corn oil (test substance was dissolved in corn oil prior to incorporation into the diet)
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Exposure duration: P males were exposed for 8 weeks during a pre-mating period and 

throughout mating; P females were exposed for 8 weeks during a pre-mating period and during

mating, gestation, lactation, and weaning of the F1 offspring (until postnatal day [PND] 21); F1

and F2 animals were exposed for the same duration as the P animals, starting at the time of 

weaning (i.e., at 3 weeks old).

Doses/concentrations tested: 0, 15, 60, 100, 500, and 3000 ppm in feed (approximately 

equivalent to 0, 1.3, 5.2, 8.6, 43, and 258 mg/kg-bw/day for males; 1.5, 5.9, 9.8, 49, and 

294 mg/kg-bw/day for females1)

Statistical methods used: Not specified

Test method/conditions remarks: “The feeding of the experimental diets was started when the 

initial animals were approximately 7 weeks of age; thereafter, the initial progenitors and 3 

successive generations of offspring were maintained exclusively on the respectively assigned 

diets. After the initial animals had fed on the respective diets for 8 weeks and were 

approximately 105 days of age, the 2 sexes were housed together, 2 females and 1 male being 

fed on the same diet per cage. The male rats were kept with the females until the pregnancy of 

each female was ascertained by a sharp increase in body weight. The pregnant females were 

placed in individual nesting cages and were observed daily thereafter. If neither female was 

evidently pregnant after 4 weeks, another male that had already sired offspring was placed with 

the females and the delinquent male was housed with females of established fertility.” [Witherup 

et al. (1969), p. 4-5]

“Shortly after birth, each pup was examined for anomalies in the form, size, and color of the 

trunk; the number, size, formation, and location of the various appendages (head, feet, limbs, 

toes, and tail), and the number and location of the rudimentary ears, eyes, nose, and mouth were 

examined also. The pups were counted and weighed as a group.” [Witherup et al. (1969), p. 5]

“The initial progenitors and 2 generations of offspring were given 2 opportunities to mate

[resulting in two litters, “a” and “b”]; the first litter of each initial female [from the P generation] 

was permitted to suckle for 7 days [or 4 days for pups from F1 females]; the pups then were 

killed and their viscera were examined for gross anatomical anomalies and pathological 

abnormalities. After an interval of 1 week, the dams again were housed with the males. The 

offspring resulting from the second period of mating were permitted to suckle for 3 weeks, the 

normal age of weaning. The pups then were grouped according to their sex and were counted 

and weighed as a group. The offspring of the [P and F1] animals fed on each diet were reduced 

in number by random selection to 20 females and 10 males [per dietary concentration]; these [F1 

and F2 animals] were continued on the same diets as had been fed to their progenitors and in turn 

were mated when about 3 months of age.” [Witherup et al. (1969), p. 5]

“Pups in the first litters of the F1 and F2 females were counted and weighed when 4 days of age; 

those of the Fl females were killed and the dams were remated. The pups in both litters of the F2 

 

1 Doses were converted from ppm to mg/kg-bw/day using the SRC Dose Calculator v1.0, with exposure duration set 

to “subchronic” and using default body weights and food consumption values for Sprague-Dawley rats because CD 

rats are Sprague-Dawley-derived. 
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females and those in the second litters of the F1 females were reduced to no more than 10 pups 

per litter and continued to suckle on the dams until they were weaned at 21 days of age.” 

[Witherup et al. (1969), pp. 5-6]

“Representative animals of the F3 generations when 21 days of age were killed by intraperitoneal 

injections of Nembutal. Their viscera were examined for gross pathological changes. Tissues of 

the brain, heart, lungs, liver, kidneys, spleen, intestinal tract and the adrenal glands were 

prepared for microscopic examination; these were fixed in neutral formalin, embedded in 

paraffin and cut in sections of 6 to 8 microns in thickness. They were stained with hematoxylin 

and eosin.” [Witherup et al. (1969), p. 6]

RESULTS

Endpoint value(s): The NOAEL for systemic and reproductive toxicity was 3000 ppm (258 and 

294 mg/kg-bw/day for males and females, respectively) based on no observed treatment-related 

effects up to the highest dietary concentration tested. Due to a potential treatment-related 

decrease in the lactation index observed only in F2b pups, results of this study are considered 

equivocal and NOAEL and LOAEL values for developmental toxicity cannot be determined.

Results remarks: There were no treatment-related effects on survival of parental rats. 

Occasional fatalities in the procreating animals were attributed to dystocia or pneumonia.

Fertility indices of the P generation (both test substance-exposed and control) were poor (53.8–

87.2%) compared to subsequent generations (80–100%). Variations in gestation index were 

noted in test substance-exposed and control animals from successive generations (75–100%). 

Variations in the viability index were noted in the first generation progeny from all groups, 

including controls (20.8–83.7%). The differences in fertility, gestation, and viability indices 

showed no dose-relationship and were attributed to the influence of extraneous diseases 

unrelated to the test substance; therefore, these effects are not considered to be treatment-related.

A dose-related decrease in survival from postpartum days 4 to 21 [i.e., the lactation index, 

calculated as: (number of pups alive on postpartum day 21/number of pups entered into the 

lactation phase on postpartum day 4) × 100%] was observed in the F2b generation at dietary 

concentrations of 100, 500, and 3000 ppm; lactation indices were 88.9, 91.9, 83.2, 67.1, 62.8, 

and 47.3% at dietary concentrations of 0, 15, 60, 100, 500, and 3000 ppm, respectively. The 

study authors did not regard the observed decrease as treatment-related because no dose-related 

changes were observed in the lactation index of F3 offspring. However, the study authors did

not provide additional information or data to evaluate other possible causes of the dose-related 

effect in the F2b generation. A relationship between the decrease in lactation index and 

treatment with the test diets cannot be ruled out; therefore, results of this study are considered 

equivocal.

There were no effects on body weights of offspring. 

Approximately 5400 rats that were progeny of animals fed on diets containing the test article 

were examined for anatomical anomalies; none were found. All of the offspring were normal in 
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appearance of the trunk and appendages, whether examined at birth, 4 days, 7 days, or 3 weeks 

of age. In the control offspring (1100), there were 5 pups in the first litter of one of the second 

generation females that were dismembered by the dam. No gross pathological changes were 

observed in the viscera of pups killed at 4, 7, or 21 days of age. Histological examination of the 

brain, heart, lungs, kidneys, liver, spleen, intestinal tract, and adrenal glands of the third

generation of offspring (F3a, b) revealed no pathology. 

CONCLUSIONS

Throughout the study, the animals were affected by extraneous diseases complicated by 

environmental factors that were unrelated to the content of test substance in the diets. The test 

substance had no effect on fertility and did not produce anatomical anomalies in offspring. The 

NOAEL for systemic and reproductive toxicity was 3000 ppm (258 and 294 mg/kg-bw/day for 

males and females, respectively) based on no observed treatment-related effects up to the highest 

dietary concentration tested. However, due to a potential treatment-related decrease in the 

lactation index observed only in F2b pups, results of this study are considered equivocal and 

NOAEL and LOAEL values for developmental toxicity cannot be determined.

STUDY RELIABILITY

[3] Not reliable. The highest dose tested did not induce toxic effects as recommended in 

guideline 40 CFR 799.9355 and was below the recommended limit dose of 1000 mg/kg-bw/day. 

In addition, results of this study are considered equivocal due to a potential treatment-related 

decrease in the lactation index in F2b pups; therefore, NOAEL and LOAEL values for 

developmental toxicity cannot be determined. Important study details that were missing from the 

study summary included test substance purity, test method/guideline followed, and statistical 

methods used. Measurements of body weights and food consumption were not conducted for the 

parental animals. Additionally, extraneous disease and underlying environmental factors 

complicate the interpretation of the results.

REFERENCE

Witherup, S., K.L. Stemmer, P. Taylor, and E.A. Pfitzer. 1969. “The Effects Exerted Upon the 

Fertility of Rats and Upon the Viability of Their Offspring by the Introduction of 4,4'-

Methylenebis (2,6-Ditertiary Butylphenol) Into Their Diets,” The Kettering Laboratory in the 

Department of Preventive Medicine and Industrial Health, College of Medicine, University of 

Cincinnati, Cincinnati, Ohio, for Ethyl Corporation. Available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2005-0033-0181 (on pp. 22-

61 of “End_Point_F2 –Mammalian_Toxicity_-_Repeated_Dose_Reproduction

_Developmental.pdf”) as of December 4, 2013.

Text in quotes was taken directly from Witherup et al., 1969.
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Study 4: Reproductive toxicity study in rats and dogs (Schering Corporation, 1966)

Title: Not provided

This study is: Adequate Not Adequate

The original study report was not available for review. This was not a standard study of 

reproductive toxicity endpoints; the endpoints assessed and numbers of animals used were 

insufficient to satisfy guideline requirements. Important study details that were missing from the 

summary included test substance purity, test methods/guidelines followed, type of oral route

used in the rat study, dog strain and sex, statistical methods used, test methods/conditions, and 

detailed results/findings. Additionally, LOAEL/NOAEL values were not reported and could not 

be determined based on the limited study details provided.

TEST SUBSTANCE 

Identity: Phenol, 4,4’-methylenebis[2,6-bis(1,1-dimethylethyl)-, CASRN 118-82-1; Synonym: 

SN# 1002, AN 702; Sch 12150

Remarks: Purity not provided

METHOD

Method/guideline followed: Not specified

Test type: Reproductive Toxicity

GLP compliant?: N/A (pre-GLP)

Year study performed: 1966

Test species: Rats (ovariectomized females) and Dogs (strain and sex not specified)

Number of animals/sex/dose: 5/group (rats); Not specified (dogs)

Route of administration: Oral or subcutaneous injection (rat studies); In capsules or as pure 

compound dissolved in edible oil and fed in the diet (dog studies)

Vehicle: Edible Oil (for dietary study in dogs)

Exposure duration: 4 weeks (rats); 20 days (dogs)

Doses/concentrations tested: 

Rats: 100 or 300 mg/kg-bw/day (subcutaneous); 100, 300, or 900 mg/kg-bw/day (oral route)

Dogs: 15, 30, or 45 mg/kg-bw/day
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Statistical methods used: Not specified

Test method/conditions remarks: “Numerous studies were conducted on AN 702 to determine 

an appropriate species for investigation of the pharmacologic activity and mechanism of action 

of AN 702. Most of the studies were performed for Ethyl Corporation by Schering Corporation 

(thus the Sch 12150 test article identification). Some endpoints relative to reproduction were 

assessed:

a. Five rats per group were ovariectomized and given AN702 orally [at 100, 300, or 900 mg/kgbw/day] or subcutaneously [at 100 or 300 mg/kg-bw/day] for four weeks to assess 

hypocholesterol effects. Effects on uterus, vagina, pituitary, and adrenal were also measured...

b. 24 dogs were administered AN 702 for 20 days as micronized, dispersible formulation in 

capsules or as pure compound dissolved in edible oil and fed in the diet. Doses were 15 mg/kg, 

30 mg/kg, and 45 mg/kg. Six weeks after discontinuing the drug, eyes, liver, kidneys, and testes 

were examined histologically.” [EPA-HQ-OPPT-2005-0033-0181, p. 8]

RESULTS

Endpoint value(s): N/A

Results remarks: 

a. Ovariectomized rats injected subcutaneously with AN 702 at 100 or 300 mg/kg-bw/day for 

4 weeks had slightly higher uterine weights than control rats (0.136 and 0.145 g at 100 and 

300 mg/kg-bw/day, respectively, versus 0.108 g in controls). Ovariectomized rats given AN 702 

orally for 4 weeks at doses of 100, 300, or 900 mg/kg-bw/day did not show a trend in uterine 

weight differences (0.117, 0.138, and 0.101 g at 100, 300, and 900 mg/kg-bw/day, respectively,

versus 0.115 g in controls). No differences from control were seen in either group in vaginal 

cytology, pituitary, or adrenal weights. 

b. No abnormalities associated with the test article were found upon histological examination of 

the eyes, liver, kidneys, and testes of dogs six weeks after discontinuing oral administration of 

AN 702. 

CONCLUSIONS

Increased uterine weights (statistical significance not indicated) were observed in ovariectomized 

rats that were injected subcutaneously with 100 or 300 mg/kg-bw/day AN 702 for a period of 

4 weeks. No effects were observed in ovariectomized rats that were dosed orally with up to 

900 mg/kg-bw/day AN 702 for 4 weeks or in dogs dosed orally with up to 45 mg/kg-bw/day AN 

702 for 20 days followed by six weeks of observation. 
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STUDY RELIABILITY

[3] Not reliable. This was not a standard study of reproductive toxicity endpoints; the endpoints 

assessed and numbers of animals used were insufficient to satisfy guideline requirements. 

Important study details that were missing from the summary (the original study report was not 

available for review) included test substance purity, test methods/guidelines followed, type of 

oral route used in the rat study, dog strain and sex, statistical methods used, test 

methods/conditions, and detailed results/findings. Additionally, LOAEL/NOAEL values were 

not reported and could not be determined based on the limited study details provided.

REFERENCE

Schering Corporation. 1966. “AN 702 (Sch 12150) Schering Corporation for Ethyl Corporation 

Taken from ‘Preclinical Information for EY 2333 (702)’ Vol. II, Book 1 of 3, 1966.” Study 

summary available online at http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT2005-0033-0181 (on pp. 7-8 of “End_Point_F2 –Mammalian_Toxicity_-

_Repeated_Dose_Reproduction_Developmental.pdf”) as of December 4, 2013.

Text in quotes is taken directly from EPA-HQ-OPPT-2005-0033-0181.
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Study 5: Teratology study in rats (Powers, 1970a)

Title: Teratology Study - Rats, EY 2333 Final Report

This study is: Adequate Not Adequate

The highest dose tested did not induce toxic effects as recommended in guideline 40 CFR 

799.9355 and was below the recommended limit dose of 1000 mg/kg-bw/day. Additionally, 

important details that were missing from the study included test method/guidelines followed and 

statistical methods used.

TEST SUBSTANCE 

Identity: Phenol, 4,4’-methylenebis[2,6-bis(1,1-dimethylethyl)-, CASRN 118-82-1; referred to 

as EY 2333 in study report

Remarks: 100% purity was assumed by study authors; Sample No. 158-70; Fine white powder 

with noticeable odor

METHOD

Method/guideline followed: Not specified

Test type: Developmental Study - Rats

GLP compliant?: N/A (pre-GLP)

Year study performed: 1970

Test species: Charles River Rats; obtained from Charles River Breeding Laboratories

Number of animals/sex/dose: 18 (control and low-dose) or 19 (high-dose); females only

Route of administration: Oral gavage

Exposure duration: 10 days (gestational days 6–15)

Doses/concentrations tested: 0 (vehicle control), 15, and 150 mg/kg-bw/day

Vehicle: Corn Oil

Statistical methods used: None specified

Test method/conditions remarks: Rats were treated by oral gavage with 0, 15, or 150 mg/kgbw/day in corn oil on gestational days 6 through 15. The control group received corn oil. Two 

females were placed with one male in a breeding cage until the day sperm were noted in a 
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vaginal smear. Females were then housed singly. From a total of 55 females in which sperm 

were noted, animals were randomly assigned to test groups, with 18 females each in the control 

and 15 mg/kg-bw/day groups and 19 females in the 150 mg/kg-bw/day group.

“Mortality, appearance, and behavior of the females were observed daily during the mating and 

gestation periods. Body weights were recorded after observation of sperm, at initiation of 

compound administration, at a five-day interval during treatment, at termination of treatment, 

and prior to sacrifice for Caesarean delivery. Food consumption was recorded for the period 

prior to treatment, at two intervals during treatment, and for the period following treatment to 

time of sacrifice” [Powers (1970a), p. 4]

“Females were sacrificed on Day 19 of gestation. Caesarean sections were performed, and the 

following observations were made: number and placement of implantation sites, resorption sites,

and live and dead fetuses; individual fetal weight and length (crown-rump); and external fetal

anatomical structure.” [Powers (1970a), p. 4] After external observations, about one-third of the 

fetuses from each litter were fixed with Bouin’s solution and evaluated for visceral changes by 

Wilson's technique. The remaining two-thirds of the fetuses were prepared for skeletal 

examination.

Examination of the visceral structures by Wilson's sectioning technique was performed on 58, 

61, and 68 fetuses from the control, low-dose, and high-dose groups, respectively.

RESULTS

Endpoint value(s): NOAEL (maternal and developmental toxicity) = 150 mg/kg-bw/day based 

on no treatment-related effects observed at the highest dose level tested.

Results remarks: 

Maternal Toxicological Data: There were no maternal deaths during the study. Pregnancy was 

observed in a total of 53 out of 55 rats (18 control, 17 low-dose, and 18 high-dose animals). One 

low-dose female and one high-dose female were not pregnant. There were no treatment-related 

changes in appearance, behavior, body weight gain, and food consumption. Incidental findings 

consisted of wheezing observed in one low-dose female on day 11 and soft feces in one highdose female on day 19. The low-dose animal which was not pregnant exhibited distended 

uterine horns with clear fluid. No other findings were noted at necropsy.

Caesarean Data: There were no effects on the number and placement of uterine implantation 

and resorption sites or the number, weight, and length of live fetuses in the test groups. One 

dead fetus, which was normally developed, was found in the control group. Three late resorption 

sites were noted in one control female, and one late resorption site was noted in a second control 

female. Two low-dose females exhibited one late resorption site each. 
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External Visceral Data: A few fetuses from all groups, including the control, appeared small in 

size. One of the small fetuses from the high-dose level and another fetus from the same dose 

group were pale in color. One fetus from the low-dose group was missing due to a technical 

error. 

Findings included clotted blood in the thoracic region in 15 control, 28 low-dose, and 18 highdose fetuses, clotted blood in the abdominal or intestinal region in 6 control fetuses and 1 highdose fetus, clotted blood in the cranial region in 1 control fetus, dilation of one or both renal 

pelves in 2 control, 3 low-dose, and 8 high-dose fetuses, dilation of the lateral ventricles of the 

brain in 1 control and 3 high-dose fetuses, and light colored areas in the liver of one control 

fetus.

Skeletal Data: “Single ribs [seen in 1 fetus at the high dose] and ribs described as ‘floating’ in 

appearance or unattached from the vertebral column [seen in 1 fetus in the low dose] were 

located at the 20th vertebra in all animals in which they were observed. The 14th pair of ribs 

noted in one control fetus was small in size. Rudimentary bone structures were observed at the 

20th vertebra on both sides of the vertebral column in one high level fetus and on the left side in 

another. The interparietal skull bone was missing or underdeveloped in two control fetuses and 

one high level fetus. Three control and two high level fetuses had the occipital skull bone 

missing or underdeveloped. The centrum of the first lumbar vertebra was split in two fetuses and 

small in one, while the centra of the 11th, 12th, and 13th thoracic vertebrae were split in one 

fetus from one control litter. A fetus from another control litter had the centrum of the 12th 

thoracic vertebra small and split.” [Powers (1970a), p. 8]

“There was no evidence of a compound-related effect among the test fetuses. There were no 

meaningful differences in the developmental indices of the test groups as compared to the 

controls. No trends toward lesser or greater skeletal development were observed.” [Powers 

(1970a), p. 9]

CONCLUSIONS

Based on the results of this study, the test substance did not produce maternal toxicity or 

developmental effects in rats. The NOAEL for maternal and developmental toxicity in rats was

150 mg/kg-bw/day (highest dose tested).

STUDY RELIABILITY

[3] Not reliable. The highest dose tested did not induce toxic effects as recommended in 

guideline 40 CFR 799.9355 and was below the recommended limit dose of 1000 mg/kg-bw/day. 

Additionally, important details that were missing from the study included test method/guidelines 

followed and statistical methods used.
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REFERENCE 

Powers, M.B. 1970a. “Teratology Study – Rats: EY 2333 Final Report,” Hazleton 

Laboratories, Inc., for Ethyl Corporation. Available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2005-0033-0181 (on pp. 84-

100 of “End_Point_F2 –Mammalian_Toxicity_-_Repeated_Dose_Reproduction_Developmental.

pdf”) as of December 4, 2013. 

Text in quotes is taken directly from Powers, 1970a.
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Study 6: Teratology study in rabbits (Powers, 1970b)

Title: Teratology Study - Rabbits, EY 2333 Final Report

This study is: Adequate Not Adequate

A method/guideline was not identified and no rationale was provided for selection of the doses 

that were tested. In addition, this study had the following deficiencies compared with guidelines 

for the conduct of a prenatal developmental toxicity test (OECD TG 414, OPPTS 870.3700, and 

40 CFR 799.9370): (1) Each test and control group should contain a sufficient number of 

females to result in approximately 20 animals with implantation sites at necropsy, whereas initial 

group size in the current study was 15 females and two maternal animals died (one at 0 mg/kgbw/day and one at 15 mg/kg-bw/day) and seven were non-pregnant (one at 15 mg/kg-bw/day and 

six at 150 mg/kg-bw/day); (2) Two doses were tested whereas at least three doses should be used 

and the highest dose should produce some evidence of developmental and/or maternal toxicity; 

(3) Statistical analyses of the results were not provided; (4) Maternal body weight and food 

consumption data were recorded but were not presented in the study report that was available; 

(5) Pre- and post-implantation loss calculations were not provided; and (6) Fetal sex ratio data 

were not presented in the study report that was available. Also, historical control data were not 

presented to allow interpretation of the following findings of this study: (1) abortion occurred in 

~20% of the control animals but not in dosed groups; and (2) 40% of the high-dose animals were 

non-pregnant compared with 0% in controls and ~7% at the low dose. 

TEST SUBSTANCE 

Identity: Phenol, 4,4’-methylenebis[2,6-bis(1,1-dimethylethyl)-, CASRN 118-82-1; Referred to 

as EY 2333 in study report

Remarks: 100% purity was assumed by study authors

METHOD

Method/guideline followed: Not specified

Test type: Prenatal Developmental Study - Rabbits

GLP compliant?: N/A (pre-GLP)

Year study performed: 1970

Test species: New Zealand White rabbits; obtained from Rowmar Rabbitry

Number of animals/sex/dose: 15 females/dose

Route of administration: Oral (gelatin capsules)
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Exposure duration: Gestational days 6–18

Doses/concentrations tested: 0 (control), 15, and 150 mg/kg-bw/day

Vehicle: None

Statistical methods used: None specified

Test method/conditions remarks: Following the injection of 45 female New Zealand White 

rabbits with pituitary luteinizing hormone to stimulate ovulation, each animal was impregnated 

by artificial insemination. “The day of insemination was designated as Day 0 of gestation.” 

[Powers (1970b), p. 3]. From the group of 45 impregnated females, 15 each were randomly 

assigned to the control, 15-, and 150-mg/kg-bw/day treatment groups. Daily, from days 6 

through 18 of gestation, the impregnated females were administered gelatin capsules “containing 

the appropriate dosage levels” [Powers (1970b), p. 4] of test material. Animals of the control 

group received empty capsules. 

“Mortality, appearance, and behavior of the animals were observed daily. Body weights were 

recorded weekly and terminally. Food consumption was recorded daily during the treatment 

period.” [Powers (1970b), p. 4]

The females were sacrificed on day 28, 29, or 30 of gestation. Pups were removed and the 

following observations were made: number of corpora lutea; number and placement of 

implantation sites, resorption sites, and live and dead fetuses; number of live and dead fetuses 

after a 24-hour incubation period; individual fetal weights and lengths; and external fetal 

anatomy. Pups were examined for visceral and skeletal abnormalities.

RESULTS

Endpoint value(s): NOAEL (maternal and developmental toxicity) = 150 mg/kg-bw/day based 

on no treatment-related effects observed at the highest dose level tested.

Results remarks: “One control doe and one low level doe died during the study; in both cases, 

it was too early in gestation to determine pregnancy. Among the surviving animals, evidence of 

abortion was noted in three control animals. One control doe delivered her litter naturally. The 

remaining control and all test litters were delivered by Caesarean section. Pregnancy was 

observed in a total of 36 of the 45 does used in this study (14 control, 13 low level, and nine high 

level animals).” [Powers (1970b), p. 1]

“Appearance, behavior, body weight gain, and food consumption were comparable among 

control and test animals. All uterine, litter, and Caesarean data were also comparable among the 

test and control groups.” [Powers (1970b), p. 1]

“There was no indication of a compound-related effect in the external appearance, visceral 

anatomy, development, and skeletal structure of the test fetuses as compared to those of the 

control fetuses.” [Powers (1970b), p. 1]
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CONCLUSIONS

The NOAEL for maternal and developmental toxicity in rabbits was 150 mg/kg-bw/day (highest 

dose tested).

STUDY RELIABILITY

[3] Not reliable. A method/guideline was not identified and no rationale was provided for 

selection of the doses that were tested. In addition, this study had the following deficiencies 

compared with guidelines for the conduct of a prenatal developmental toxicity test (OECD TG 

414, OPPTS 870.3700, and 40 CFR 799.9370): (1) Each test and control group should contain a 

sufficient number of females to result in approximately 20 animals with implantation sites at 

necropsy, whereas initial group size in the current study was 15 females and two maternal 

animals died (one at 0 mg/kg-bw/day and one at 15 mg/kg-bw/day) and seven were non-pregnant

(one at 15 mg/kg-bw/day and six at 150 mg/kg-bw/day); (2) Two doses were tested whereas at 

least three doses should be used and the highest dose should produce some evidence of 

developmental and/or maternal toxicity; (3) Statistical analyses of the results were not provided; 

(4) Maternal body weight and food consumption data were recorded but were not presented in 

the study report that was available; (5) Pre- and post-implantation loss calculations were not 

provided; and (6) Fetal sex ratio data were not presented in the study report that was available. 

Also, historical control data were not presented to allow interpretation of the following findings 

of this study: (1) abortion occurred in ~20% of the control animals but not in dosed groups; and 

(2) 40% of the high-dose animals were non-pregnant compared with 0% in controls and ~7% at 

the low dose. 

REFERENCE 

Powers, M.B. 1970b. “Teratology Study - Rabbits, EY 2333 Final Report,” Hazleton 

Laboratories, Inc., for Ethyl Corporation. Available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2005-0033-0181 (on pp. 62-

83 of “End_Point_F2 –Mammalian_Toxicity_-_Repeated_Dose_Reproduction_

Developmental.pdf”) as of December 4, 2013.

Text in quotes is taken directly from Powers, 1970b.
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D. SUPPLEMENTAL DATA (NON-SIDS ENDPOINTS)

D-1. Carcinogenicity

Study 1: Combined repeated-dose and carcinogenicity toxicity study (ECHA website, Exp Key 

Carcinogenicity.001)

Title: Not provided

TEST SUBSTANCE 

Identity: Phenol, 4,4’-methylenebis[2,6-bis(1,1-dimethylethyl)-, CASRN 118-82-1; referred to 

as 4,4'-Methylenebis(2,6-di-tert-butylphenol) in the study report (other synonyms: BHD, AN2, 

Ethyl Antioxidant 702).

Remarks: Purity not specified; crystalline solid 

METHOD

Method/guideline followed: Similar to OECD TG 453 “Combined Chronic 

Toxicity/Carcinogenicity Studies”

Test type: Combined Repeated-Dose Toxicity/Carcinogenicity Study

GLP compliant?: N/A (Pre-GLP)

Year study performed: 1969

Test species: Crj:CD(SD) rat; source: Charles River Breeding Laboratories Inc., Brookline, 

MA; age: weanlings at initiation; weight (average during week 1): 394–436 g (males) and 248–

269 g (females).

Number of animals/sex/dose: 25 in the test groups; 50 in the control group

Route of administration: Oral - Diet

Vehicle: Mazola corn oil (reported to be free of any antioxidants)

Exposure duration: 2 years

Doses/concentrations tested: 0, 15, 60, 100, 500, and 3000 ppm (corresponding approximate 

dose levels were 0, 0.55, 2.14, 3.56, 18.1, and 107 mg/kg-bw/day, respectively, for males and 0, 

0.67, 2.63, 4.35, 22.2, and 138 mg/kg-bw/day, respectively, for females)

Statistical methods used: Not specified
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Test method/conditions remarks: 

Dosing and Environmental Conditions: Rats (25/sex/dose) were fed diets containing the test 

substance at various concentrations for up to 2 years. A control group of 50 rats was fed a diet 

containing only the corn oil vehicle. Four rats per sex were assigned to subgroups of each dose 

group for interim kills at 13, 26, 52, and 78 weeks (3, 6, 12, and 18 months). Terminal sacrifice 

was performed at 104 weeks (24 months). 

The animals were acclimated for 2 weeks and were not fasted prior to the start of the study. The 

test animals were housed three males and four females per cage prior to the start of the study. 

Diet and water were provided ad libitum throughout the study. During the study, the test 

temperature was maintained at 76 ± 2°F and the humidity was not controlled.

Preparation of Test Diets: For preparation of the test diets, “1 kg of corn oil was added to 19 kg 

of the base diet with or without the test substance. The mixture was stirred for 20 min in a 

Hobart mixer (stainless steel). [Five] diets containing the test item were prepared... The diet was 

freshly prepared every week.” [ECHA]

The test diets were prepared by “mixing appropriate amounts with [a] special preparation of 

Rockland Mouse and Rat diet containing no more than 1% of fat.” [ECHA] The rodent diet food 

was obtained fresh once each month.

Clinical Observations, Body Weights, and Food Consumption: Cage-side observations were 

made on a daily basis for general appearance and behavior. Animals were observed weekly for a 

detailed assessment of clinical signs. Body weight and food consumption were recorded weekly. 

Mean test substance intake for males and females in each group was calculated by an unspecified 

method. 

Ophthalmological examinations were performed weekly and all groups were examined for the 

presence of cataracts. 

Hematology and Clinical Chemistry Measurements: Hematological examinations were 

performed on unfasted treated and control animals (10 rats/sex/dose) at 1 week before the start of 

dosing, and again at 13, 26, 52, 78, and 104 weeks. Hematological parameters examined 

included the following: prothrombin time, hemoglobin, packed cell volume of erythrocytes, and 

total and differential leukocytes. Clinical chemistry investigations were performed on blood 

samples collected at scheduled sacrifice of all test animals. Clinical chemistry parameters 

examined included the following: total lipids, cholesterol, and alkaline phosphatase. 

No urinalysis assessments were performed.

Gross Necropsy and Histopathology: It appears that organ weights were recorded, but, with the 

exception of the liver, the organs weighed were not specified in the ECHA study summary (the 

full study report was not available for review). Pathological examinations were performed on 

the following organs at gross necropsy: liver, heart, lung, kidneys, spleen, brain, gonads, 

pituitary, adrenals, and thyroid. Histological examinations were performed on selected tissues, 

including the heart, aorta, lung, liver, spleen, kidney, stomach, duodenum, pylorus, small 
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intestine (ileum), large intestine (colon), mesenteric lymph nodes, urinary bladder, prostate, 

testes, ovaries, uterus, pancreas, thyroid, thymus, adrenals, pituitary, brain, spinal cord, eye, bone 

marrow, bone, skeletal muscle, and skin.

RESULTS

Endpoint value(s): No evidence of carcinogenicity

Results remarks: 

“Neoplastic lesions observed in the study were subcutaneous or in females mammary gland 

adeno[fibroma], pituitary adenoma, one adenoma in the parathyroid, thyroid adenoma, adrenal 

adenoma, [and] liver angioma that were all benign tumors. Leukemia, [s]eminoma and ovary 

carcinoma were single findings in control animals only. No test substance related increased 

incidence of any tumor was observed and it can be concluded that the test substance did not 

induce neoplastic lesions under the conditions of this study.” [ECHA]

“The histopathology findings were confirmed in a second reading of the data with independent 

pathologists and reported in a supplementary report of the same laboratory...” [ECHA]

CONCLUSIONS

The study summary concluded that the test substance did not induce an increase in tumor 

incidence under the conditions of the study.

STUDY RELIABILITY

[2] Reliable with restrictions. When compared to the TSCA 799.9430 guideline, the following 

deviations were noted: (1) the total number of animals at test initiation was not specified; (2) the 

lot or batch number of the test substance was not provided; (3) the environmental test 

temperature (76 ± 2°F) exceeded the recommended temperature of 22 ± 3°C (71.6°F); (4) 

humidity was not controlled; (5) limited hematology and clinical chemistry parameters were 

examined; (6) urinalysis was not conducted; (7) organs weighed at necropsy were not specified;

and (8) group mean data for endpoints other than histopathological lesions were not reported. 

REFERENCE

Study summary available from the ECHA website, 

http://apps.echa.europa.eu/registered/data/dossiers/DISS-dcee47d2-62ad-0171-e044-

00144f67d031/AGGR-b809391a-f592-4fd4-b07c-046e2cb18268_DISS-dcee47d2-62ad-0171-

e044-00144f67d031.html#AGGR-b809391a-f592-4fd4-b07c-046e2cb18268. The data source is 

referred to as a “study report”, with a report date of 1969-07-08. No other reference information 

is provided. 

Text in quotes is taken directly from the ECHA website.
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Copyright Notice: © European Chemicals Agency, 2007-2011. Reproduction is authorised 

provided the source is fully acknowledged in the form “Source: European Chemicals Agency, 

http://echa.europa.eu/”, and provided written notification is given to the ECHA Communication 

Unit (info (at) echa.europa.eu).
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D-2. Skin Irritation

Study 1: Acute dermal toxicity study in rabbits (Carter and Dharmarajan, 1976)

Title: A Report on the Acute Toxicity of AN-702

TEST SUBSTANCE 

Identity: Phenol, 4,4’-methylenebis[2,6-bis(1,1-dimethylethyl)-, CASRN 118-82-1; referred to 

as AN-702 in study report

Remarks: Purity not provided

METHOD

Method/guideline followed: Not specified

Test type: Acute Dermal - Rabbit

GLP compliant?: Not specified

Year study performed: 1976

Test species: New Zealand White rabbits; 2.3–3.0 kg

Number of animals/sex/dose: 4 (sex not specified)

Route of administration: Dermal

Vehicle: Peanut Oil

Doses/concentrations tested: 1500 mg/kg-bw

Statistical methods used: No information available

Test method/conditions remarks: In an acute dermal toxicity test, the test substance (10% w/v 

solution in peanut oil) was injected under a plastic sleeve wrapped around the clipped trunks of 

two intact and two abraded rabbits for an exposure period of 24 hours. Evaluations of erythema 

and edema were made 24 and 72 hours after application of the test material. After removal of 

the sleeves, the animals were observed for 14 days.

RESULTS

Endpoint value(s): Irritating
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Results remarks: No mortalities occurred. Well defined erythema was observed on the first 

day of observation.

CONCLUSIONS

The test substance was irritating to rabbit skin.

STUDY RELIABILITY

[2] Reliable with restrictions. Important study details that were missing from the study summary 

included test substance purity, methods/guidelines followed, GLP compliance, sex of test 

animals, statistical methods, detailed findings, and numerical results. 

REFERENCE

Carter, G. and V. Dharmarajan. 1976. “A Report on the Acute Toxicity of AN-702,” Tulane 

University School of Medicine, for Ethyl Corporation. Submitted by Albemarle Corporation. 

Study summary available online at http://www.regulations.gov/#!documentDetail;D=EPA-HQOPPT-2005-0033-0181 (on pp. 7-13 of the document “End_Point_D –

Mammalian_Toxicity_Acute.pdf”) as of December 4, 2013.
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Study 2: Skin irritation study in rabbits (Ethyl Petroleum Additives International, 1991)

Title: Acute Dermal Irritation/Corrosion Test in the Rabbit

TEST SUBSTANCE 

Identity: Phenol, 4,4’-methylenebis[2,6-bis(1,1-dimethylethyl)-, CASRN 118-82-1

Remarks: Purity not provided

METHOD

Method/guideline followed: OECD 404 “Acute Dermal Irritation/Corrosion”

Test type: Primary Dermal Irritation - Rabbit

GLP compliant?: Yes

Year study performed: 1991

Test species: New Zealand White rabbits

Number of animals/sex/dose: 3 males

Route of administration: Dermal

Vehicle: None specified

Doses/concentrations tested: 0.5 mL

Statistical methods used: None specified

Test method/conditions remarks: Three male rabbits were exposed to the test substance for 

4 hours and observed for 16 days thereafter. 

RESULTS

Endpoint value(s): Irritating 

Results remarks: No deaths were observed. Well-defined to moderate erythema was observed 

in all animals during the first 9 days following treatment. Slight erythema was noted in all 

animals 12 days after treatment and in one animal at study termination. Very slight or slight 

edema was noted in two animals during the first 4 days following treatment. Very slight edema 

was noted at all test sites 2 days later, and persisted in one animal up to 12 days. Two animals 

exhibited exfoliation on days 6, 9, and/or 12 post-exposure. 
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CONCLUSIONS

The test substance was irritating to rabbit skin.

STUDY RELIABILITY

[2] Reliable with restrictions. Only the study summary was available. Important study details 

that were missing from the study summary included test substance purity, concentration of test 

solution, interpretation of irritation scores, and detailed numerical results. 

REFERENCE

Ethyl Petroleum Additives International. 1991. Acute Dermal Irritation/Corrosion Test in the 

Rabbit. Study No. 90/ETP012/1368. Study conducted by Life Science Research Limited. 

(Cited in IUCLID Dossier, European Chemical Bureau, 2000). Study summary available online 

at http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2005-0033-0181 (on p. 71

of the document “End_Point_D – Mammalian_Toxicity_Acute.pdf) as of December 4, 2013.
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Study 3: Dermal irritation study in rabbits (Carter and Dharmarajan, 1976)

Title: A Report on the Acute Toxicity of AN-702

TEST SUBSTANCE 

Identity: Phenol, 4,4’-methylenebis[2,6-bis(1,1-dimethylethyl)-, CASRN 118-82-1; referred to 

as AN-702 in study report

Remarks: Purity not provided

METHOD

Method/guideline followed: Not specified

Test type: Primary Dermal Irritation - Rabbit

GLP compliant?: Not specified

Year study performed: 1976

Test species: New Zealand White rabbits

Number of animals/sex/dose: 6 (sex not specified)

Route of administration: Dermal

Vehicle: None

Doses/concentrations tested: 0.5 g

Statistical methods used: None specified

Test method/conditions remarks: The test substance was applied as a neat powder to four 

clipped skin areas of each animal, two of which were abraded. The areas were covered with 

gauze pads for 24 hours. Observations for erythema and edema were made at 24 and 72 hours 

following exposure. 

RESULTS

Endpoint value(s): Not irritating 

Results remarks: No erythema or edema was observed (Draize score of 0). 
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CONCLUSIONS

The test substance was not irritating to rabbit skin when applied as a neat powder. However, the 

investigators noted that when the test substance was applied as a 10% w/v solution in peanut oil 

there was defined erythema.

STUDY RELIABILITY

[4] Not assignable. Important study details that were missing from the study summary included 

test substance purity, methods/guidelines followed, GLP compliance, and sex of test species. In 

addition, the results conflict with a different test conducted with the same chemical.

REFERENCE

Carter, G. and V. Dharmarajan. 1976. “A Report on the Acute Toxicity of AN-702,” Tulane 

University School of Medicine, for Ethyl Corporation. Submitted by Albemarle Corporation. 

Study summary available online at http://www.regulations.gov/#!documentDetail;D=EPA-HQOPPT-2005-0033-0181 (on pp. 7-13 of the document “End_Point_D – Mammalian_Toxicity_

Acute.pdf) as of December 4, 2013.
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D-3. Eye Irritation

Study 1: Eye irritation study in rabbits (Carter and Dharmarajan, 1976)

Title: A Report on the Acute Toxicity of AN-702

TEST SUBSTANCE 

Identity: Phenol, 4,4’-methylenebis[2,6-bis(1,1-dimethylethyl)-, CASRN 118-82-1; referred to 

as AN-702 in study report

Remarks: Purity not provided

METHOD

Method/guideline followed: Not specified

Test type: Primary Eye Irritation - Rabbit

GLP compliant?: Not specified

Year study performed: 1976

Test species: New Zealand White rabbits

Number of animals/sex/dose: 6 (sex not specified)

Route of administration: Ocular

Vehicle: None

Doses/concentrations tested: 0.1 g

Statistical methods used: None specified

Test method/conditions remarks: One eye of each of six New Zealand White rabbits was 

treated with 0.1 g of test substance as a neat powder. The other untreated eye served as the 

control. Observations for damage and irritation were made at 1, 2, 3, and 7 days following 

administration. 
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RESULTS

Endpoint value(s): Not Irritating

Results remarks: Slight corneal opacity was observed in one rabbit and slight conjunctival 

irritation was observed in five rabbits on the first day of observation. Mean scores for cornea, 

iris, and conjunctivae at 1, 2, 3, and 7 days post-exposure, respectively, were 3.5, 1.5, 0.8, and 

0 out of a total of 110. 

CONCLUSIONS

The test substance caused slight corneal opacity and slight conjunctival irritation.

STUDY RELIABILITY

[4] Not assignable. Important study details that were missing from the study summary included 

test substance purity, methods/guidelines followed, GLP compliance, sex of test species, and 

definition of the scoring system. 

REFERENCE

Carter, G. and V. Dharmarajan. 1976. “A Report on the Acute Toxicity of AN-702,” Tulane 

University School of Medicine, for Ethyl Corporation. Study summary available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2005-0033-0181 (on pp. 7-13

of the document “End_Point_D – Mammalian_Toxicity_Acute.pdf) as of December 4, 2013.
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E. DATA ADEQUACY SUMMARY TABLE

Endpoint Study Information 

Available

Acceptable Data Gap

Aquatic Effects

Acute Toxicity to Fish1 Yes No No

Acute Toxicity to Aquatic Invertebrates1 Yes No No

Chronic Toxicity to Aquatic Invertebrates2 Yes Yes No

Toxicity to Aquatic Plants2 Yes Yes No

Health Effects

Acute Toxicity Yes Yes No

Repeated-Dose Toxicity Yes Yes No

Gene Mutation Yes Yes No

Chromosomal Aberrations Yes Yes No

Reproductive Toxicity3 Yes Yes No

Developmental Toxicity3 Yes Yes No

1 Acute toxicity testing in fish and aquatic invertebrates is not required under the HPV2 test rule 

because the log Kow of CASRN 118-82-1 is ≥ 4.2. (The log Kow value of CASRN 118-82-1 was 

experimentally determined to be > 6.50 by SafePharm Laboratories [study submitted to EPA by 

Albemarle Corporation on May 12, 2006, available online at http://www.regulations.gov/

#!documentDetail;D=EPA-HQ-OPPT-2005-0033-0163 as of December 5, 2013; “4,4’-

Methylene bis(2,6-di-tert-butylphenol): Determination of Water Solubility and Partition 

Coefficient”, SPL Project Number 1193/011, study completed on July 18, 2005].) 

2 The HPV2 test rule, “Testing of Certain High Production Volume Chemicals; Second Group of 

Chemicals” (76 FR 1067, January 17, 2011), requires C1 aquatic toxicity testing for Phenol, 4,4'-

methylenebis[2,6-bis (1,1-dimethylethyl)- (CASRN 118-82-1). The log Kow for CASRN 118-

82-1 is > 6.50, indicating that testing under Test Group 2, which applies to chemicals with log 

Kow ≥ 4.2, is required. Test Group 2 includes the following tests: (1) Chronic Toxicity to 

Daphnia (ASTM E 1193-97) and (2) Toxicity to Plants (Algae; ASTM E 1218-04). 

3 Adequate data were available for an acceptable supporting compound (2,6-di-tert-butylphenol; 

CASRN 128-39-2).