SRC TR-14-020

HPV3 TEST RULE DATA ADEQUACY REVIEW (FINAL)

1-Decene, sulfurized CASRN 72162-15-3

Prepared by:

Amy Nguyen, Teresa Manyin, Ashlee Aldridge, Christopher Janson, Megan Nelson, 

William L. Richards, and Gary Diamond

Defense and Environmental Solutions Division

SRC, Inc.

7502 Round Pond Road

North Syracuse, NY 13212

Contract No. EP-W-09-027

Task FG007.4.009.006

Submitted to:

OCSPP/OPPT/RAD (7403M)

U.S. Environmental Protection Agency

1200 Pennsylvania Avenue

Washington, D.C. 20460

Bryan Lobar, Project Officer

David Brooks, Work Assignment Manager

Amy Benson, Alternate Work Assignment Manager

July 17, 2014
Final HPV3 Test Rule Data Adequacy Review (July 2014) CASRN 72162-15-3

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TABLE OF CONTENTS

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

B. EVALUATING DATA ADEQUACY ..................................................................................... 2

C. SUMMARY OF DATA ADEQUACY FOR TESTING REQUIRED BY TEST RULE ........ 2

D. DATA FOR SIDS ENDPOINTS.............................................................................................. 4

D-1. Acute Toxicity to Fish ....................................................................................................... 4

D-2. Acute Toxicity to Aquatic Invertebrates.......................................................................... 10

D-3. Chronic Toxicity to Aquatic Invertebrates....................................................................... 13

D-4. Toxicity to Aquatic Plants ............................................................................................... 19

D-5. Acute Toxicity.................................................................................................................. 22

D-6. Repeated-Dose, Reproductive, and Developmental Toxicity.......................................... 39

D-7. Genetic Toxicity – Gene Mutation................................................................................... 62

D-8. Genetic Toxicity – Chromosomal Aberrations................................................................ 68

E. DATA ADEQUACY SUMMARY TABLE .......................................................................... 75
Final HPV3 Test Rule Data Adequacy Review (July 2014) CASRN 72162-15-3

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A. SOURCE MATERIAL USED FOR ASSESSMENT

This document provides a review of data describing the toxicity of 1-decene, sulfurized (CASRN 

72162-15-3). In addition to data that were previously assessed for adequacy by EPA1, this 

document evaluates a newly submitted combined repeated-dose toxicity study with the 

reproduction/developmental toxicity screening test for CASRN 72162-15-3, which was 

submitted on May 9, 2014 by Dover Chemical Corporation to EPA in response to the final test 

rule, “Testing of Certain High Production Volume Chemicals; Third Group of Chemicals” (76 

FR 65385, October 21, 2011; referred to herein as HPV3 test rule; available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0112-0086 as of July 

17, 2014). 

The HPV3 test rule requires the following toxicological testing for CASRN 72162-15-3: C1 

(consisting of Test Group 1 [acute toxicity to fish, acute toxicity to Daphnia, and toxicity to 

algae tests] if log Kow < 4.2 or Test Group 2 [chronic toxicity to Daphnia and toxicity to algae 

tests] if log Kow ≥ 4.2), D (acute mammalian toxicity test), E1 (bacterial reverse mutation test), 

E2 (chromosomal aberration or micronucleus test), and F1 (combined repeated-dose toxicity 

study with the reproduction/developmental toxicity screening test). Based on a measured log 

Kow of > 8.02, Test Group 2 testing is required to determine the toxicity of CASRN 72162-15-3 

to aquatic organisms. 

Previous submissions in response to the toxicity testing requirements included the following:

(1) On February 20, 2012, Dover Chemical Corporation submitted a test plan (available 

online at http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0112-

0112 as of July 17, 2014) that proposed the use of data from toxicity tests of three 

analogs (1-propene, 2-methyl-, sulfurized, CASRN 68511-50-2; pentene, 2,4,4-

trimethyl-, sulfurized, CASRN 68515-88-8; and alkenes, C15-18 alpha-, sulfurized, 

CASRN 67762-55-4) to satisfy some of the required toxicity testing requirements for 

CASRN 72162-15-3. 

(2) On April 4, 2014, Dover Chemical Corporation supplied data for the chronic toxicity 

to Daphnia (component of C1) testing requirement (submission available online at

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0112-0265 as of 

July 17, 2014). 

 

1 Data for CASRN 72162-15-3 were previously reviewed in the following reports: “HPV3 Test Plan and Data 

Adequacy Review, 1-Decene, sulfurized, CASRN 72162-15-3” (SRC Technical Report #12-034, dated March 14, 

2012; available online at http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0112-0185 as of 

July 17, 2014) and “HPV3 Test Rule Data Adequacy Review: Chronic Toxicity to Daphnia, 1-Decene, sulfurized, 

CASRN 72162-15-3” (SRC Technical Report #14-012, dated May 1, 2014; not available online as of July 17, 2014).

2 The log Kow was experimentally determined by WIL Research Europe B.V. (Oudhoff, K.A. 2013. Determination 

of physico-chemical properties of 1-decene, sulfurized. WIL Research Europe B.V., ‘s-Hertogenbosch, The 

Netherlands. Project No. 502505.) Study submitted by Dover Chemical Corporation to EPA on September 25, 

2013 and available online at http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0112-0161 as 

of July 17, 2014.
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EPA evaluated the above submissions and determined that the available data were adequate to 

satisfy the C1, D, E1, and E2 testing requirements and the repeated-dose toxicity component of 

F1 testing requirement.

On May 9, 2014, Dover Chemical Corporation submitted the following study report to address 

the remaining components of the F1 testing requirement:

 McConnachie, K. (Study Director). 2014. A combined repeated dose toxicity study with 

a reproduction/developmental toxicity screening of sulfurated decene by oral gavage in 

rats. Charles River Laboratories, Edinburgh, UK. Study No. 496486. 1

Data from all studies submitted by Dover Chemical Corporation, as well as data identified for the 

proposed analogs, are summarized and evaluated for adequacy in Section D. The newly 

submitted repeated-dose toxicity study with the reproduction/developmental toxicity screening 

test is evaluated on pp. 39-48 of this review.

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 these 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 an 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. 

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

Evaluation of Toxicological Data Submitted on May 9, 2014:

Data submitted by Dover Chemical Corporation and reviewed in this document are adequate to 

satisfy the F1 (combined repeated-dose toxicity study with the reproduction/developmental

toxicity screening) testing requirement for CASRN 72162-15-3 promulgated in the final HPV3 

test rule. 

Evaluation of Previously Submitted Toxicological Data:

In its submission dated February 20, 2012, Dover Chemical Corporation proposed the use of data 

from three analogs (CASRNs 68511-50-2, 68515-88-8, and 67762-55-4) to satisfy some of the 

required toxicity testing requirements. EPA accepted the use of the proposed analogs because 

they are structurally similar to the test rule chemical substance2 with similar functional groups 

 

1 Submitted via email from Tim Nowicki (Dover Chemical Corporation) to John Schaeffer (EPA).

2

In its test plan, Dover erroneously states that the analogs and test rule chemical substance are all composed of 

highly branched alkanes (on pp. 3 and 9 of Dover’s test plan). Dover’s test plan indicates that the test rule chemical 

“is manufactured by reacting molten sulfur with 1-decene” and that the “decene feedstock is a mono-unsaturated 

highly branched C10 hydrocarbon” (on p. 1 of Dover’s test plan). However, according to Chemical Abstract 
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and all four chemicals share similar physicochemical characteristics,1including high partition 

coefficient (log Kow), low water solubility, low vapor pressure and high boiling point. 

Additionally, the molecular weights of the analogs and test rule chemical are generally similar. 

Toxicological data from the analogs2 were determined to be adequate to satisfy the toxicity to 

algae (component of C1), acute mammalian toxicity (D), bacterial reverse mutation (E1), 

chromosomal aberration (E2), and repeated-dose toxicity (component of F1) testing requirements 

for CASRN 72162-15-3 promulgated in the final HPV3 test rule.

Dover Chemical Corporation’s April 4, 2014 submission supplied data for the chronic toxicity to 

Daphnia (component of C1) testing requirement. These data were determined to be adequate to 

satisfy the chronic toxicity to Daphnia component of the C1 testing requirement for CASRN 

72162-15-3 promulgated in the final HPV3 test rule.

Summary of Data Adequacy for Toxicological Endpoints:

Adequate data are available to satisfy all HPV3 toxicity testing requirements for CASRN 72162-

15-3. There are no outstanding HPV test rule requirements for toxicity testing of CASRN 

72162-15-3.

 

Service nomenclature conventions, both the 1-decene feedstock and the test rule chemical, 1-decene, sulfurized 

(CASRN 72162-15-3), are non-branched. Of the three chemicals that Dover proposes as analogs of the test rule 

chemical, only CASRNs 68511-50-2 and 68515-88-8 are composed of highly branched alkanes (as shown in Figure 

1 of the initial test plan for the Alkyl Sulfide Category, submitted by the Health, Environmental, and Regulatory 

Task Group of the Chemical Manufacturers Association Petroleum Additives Panel under the HPV Challenge 

Program and available online at http://www.epa.gov/hpv/pubs/summaries/alkylsul/c12549tp.pdf as of July 17, 

2014). Although only two of the four chemicals of the Dover test plan (the test rule chemical and three analogs) are 

composed of highly branched alkanes, all four chemicals possess sufficient structural similarities to allow read 

across of toxicity values.

1 Values estimated using EPISuite (p. 4 of Dover’s test plan).

2 Data for analogs were obtained from the Final Submission for the Alkyl Sulfides Category, submitted to EPA 

under the HPV Challenge Program by the Health, Environmental, and Regulatory Task Group of the American 

Chemistry Council’s Petroleum Additives Panel on May 19, 2005. EPA comments on the initial test plan for the 

Alkyl Sulfides Category are available online at http://www.epa.gov/oppt/chemrtk/pubs/summaries/alkylsul/

c12549ct.pdf as of July 17, 2014. The final submission of robust summaries for the Alkyl Sulfide Category is 

available online at http://www.epa.gov/hpv/pubs/summaries/alkylsul/c12549rr2.pdf as of July 17, 2014. The 

proposed analog chemicals were classified as the “Alkyl Polysulfides” Subcategory in EPA’s Hazard 

Characterization of the Alkyl Sulfides Category; EPA’s Hazard Characterization of the Alkyl Sulfides Category, 

dated September 2009, is available online at http://www.epa.gov/chemrtk/hpvis/hazchar/Category_Alkyl%20

Sulfides_Sept2009.pdf as of July 17, 2014.
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D. DATA FOR SIDS ENDPOINTS

D-1. Acute Toxicity to Fish

Summary of endpoint: Adequate

Study 1: Acute toxicity study of CASRN 68511-50-2 in fathead minnow (Ward, 1993)

Title: Acute Toxicity of the Water Accommodated Fractions (WAFs) of CMA 613 to the 

Fathead Minnow, Pimephales promelas

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 1-Propene, 2-methyl-, sulfurized, CASRN 68511-50-2

Remarks: Purity not provided

METHOD

Method/guideline followed: US EPA Toxic Substances Control Act Test Guideline #797.1400 

(1985), OECD TG 203, Fish Acute Toxicity Test (1984)

Test type: Fish acute toxicity test (WAF static renewal)

GLP compliant?: Yes

Year study was performed: 1993

Test species: Fathead minnow (Pimephales promelas)

Analytical monitoring: Yes; total organic carbon (TOC) measurements of initial test solutions 

and control (0-hour) and after one day on test (24-h) before daily renewal of fresh test solution.

Exposure duration: 96 hours

Concentrations tested: 0, 100, 300, or 1000 mg/L (nominal loading rates)

Test method/conditions remarks: Test Organisms: source – Aquatic Research Organisms, 

Hampton, New Hampshire; age – juvenile; total length – 25 mm average (longest fish not more 

than twice the shortest fish); wet weight – 0.1 g average (no range reported). Loading: < 0.5 g 

biomass/L; pretreatment: none, fish held for a minimum of 14 days before testing. No feeding 

occurred during the test. 
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Test System: Individual WAFs were prepared for each test level and renewed daily. A 

measured weight of test material was added to a measured volume of dilution water (30 L) in a 

glass vessel and stirred for 24 hours. Stirring was accomplished using a Telfon-coated magnetic 

stir bar. Mixing speed was adjusted such that a vortex formed between 30 to 50% of the distance 

to the bottom. Following the mixing period, the test solutions were allowed to stand for 1 hour 

before the water phase was removed. To avoid removing test material from the surface or 

bottom, a siphon was placed in the mixing vessel prior to addition of water and test substance 

with the lower end 1-2 inches off the bottom. The siphoned water phase (i.e., WAF) was used in 

the aquatic toxicity test. About 80% of the solution in each test level was renewed daily after 24, 

48, and 72 hours. Two 15-L replicates were used per treatment, and 10 fish were used per 

replicate (for a total of 20 per treatment). Test vessels were loosely covered to reduce entry of 

dust, etc. 

Dilution Water: Filtered well water collected at Hampton, New Hampshire and adjusted to the 

appropriate hardness of 176 mg/L as CaCO3. The water was passed through activated carbon, a 

particle filter, and then an ultraviolet sterilizer, and then it was stored in a polyethylene tank 

where it was aerated. The water was characterized as moderately hard water. Alkalinity was not 

reported. Dissolved oxygen: 7.1 to 8.7 mg/L; pH: 7.8 to 8.5; conductivity: 570 to 650 

μmhos/cm; temperature: 21.4 to 22.8 °C. TOC levels were between 2 to 3 mg/L in the control, 3 

mg/L at 100 mg/L test level, between 3 to 4 mg/L at 300 mg/L test level, and 5 mg/L at 1000 

mg/L test concentration level. 

Test Levels: Control, 100, 300, & 1000 mg/L WAF loading rates. 

Calculation of LL50s: Statistical analysis of survival data was not warranted. 

Test Substance: No undissolved test material was seen on the surface of the test vessels during 

the entire aquatic toxicity test. 

Reference Substance: No 

RESULTS

Endpoint value(s): 96-h LL50 > 1000 mg/L (nominal); 96-h LOEC = 1000 mg/L (nominal)

based on lethargy and erratic swimming; 96-h NOEC = 300 mg/L (nominal)

Results remarks: Test Findings: No mortality was observed in all treatments and the control 

throughout the entire test and no signs of toxicity were noted in all treatments throughout 72 

hours. At 96 hours, all 20 fish in the 1000 mg/L test level were lethargic and exhibited erratic 

swimming, but no signs of toxicity were observed in the lower test levels and control. 

TOC measurements at 1000 mg/L were 5 mg/L compared to 2 to 3 mg/L in the control. 

The TOC in dilution water at the beginning and end of the test was greater than 2 mg/L rather 

than < 2 mg/L. It could not be verified that water samples were passed through a 0.45 micron 
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filter prior to TOC analysis, that the test vessels were covered, or that the continuous temperature 

measurement was made in a control vessel during the study.

EPA does not consider the loading rate as the no-effect concentration when the concentration 

exceeds the water solubility of the substance. Assuming that the exposure concentration in the 

WAF is the water solubility limit (saturation) for 1-propene, 2-methyl-, sulfurized, the no-effect 

concentration for lethality would be approximately 2.7 mg/L. (No lethal effects were observed at 

saturation.)

CONCLUSIONS

No mortality was observed in all treatments and the control throughout the entire test and no 

signs of toxicity were noted in all treatments throughout 72 hours. At 96 hours, all 20 fish in the 

1000 mg/L test level were lethargic and exhibited erratic swimming, but no signs of toxicity 

were observed in the lower test levels and control. 

STUDY RELIABILITY

[2] Reliable with restrictions. The TOC in dilution water at the beginning and end of the test was 

greater than 2 mg/L rather than < 2 mg/L. The pH range extended above 8.0. It could not be 

verified that water samples were passed through a 0.45 micron filter prior to TOC analysis, that 

the test vessels were covered, or that the continuous temperature measurement was made in a 

control vessel during the study.

REFERENCE

Ward, T.J. 1993. Acute Toxicity of the Water Accommodated Fractions (WAFs) of CMA 613 

to the Fathead Minnow, Pimephales promelas. T.R. Wilbury Study #9176-CMA/ESI-613. As 

summarized in the Robust Summaries for the Alkyl Sulfides Category (submitted to EPA under 

the HPV Challenge Program by the Health, Environmental, and Regulatory Task Group of the 

American Chemistry Council’s Petroleum Additives Panel on May 19, 2005).
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Study 2: Acute toxicity study of CASRN 68511-50-2 in sheepshead minnow (Nicholson, 1986)

Title: Acute toxicity of CMA Test Material Code 525 to Sheepshead Minnow, Cyprinodon 

variegatus

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 1-Propene, 2-methyl-, sulfurized, CASRN 68511-50-2

Remarks: Purity not provided

METHOD

Method/guideline followed: Test protocol followed OECD TG 203, Fish Acute Toxicity Test 

(1984)

Test type: Fish acute toxicity test (WSF static renewal test)

GLP compliant?: Yes

Year study was performed: 1986

Test species: Sheepshead minnow (Cyprinodon variegatus)

Analytical monitoring: Yes; total organic carbon (TOC) measurements of each freshly 

prepared test solution and control and after 24-h on test just before daily renewal with fresh test 

solution.

Exposure duration: 96 hours

Concentrations tested: 0 or 10,000 mg/L (nominal loading rates)

Test method/conditions remarks: Test Organisms: source – a commercial supplier in New 

Hampshire; age – 24 to 29 days old; total length – 20.3 mm average (range = 13 to 30 mm; n =

18); wet weight – 0.16 g average (range = 0.03 to 0.039 g; n = 18). Loading – 0.32 g biomass/L;

pretreatment – none, fish held for a minimum of 20 days before testing. No feeding occurred 

during the test. 

Test System: Individual WSFs were prepared for each daily renewal of the 10,000 mg/L test 

level. A measured weight of test material was added to a measured volume of dilution water 

(15-L) in a glass vessel and stirred for 16 to 24 hours. Stirring was accomplished using a Telfoncoated magnetic stir bar. Mixing speed was adjusted such that a vortex formed between 30 to 

50% of the distance to the bottom. Following the mixing period, the test solutions were allowed 

to stand for 2 hours before the water phase was removed. To avoid removing test material from 
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the surface or bottom, a siphon was placed in the mixing vessel prior to addition of water and test 

substance with the lower end approximately midway between bottom and surface. The siphoned 

water phase, designated water soluble fraction (WSF), was used for the aquatic toxicity test.

About 90% of the test solution in each test vessel was renewed daily after 24, 48, and 72 hours. 

Two 5-L replicates were used per treatment, and 10 fish were used per replicate (for a total of 20 

per treatment). Test vessels were loosely covered to reduce entry of dust, etc. 

Dilution Water: Natural seawater collected from Cape Cod Canal, Bourne, Massachusetts which 

derives water from Buzzards Bay or Massachusetts Bay. The water was filtered through 0.5-

micron polypropylene core filter and activated carbon, then stored for 1 to 4 days prior to use 

while being constantly aerated. During storage, the water had a salinity of 32 to 33 ppt and pH 

of 7.7 to 7.8. During the test: dissolved oxygen – 5.6 mg/L to above 100% saturation (7.5 mg/L), 

pH – 6.9 to 8.0, salinity – 32, temperature – 20 to 22 °C. Mean measured TOC levels in the 

control and 10,000 mg/L WSF test level were 2.7 mg/L (range = 1.2 to 6.0) and 4.5 mg/L (range 

= 3.0 to 5.6), respectively.

Test Levels: Control & 10,000 mg/L WSF loading rate. 

Calculation of LL50s: Statistical analysis of survival data not warranted. 

Test Substance: No undissolved test material was seen on the surface of the test vessels during 

the entire aquatic toxicity test. 

Reference Substance: Sodium lauryl sulfate (SLS). The 96-h LC50 was 1.2 mg/L. No 

information was provided on method of calculation. 

RESULTS

Endpoint value(s): Nominal concentrations: 96-h LL50 > 10,000 mg/L; 96-h NOEC = 10,000 

mg/L (no mortality or toxic signs noted). 

Results remarks: No mortality or signs of toxicity were noted in the 10,000 mg/L WSF test 

level and the control throughout the entire test. 

Mean measured TOC in the 10,000 mg/L WSF test level was 4.5 mg/L compared to 2.7 mg/L in 

the control.

Statistical results: Statistical analysis of survival data not warranted.

EPA does not consider the loading rate as the no-effect concentration when the concentration 

exceeds the water solubility of the substance. Assuming that the exposure concentration in the 

WSF is the water solubility limit (saturation) for 1-propene, 2-methyl-, sulfurized, the no-effect 

concentration would be approximately 2.7 mg/L. (No effects were observed at saturation.) 
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CONCLUSIONS

No mortality or signs of toxicity were noted in the 10,000 mg/L WSF test level and the control 

throughout the entire test.

STUDY RELIABILITY

[2] Reliable with restrictions. The TOC in dilution water at the beginning and end of the test was 

greater than 2 mg/L rather than < 2 mg/L. 

REFERENCE

Nicholson, R.B. 1986. Acute toxicity of CMA Test Material Code 525 to Sheepshead Minnow, 

Cyprinodon variegatus. Springborn Bionomics Study #10823-0186-6100-500-525, Report 

#BW-86-04-2004. As summarized in the Robust Summaries for the Alkyl Sulfides Category 

(submitted to EPA under the HPV Challenge Program by the Health, Environmental, and 

Regulatory Task Group of the American Chemistry Council’s Petroleum Additives Panel on 

May 19, 2005).
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D-2. Acute Toxicity to Aquatic Invertebrates

Summary of endpoint: Adequate

Study 1: Acute toxicity study of CASRN 68511-50-2 in Daphnia magna (Ward, 1993)

Title: Acute Toxicity of the Water Accommodated Fractions (WAFs) of CMA #613 to the 

Daphnid, Daphnia magna

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 1-Propene, 2-methyl-, sulfurized, CASRN 68511-50-2

Remarks: Purity not provided

METHOD

Method/guideline followed: U.S. EPA 797.1300 (1985, 1987), OECD TG 202 (1984)

Test type: Daphnia sp., Acute Immobilization Test (Static)

GLP compliant?: Yes

Year study was performed: 1993

Test species: Water flea (Daphnia magna), less than 24-hours old, bred at lab facility

Analytical monitoring: Yes; total organic carbon (TOC) measurements.

Exposure duration: 48 hours

Concentrations tested: 0, 100, 300, and 1000 mg/L WAF loading rates

Test method/conditions remarks: A static non-renewal test was conducted using water 

accommodated fractions (WAF) of the test material at 100, 300, and 1000 mg/L loading rates. 

WAFs were prepared by adding a measured weight of the test material to a measured volume of 

the dilution water and stirring for 24 hours with a magnetic stir bar. The test solutions were 

allowed to stand for 1 hour before the water phase (WAF) was siphoned off. 

Test conditions: Two 250-mL glass beakers that contained 200 ml of test solution were used per 

treatment. The 250-mL test vessels were loosely covered to reduce entry of dust, etc. 

Test temperature range: 20 ± 1 oC 
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Dilution water: Filtered well water collected at Hampton, New Hampshire and adjusted to the 

appropriate hardness 168 to 172 mg/L as CaCO3. The water was passed through activated 

carbon, a particle filter, and then an ultraviolet sterilizer and stored in a polyethylene tank where 

it was aerated. TOC levels were 2 mg/L at the beginning and end of the test, and 10 mg/L total 

suspended solids (TSS) at the beginning and < 10 mg/L at the end of the test. 

Lighting: A 16 hour light and 8 hour dark photoperiod was maintained with cool-white

fluorescent lights with an intensity of 20 μEin-1m-2. 

Water chemistry: Dissolved oxygen – 8.2 to 8.7 mg/L; pH – 7.8 to 8.2; conductivity – 570 to 

640 μmhos/cm; temperature – 20.5 to 20.9 °C. 

Element: Immobilization 

Test design: Control, 100, 300, & 1000 mg/L WAF loading rates. 10 daphnids per replicate (20 

per treatment). Method of calculating mean measured concentrations: not applicable. Exposure 

period: 48 hours. Analytical monitoring: Total organic carbon (TOC) measurements of initial 

test solutions and control (0-hour) and at test termination (48-h). TOC levels were 2 mg/L in the 

control, 3 mg/L at the 100 mg/L and 300 mg/L test levels; and 4 to 5 mg/L at the 1000 mg/L test 

vessel. TOC levels were not considered to be indicative of actual test material concentrations 

and results are therefore based on nominal loading rates.

Statistical methods were not utilized because there was greater than 50% survival in all test 

vessels. 

RESULTS

Endpoint value(s): 48-hour and 24-hour EC50 > 1000 mg/L (based on nominal loading rates); 

48-hour and 24-hour NOEC = 1000 mg/L (based on nominal loading rates).

Results remarks: Effect concentrations were based on nominal loading rates.

No immobilization was seen at the highest test concentration of 1000 mg/L (WAF).

Control response was satisfactory.

EPA does not consider the loading rate as the no-effect concentration when the concentration 

exceeds the water solubility of the substance. Assuming that the exposure concentration in the 

WAF is the water solubility limit (saturation) for 1-propene, 2-methyl-, sulfurized the no-effect 

concentration would be approximately 2.7 mg/L. (No effects were observed at saturation.) 

CONCLUSIONS

The WAFs of the test material were not toxic to daphnids at the concentrations tested. Ninetyfive to 100% survival occurred at all test concentrations. No sublethal effects were noted during 

the test. 
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STUDY RELIABILITY

[1] Reliable without restrictions. 

REFERENCE

Ward, T.J. 1993. Acute Toxicity of the Water Accommodated Fractions (WAFs) of CMA #613 

to the Daphnid, Daphnia magna. T.R. Wilbury Study #9178-CMA/ESI-613. As summarized in 

the Robust Summaries for the Alkyl Sulfides Category (submitted to EPA under the HPV 

Challenge Program by the Health, Environmental, and Regulatory Task Group of the American 

Chemistry Council’s Petroleum Additives Panel on May 19, 2005).
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D-3. Chronic Toxicity to Aquatic Invertebrates

Summary of endpoint: Adequate

Study 1: Reproduction test in Daphnia magna (Beattie, 2014)

Title: The effect of sulfurated decene on the reproductive output of Daphnia magna over a 21 

day exposure period

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 1-Decene, sulfurized, CASRN 72162-15-3; referred to as sulfurated decene or Mayco 

Base 1540 in the study report.

Remarks: Dark brown oily liquid; Batch No. S-31343; Typical purity: 90-100% (referenced 

from an MSDS). Certificate of analysis provided in the study report indicates a sulfur content of 

38.1% for Batch No. S-31343, but does not provide confirmation of the purity referenced from 

an MSDS and does not provide the percentage of other components of the test substance.

METHOD

Method/guideline followed: OECD TG 211 (2012) “Daphnia magna Reproduction Test”

Test type: Reproduction toxicity test in daphnids (semi-static)

GLP compliant?: Yes

Year study was performed: 2013-2014

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

(Brixham Environmental Laboratory) and Smithers Viscient

Analytical monitoring: No

Exposure duration: 21 days
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Concentrations tested: Solutions of the test item, a multi-constituent substance with low 

aqueous solubility (measured: ≤ 10 μg/L for the test item1; estimated: 6.15 x 10-6 mg/L for 

CASRN 72162-15-3

2

), were prepared as water accommodated fractions (WAFs).

Range-Finding Test: Nominal loading rates: 0 (control), 0.01, 0.1, 1.0, and 10 mg/L

Definitive Test 1: Nominal loading rates: 0 (control), 0.2, 0.5, 1.3, 3.2, and 8 mg/L

Definitive Test 2: Nominal loading rates: 0 (control), 0.5, 1.3, 3.2, 8, and 20 mg/L

Test method/conditions remarks: 

Test Medium: 

The test medium was Elendt M4 media, which contained trace elements, macronutrients, and 

vitamins. The medium had a hardness of 199.88 mg CaCO3/L (calculated from the chemical 

composition), with pH stabilized between 6 and 9. 

Preparation of Test Solutions: 

Range-Finding Test:

No information was provided regarding the preparation of test item solutions for the rangefinding test. 

Definitive Test:

Two definitive tests were sequentially conducted. The first definitive test was terminated early 

because the number of neonates produced in the control group was too few and inhibition was 

less than expected at the highest nominal loading rate of 8 mg/L. No further details regarding 

this test were provided in the study report.

For the second definitive test: “The stock solutions were prepared in dilution water (Elendt M4 

daphnia media) three times weekly. A stock of 20 mg/L was prepared by adding 0.1 g of sample 

into 5000 mL of dilution water. This was prepared as a Water Accommodated Fraction (WAF) 

by stirring for 48 ± 3 hours and being left to settle for a minimum of 4 hours.3 This was diluted 

to give [nominal] loading rates of 0.5, 1.3, 3.2, 8, and 20 mg/L... by adding 50, 130, 320, and 

800 mL of the appropriate stock to the corresponding 2 litres volumetric flask and making the 

volume [i.e., filling the volumetric flask] with Elendt M4 daphnia media.” [Beattie 2014, p. 8] 

No further details, such as whether the aqueous phase of the WAF was siphoned off for use as 

the test solution, were provided.

 

1 The water solubility limit of the test item was experimentally determined to be ≤ 10 μg/L at 20 °C by WIL 

Research Europe B.V. (Oudhoff, K.A. 2013. Determination of physico-chemical properties of 1-decene, 

sulfurized. WIL Research Europe B.V., ‘s-Hertogenbosch, The Netherlands. Project No. 502505.) Study submitted 

by Dover Chemical Corporation to EPA on September 25, 2013 and available online at http://www.regulations.gov/

#!documentDetail;D=EPA-HQ-OPPT-2009-0112-0161 as of July 17, 2014. 

2 The water solubility limit of CASRN 72162-15-3 was estimated to be 6.15 x 10-6 mg/L at 25 °C using EPI Suite 

v4.11. (U.S. EPA. 2012. Estimation Programs Interface SuiteTM (EPI Suite) for Microsoft® Windows, v4.11. U.S. 

Environmental Protection Agency, Washington, DC, USA. Available online at http://www.epa.gov/opptintr/

exposure/pubs/episuitedl.htm as of July 17, 2014.)

3

In the “Deviations” section, the study report states: “Some of the Water Accommodated Fractions were only 

allowed to settle for 3 hours and 45 minutes rather than the minimum 4 hours specified.” [Beattie 2014, p. 8]
Final HPV3 Test Rule Data Adequacy Review (July 2014) CASRN 72162-15-3

15

Test Procedures:

Range-Finding Test Procedure: 

The test was conducted using two replicates for the control group and each test concentration 

(0.01, 0.1, 1.0, and 10 mg/L). Each test chamber contained 100 mL test solution. The duration 

of the exposure was not specified. The study report states that the “range finding study did not 

have a solvent control” [Beattie 2014, p. 8], but does not indicate elsewhere that a solvent was 

used in the range-finding test. The study report also states that “the replacement days differed” 

[Beattie 2014, p. 8] in the range-finding test, but no further details are provided.

Definitive Test Procedure:

The first definitive test (using nominal loading rates of 0, 0.2, 0.5, 1.3, 3.2, and 8 mg/L) was 

terminated early, as described above. No further details regarding the test procedure for this test 

were provided in the study report.

In the second definitive test, neonate daphnids (10 replicates/concentration1; 1 daphnid/replicate) 

were exposed to WAFs of the test item at nominal loading rates of 0 (control), 0.5, 1.3, 3.2, 8, 

and 20 mg/L for 21 days under semi-static conditions with test solution renewal 3 times per week 

(i.e., renewal on study days 3, 5, 7, 10, 12, 14, 17, and 19). The test vessels were 120-mL glass 

bottles, each containing 100 mL of control or test medium. The loading rate was 10 daphnids/L. 

The test vessels were placed in an incubator at a temperature of 19.5 to 21.1 °C and were covered 

with a transparent Perspex sheet to prevent aerial contamination of the vessels and to reduce 

evaporative loss of liquid during the exposure period. The photoperiod was 16 hours of light and 

8 hours of darkness with a light intensity of 1000-1400 lux. The daphnids were fed 0.5 mL of an 

algal suspension daily, corresponding to 0.14 mg organic carbon per liter of test solution. The 

test vessels were not aerated during the exposure period.

Parents were examined daily for mortality and the number of offspring (neonates). The presence 

of aborted eggs or dead offspring was also recorded and these were removed daily. Wet weights 

of each surviving adult were recorded on day 21. Dissolved oxygen and pH were recorded at the 

start and end of the test, and just before and after renewal of the test solutions. Temperature in 

the incubator was recorded daily. 

Analytical Monitoring: 

The study authors stated: “It was not possible to conduct analysis to confirm exposure 

concentrations. Attempts were made outside of this study including the use of GC/MS but a 

combination of the low solubility and Sulfurated decene containing multiple constituents meant 

that these attempts were unsuccessful.” [Beattie 2014, p. 6]

Statistical Methods:

Toxcalc v5.0 statistics program was used to determine the EL50 (the loading rate resulting in a 

50% effect level), LOELR (lowest observed effect loading rate), and NOELR (no observed 

effect loading rate) for the following parameters: adult mortality, adult growth (based on wet 

weight of adults on day 21), fecundity (number of offspring produced per surviving adult), and 

intrinsic rates of natural increase (measurement of population growth calculated using the Euler-

 

1 Although the discussion section (p. 12) states that there were 20 daphnids in the control group, the methods section 

(p. 7) and results tables indicate that there were only 10 daphnids in the control group. 
Final HPV3 Test Rule Data Adequacy Review (July 2014) CASRN 72162-15-3

16

Lotka equation). “Fecundity, growth, and intrinsic rates of natural increase were determined, 

following tests for normality of distribution and equality of variance, using the Maximum 

Likelihood-Probit-test since the numbers of replicates differed between treatments. The parent 

survival used linear interpolation as the numbers of replicates were the same between treatments. 

LOEL[R] and NOEL[R] for adult survival were calculated using the Dunnett’s test with the top 

concentration of 20 mg/L removed as there was only [one] replicate remaining [at this 

concentration] and more than one replicate is required [for this test].” [Beattie 2014, p. 8]

RESULTS

Endpoint value(s): The following endpoint values and 95% confidence intervals (CIs) were 

based on nominal loading rates1:

Parameter

EL50 value (mg/L) 

(95% CI) NOELR (mg/L) LOELR (mg/L)

Fecundity 0.830

(0.397-1.29) < 0.5 0.5

Weight (mg) 1.14

(0.952-3.97) < 0.5 0.5

Parental Survival 12.0

(6.93-15.2) 8 20

Intrinsic Rate of 

Natural Increase

3.81

(2.0-13.3) < 0.5 0.5

Results remarks: 

Range-Finding Test Results: 

The study author reported that the EL50 for the range-finding test was determined to be between 

0.1 and 1 mg/L. The parameter (e.g., fecundity, weight, survival, etc.) used as the basis for the 

EL50 was not specified. No further results were reported for the range-finding test. 

Definitive Test Results:

Results were not provided for the first definitive test, which was terminated early. 

In the second definitive test, cumulative mortality rates for the exposure period (days 0-21) were 

0, 10, 20, 0, 30, and 90% at nominal loading rates of 0, 0.5, 1.3, 3.2, 8, and 20 mg/L, 

respectively. Times to first death were 5, 3, 3, and 3 days in the 0.5, 1.3, 8, and 20 mg/L groups, 

respectively. 

Average body weights of surviving parental daphnids on day 21 were 8.0, 5.1, 3.7, 3.8, 3.1, and 

2.1 mg at 0, 0.5, 1.3, 3.2, 8, and 20 mg/L, respectively. The corresponding percentage inhibition 

of body weight values was 35.9, 53.8, 53.2, 61.6, and 73.9% for the 0.5, 1.3, 3.2, 8, and 20 mg/L 

groups, respectively. 

 

1 Endpoint values are based on nominal loading rates of WAFs because analytical measurement of test 

concentrations was not performed.
Final HPV3 Test Rule Data Adequacy Review (July 2014) CASRN 72162-15-3

17

The cumulative number of offspring on day 21 was 1396, 879, 292, 281, 199, and 10 at 0, 0.5, 

1.3, 3.2, 8, and 20 mg/L, respectively. The average number of offspring per surviving adult 

(fecundity) was 140, 98, 37, 28, 28, and 10 in the 0, 0.5, 1.3, 3.2, 8, and 20 mg/L groups, 

respectively. The corresponding percentage inhibition of fecundity was 30.0, 73.9, 79.9, 79.6, 

and 92.8% for the 0.5, 1.3, 3.2, 8, and 20 mg/L groups, respectively. 

The number of days to first brood was 8-9, 9-11, 13-14, 12-18, 14-19, and 21 days for the 0, 0.5, 

1.3, 3.2, 8, and 20 mg/L groups, respectively. The number of days to second brood was 10-13, 

12-14, 14-19, 14-21, 17-21, and > 21 (no second brood) for the same groups, respectively. The 

mean number of broods per adult were 5, 4.4, 2.9, 2.4, 2.1, and 1.0 in the 0, 0.5, 1.3, 3.2, 8, and 

20 mg/L groups, respectively. 

The mean intrinsic rates of natural increase over the 21-day exposure were 0.473, 0.446, 0.2841, 

0.226, 0.214, and 0.110 at 0, 0.5, 1.3, 3.2, 8, and 20 mg/L, respectively. The corresponding 

percentage inhibition values for these groups were 0, 5.7, 40.0, 52.2, 54.8, and 76.7%, 

respectively. 

During the study, dissolved oxygen concentrations ranged from 4.0 to 9.2 mg/L and pH values 

ranged from 6.9 to 8.1. Temperature ranged from 19.5 to 21.1 °C.

“None (0%) of the... control parent Daphnia died during the study and no Daphnia were trapped 

at the surface of the water. The number of live offspring produced per parent animal surviving to 

the end of the test in the control vessels was 110 to 161, with a mean value of 139.6. The 

coefficient of variation for control fecundity was calculated as 11.8%. This fulfils the validity 

criteria of the study which states that adult control mortality must not exceed 20% at the end of 

the test, that the average fecundity per parent Daphnia surviving at the end of the test will be 

equal to or greater than 60, and that the coefficient of variation around the mean number of living 

offspring produced per parent Daphnia would be ≤ 25%.” [Beattie 2014, p. 12]

CONCLUSIONS

Based on nominal loading rates, the 21-day NOELR for fecundity, body weight, and intrinsic 

rate of natural increase was < 0.5 mg/L; the LOELR was 0.5 mg/L (lowest loading rate tested). 

The 21-day NOELR for parental survival was 8 mg/L; the LOELR was 20 mg/L. 

The 21-day EL50 values for fecundity, body weight, parental survival, and intrinsic rate of natural 

increase were 0.83 mg/L (95% CI: 0.397-1.29 mg/L), 1.14 mg/L (95% CI: 0.952-3.97), 12.0 

mg/L (95% CI: 6.93-15.2 mg/L), and 3.81 mg/L (95% CI: 2.0-13.3 mg/L), respectively.

STUDY RELIABILITY

[2] Reliable with restrictions. Typical purity (90-100%) of 1-decene, sulfurized was referenced 

from an MSDS, but was not provided in the certificate of analysis included in the study report. 

In addition, the certificate of analysis does not provide the percentage of all components of the 

test substance. The method of preparation of the WAF stock solution is not adequately 

 

1 This value is reported as 0.284 on p. 10 of the study report, but as 0.282 on p. 17.
Final HPV3 Test Rule Data Adequacy Review (July 2014) CASRN 72162-15-3

18

described; the study report does not specify whether the aqueous fraction of the stock solution 

was collected and used for testing or if the solution was used directly after the settling period. 

Although analytical monitoring attempts were unsuccessful, given the low water solubility of the 

test item (measured: ≤ 10 μg/L for the test item1; estimated: 6.15 x 10-6 mg/L for CASRN 72162-

15-3

2

) and the limit of detection of the validated analytical method3(10 μg/L), it is unlikely that 

the test item would have been detected in the aqueous phase of the test solutions. The reported 

LOELR was 0.5 mg/L, based on effects on fecundity, body weight, and intrinsic rate of natural 

increase at the lowest loading rate tested; however, the LOEC (lowest observed effect 

concentration) could be as low as the water solubility limit of the test item, given that the test 

substance concentrations were not analytically measured. 

REFERENCE

Beattie, H. (Study Director). 2014. The effect of sulfurated decene on the reproductive output of 

Daphnia magna over a 21 day exposure period. Chemex Environmental International Limited, 

Cambridge, UK. Study No. 10259/130506. Sponsor: Dover Chemical Corporation, Dover, OH. 

 

Text in quotes is taken directly from Beattie (2014).

 

1 The water solubility limit of the test item was experimentally determined to be ≤ 10 μg/L at 20 °C by WIL 

Research Europe B.V. (Oudhoff, K.A. 2013. Determination of physico-chemical properties of 1-decene, 

sulfurized. WIL Research Europe B.V., ‘s-Hertogenbosch, The Netherlands. Project No. 502505.) Study submitted 

by Dover Chemical Corporation to EPA on September 25, 2013 and available online at http://www.regulations.gov/

#!documentDetail;D=EPA-HQ-OPPT-2009-0112-0161 as of July 17, 2014. 

2 The water solubility limit of CASRN 72162-15-3 was estimated to be 6.15 x 10-6 mg/L at 25 °C using EPI Suite 

v4.11. (U.S. EPA. 2012. Estimation Programs Interface SuiteTM (EPI Suite) for Microsoft® Windows, v4.11. U.S. 

Environmental Protection Agency, Washington, DC, USA. Available online at http://www.epa.gov/opptintr/

exposure/pubs/episuitedl.htm as of July 17, 2014.)

3 WIL Research Europe B.V. validated a gas chromatographic method with mass spectrometric detection (GC-MS) 

for measurement of CASRN 72162-15-3. (Oudhoff, K.A. 2013. Determination of physico-chemical properties of 

1-decene, sulfurized. WIL Research Europe B.V., ‘s-Hertogenbosch, The Netherlands. Project No. 502505.) Study 

submitted by Dover Chemical Corporation to EPA on September 25, 2013 and available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0112-0161 as of July 17, 2014.
Final HPV3 Test Rule Data Adequacy Review (July 2014) CASRN 72162-15-3

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D-4. Toxicity to Aquatic Plants

Summary of endpoint: Adequate

Study 1: Toxicity of CASRN 68511-50-2 to Pseudokirchneriella subcapitata

Title: Not available

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 1-Propene, 2-methyl-, sulfurized, CASRN 68511-50-2 

Remarks: Purity not provided

METHOD

Method/guideline followed: Test protocol followed US EPA Toxic Substances Control Act 

Test Guideline #797.1050 (1985, 1987), OECD TG 201 Alga, Growth Inhibition Test (1984). 

Test type: Alga, Growth Inhibition Test (WAF static)

GLP compliant?: Yes

Year study was performed: 1994

Test species: Freshwater green algae (Pseudokirchneriella subcapitata)

Analytical monitoring: Yes. Total organic carbon (TOC) measurements of initial test solutions 

and control (0-hour) and at test termination (96-h) (EPA Method 415.1 (1979)). Water samples 

were passed through a 0.45 micron filter prior to TOC analysis. 

Exposure duration: 96 hours

Concentrations tested: Control, 1.0, 5.0, 10, 50, 100 mg/L (nominal loading rates)

Test method/conditions remarks: Test Organisms: source – T.R. Wilbury in-house culture 

originally purchased from the University of Texas at Austin algae collection. 

Test System: Individual WAFs were prepared for each test level and renewed daily. A measured 

weight of test material was added to a measured volume of dilution water (1-L) in a glass vessel 

and stirred for 24 hours. Stirring was accomplished using a Telfon-coated magnetic stir bar. 

Mixing speed was adjusted such that a vortex formed between 30 to 50% of the distance to the 

bottom. Following the mixing period, the test solutions were allowed to stand for 1 hour before 

the water phase was removed. To avoid removing test material from the surface or bottom, a 
Final HPV3 Test Rule Data Adequacy Review (July 2014) CASRN 72162-15-3

20

siphon was placed in the mixing vessel prior to addition of water and test substance with the 

lower end 1-2 inches off the bottom. The siphoned water phase (i.e., WAF) was used for the 

aquatic toxicity test. A static test was conducted; i.e., there was no daily renewal of test solution. 

Three 100-mL replicates were used per treatment, with an inoculum of ~10,000 cells/mL. The 

250-mL Erlenmeyer flasks were stoppered with foam plugs to reduce entry of dust, etc. During 

the test, all treatment and control flasks were randomly placed on an orbital shaker adjusted to 

approximately 100 cycles per minute under constant light (24 hours/day). Daily cell counts were 

made visually by means of direct microscopic examination with a hemocytometer. Cool-white 

fluorescent lights provided a light intensity of 47 to 50 μEin/m2sec. 

At the conclusion of the 96-h test, a 0.5 mL subsample of test media from each 100 mg/L test 

flask was combined with 100 mL of fresh untreated alga media and incubated for up to 9 days or 

as soon as growth occurs. This was done to determine if growth inhibition was algistatic or 

algicidal. 

Dilution Water: Sterile enriched alga growth media (US EPA, 1978, T.R. Wilbury SOP #6) 

adjusted to pH 7.5. Measured TOC and total suspended solids in fresh untreated alga media 

were < 1.0 and < 10 mg/L, respectively. Test temperature – 23.4 to 23.6 C; pH – 7.0 to 7.1 at 0-

hour and 8.6 to 10.2 after 96 hours. TOC measurements were only made on the lowest and 

highest test levels and control at the beginning and end of the test. TOC levels were < 1.0 mg/L 

in the control and 1.0 mg/L WAF test level and 3 mg/L at 100 mg/L. 

Test Levels: Control, 1.0, 5.0, 10, 50, 100 mg/L WAF loading rates. 

Calculation of EL50s and NOELRs: Moving average and probit methods (Stephan, 1983) were 

used to calculate EC50s (i.e., EL50s). A parametric one-way analysis of variance (ANOVA) and 

Dunnett’s test were used to calculate the no-observed effect concentration (i.e., EL0s) when data 

were normally distributed and a non-parametric Kruskal and Wallis test was used if data were 

not normally distributed. 

Test Substance: No undissolved test material was seen on the surface of the test vessels during 

the entire aquatic toxicity test. 

Reference Substance: No 

RESULTS

Endpoint value(s): Based on nominal loading rates: 72-hour EL50 (cell density) = 26 (21-32) 

mg/L; 72-hour NOELR (cell density) = 5.0 mg/L; 96-hour EL50 (cell density) = 34 (29-39) 

mg/L; 96-hour NOELR (cell density) = 10 mg/L; 72-hour EL50 (growth rate) > 100 mg/L; 72-

hour NOELR (growth rate) = 5.0 mg/L; 96-hour EL50 (growth rate) > 100 mg/L; 96-hour 

NOELR (growth rate) = 10 mg/L

Results remarks: Test concentrations for the definitive test were not specified in a protocol 

amendment and the pH of the sterile media at the start of the test was 7.0 rather than 7.5. These 

deviations did not compromise the study. 
Final HPV3 Test Rule Data Adequacy Review (July 2014) CASRN 72162-15-3

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72-hour EL50 for cell density was determined by moving average method. 96-hour EL50 for cell 

density was determined by Probit method. 96-hour NOELRs for cell density and growth rate 

were determined by hypothesis analysis tests.

Re-growth of inhibited cultures from the 100 mg/L test level revealed the effect was algistatic 

rather than algicidal.

CONCLUSIONS

After 96 hours of exposure, reductions in cell density and growth rate were observed at a loading 

rate > 10 mg/L.

STUDY RELIABILITY 

[1] Reliable without restrictions.

REFERENCE

Reference information is not available. This study was obtained from the Robust Summaries for 

the Alkyl Sulfides Category (submitted to EPA under the HPV Challenge Program by the 

Health, Environmental, and Regulatory Task Group of the American Chemistry Council’s 

Petroleum Additives Panel on May 19, 2005).
Final HPV3 Test Rule Data Adequacy Review (July 2014) CASRN 72162-15-3

22

D-5. Acute Toxicity

Summary of endpoint: Adequate

Study 1: Acute oral toxicity study of CASRN 68511-50-2 in rats

Title: Not available

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 1-Propene, 2-methyl-, sulfurized, CASRN 68511-50-2 

Remarks: Purity not provided

METHOD

Method/guideline followed: Not specified

Test type: Acute Oral Toxicity (LD50)

GLP compliant?: Pre-GLP

Year study performed: 1975

Test species: Sherman/Wistar rat (young adult)

Number of animals/sex/dose: 5 males/dose

Route of administration: Oral gavage

Vehicle: None

Doses/concentrations tested: 2.0, 4.0, 8.0, 16.0, or 32.0 mL/kg

Statistical methods used: Not specified

Test method/conditions remarks: Rats were fasted 24 hours prior to dosing. Test material was 

administered by gavage in a single oral dose at concentrations of 2.0, 4.0, 8.0, 16.0, or 32.0 

mL/kg. Animals observed for 14 days post-dosing for signs of toxicity or mortality. Body 

weights were not taken; gross necropsies and histopathology were not performed.
Final HPV3 Test Rule Data Adequacy Review (July 2014) CASRN 72162-15-3

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RESULTS

Endpoint value(s): LD50 = 5.7 mL/kg; 19/20 confidence limits = 4-8 mL/kg

Results remarks: No deaths were observed at 2.0 or 4.0 mL/kg. At 8 mL/kg, 4/5 were dead at 

day 1 post dosing and 1/5 were dead at day 2. 5/5 rats were dead at day 1 in groups given 16.0 

or 32.0 mL/kg. No data were presented on parameters other than mortality. 

CONCLUSIONS

The oral LD50 is 5.7 mL/kg (~5700 mg/kg) for male rats.

STUDY RELIABILITY

[2] Reliable with restrictions. Details of study design and observations during study were not 

presented. Body weights were not measured; gross necropsies and histopathology were not 

performed.

REFERENCE

Reference information is not available (the underlying study contains confidential business 

information). This study was obtained from the Robust Summaries for the Alkyl Sulfides 

Category (submitted to EPA under the HPV Challenge Program by the Health, Environmental, 

and Regulatory Task Group of the American Chemistry Council’s Petroleum Additives Panel on 

May 19, 2005).
Final HPV3 Test Rule Data Adequacy Review (July 2014) CASRN 72162-15-3

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Study 2: Acute oral toxicity study of CASRN 68515-88-8 in rats

Title: Not available

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Pentene, 2,4,4,-trimethyl, sulfurized, CASRN 68515-88-8

Remarks: Purity not provided

METHOD

Method/guideline followed: Consistent with guidelines outlined in OECD TG 401

Test type: Acute Oral Toxicity

GLP compliant?: Yes

Year study performed: 1987

Test species: Albino rats of the outbred Sprague-Dawley strain

Number of animals/sex/dose: 5/sex/dose

Route of administration: Oral gavage

Vehicle: Mineral oil-based material

Doses/concentrations tested: 5.0 g/kg

Statistical methods used: Not specified

Test method/conditions remarks: The animals were fasted overnight before dosing. The 

sample was administered as supplied at a limit dose of 5.0 g/kg via oral gavage with a syringe 

and Nelaton catheter. Following administration, the animals were allowed food and water for the 

15-day observation period. The animals were observed three times on the day of dosing and 

twice on study day two and daily thereafter. Individual weights were recorded on the day of 

dosage and on the day of termination (day 15). The animals were euthanized by carbon dioxide 

at the conclusion of the observation period. Gross autopsies were performed on all animals that 

died during the observation period and on all survivors after day 15.
Final HPV3 Test Rule Data Adequacy Review (July 2014) CASRN 72162-15-3

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RESULTS

Endpoint value(s): LD50 > 5.0 g/kg

Results remarks: All animals survived to termination of the experiment (day 15). Decreased 

activity (3/5 females), diarrhea (1/5 males), salivation (1/5 males and 1/5 females), and apparent 

urinary incontinence (3/5 females) were noted following dose administration. All animals 

appeared normal on study days 5-15. Test material did not cause an adverse effect on mean body 

weight in either sex. 

CONCLUSIONS

The oral LD50 is greater than 5.0 g/kg for male and female rats.

STUDY RELIABILITY

[1] Reliable without restrictions. 

REFERENCE

Reference information is not available (the underlying study contains confidential business 

information). This study was obtained from the Robust Summaries for the Alkyl Sulfides 

Category (submitted to EPA under the HPV Challenge Program by the Health, Environmental, 

and Regulatory Task Group of the American Chemistry Council’s Petroleum Additives Panel on 

May 19, 2005).
Final HPV3 Test Rule Data Adequacy Review (July 2014) CASRN 72162-15-3

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Study 3: Acute dermal toxicity study of CASRN 68515-88-8 in rabbits

Title: Not available

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Pentene, 2,4,4-trimethyl, sulfurized, CASRN 68515-88-8

Remarks: 97% purity

METHOD

Method/guideline followed: FHSA Regulations 16 CFR 1500.40

Test type: Acute Dermal Toxicity

GLP compliant?: Yes

Year study performed: 1986

Test species: New Zealand White rabbits (young adults)

Number of animals/sex/dose: 5/sex/dose

Route of administration: Dermal

Vehicle: Mineral oil-based material dosed undiluted

Doses/concentrations tested: 2.0 g/kg

Statistical methods used: Not specified

Test method/conditions remarks: The sample was applied to unabraded shaved skin under 

impervious occlusion for 24 hours at a limit dose of 2.0 g/kg. At the end of the 24-hour exposure 

period, the wrapping was removed and any unabsorbed test material remaining on the skin was 

removed by gentle sponging using a paper towel moistened with mineral oil. The animals were 

observed for signs of toxicity or behavioral changes frequently on the day of treatment. 

Thereafter, all surviving rabbits were examined for outward signs of toxicity once per day, for 

the entire 14-day observation period. Individual weights were recorded on the day of dosage, 

weekly thereafter and prior to sacrifice. The surviving animals were euthanized at the 

conclusion of the observation period. Gross autopsies were performed on all animals after 14 

days. 
Final HPV3 Test Rule Data Adequacy Review (July 2014) CASRN 72162-15-3

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RESULTS

Endpoint value(s): LD50 > 2.0 g/kg

Results remarks: One rabbit died on day 14 of the study. No other deaths were observed during 

the 14-day observation period. In the male group, mild skin erythema and mild-to-moderate 

edema were observed after unwrapping at 24 hours. Slight to mild skin irritation noted at day 7 

was completely resolved by day 14. A loss of body weight was noted for 1/5 male animals at 

day 7. The same animal was found dead on day 14 after experiencing a bloated appearance. 

Signs of dehydration and no formed fecal material in the intestinal tract were noted for the one 

mortality. Other than the previous observation, all animals appeared normal throughout the 14-

day observation period. In the females, skin reactions were typical of those observed with the 

male group. A loss of body weight was noted in one female at day 7. Gross pathological 

examination of the female rabbits revealed no remarkable findings. 

CONCLUSIONS

The test article, when dosed as supplied and studied in 5 males and 5 female albino rabbits, 

appears to have an acute dermal LD50 greater than 2.0 g/kg. 

STUDY RELIABILITY

[1] Reliable without restrictions. 

REFERENCE

Reference information is not available (the underlying study contains confidential business 

information). This study was obtained from the Robust Summaries for the Alkyl Sulfides 

Category (submitted to EPA under the HPV Challenge Program by the Health, Environmental, 

and Regulatory Task Group of the American Chemistry Council’s Petroleum Additives Panel on 

May 19, 2005).
Final HPV3 Test Rule Data Adequacy Review (July 2014) CASRN 72162-15-3

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Study 4: Acute dermal toxicity study of CASRN 67762-55-4 in rabbits

Title: Not available

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Alkenes, C15-18 alpha-, sulfurized, CASRN 67762-55-4

Remarks: Purity not provided

METHOD

Method/guideline followed: Not indicated

Test type: Acute Dermal Toxicity

GLP compliant?: Yes

Year study performed: 1996

Test species: New Zealand White rabbits

Number of animals/sex/dose: 10/sex/dose

Route of administration: Dermal

Vehicle: None

Doses/concentrations tested: 2000 mg/kg

Statistical methods used: Not specified

Test method/conditions remarks: One dermal, semi-occluded patch of test article at 2000 

mg/kg was applied to clipped dorsal skin of each animal. The patches were removed after 24 

hours. All animals were observed daily for 14 days following test article administration. 

RESULTS

Endpoint value(s): LD50 > 2000 mg/kg

Results remarks: No clinical signs were observed during the study. Erythema and/or edema of 

the skin at application site were observed on Day 1 in some animals. There was an increase in 

mean body weight during the study. None of the animals died during the study. No visible 

lesions were observed in any animal at terminal necropsy. 
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CONCLUSIONS

The dermal LD50 is greater than 2000 mg/kg.

STUDY RELIABILITY

[1] Reliable without restrictions. 

REFERENCE

Reference information is not available (the underlying study contains confidential business 

information). This study was obtained from the Robust Summaries for the Alkyl Sulfides 

Category (submitted to EPA under the HPV Challenge Program by the Health, Environmental, 

and Regulatory Task Group of the American Chemistry Council’s Petroleum Additives Panel on 

May 19, 2005).
Final HPV3 Test Rule Data Adequacy Review (July 2014) CASRN 72162-15-3

30

Study 5: Acute inhalation toxicity study of CASRN 68511-50-2 in rats

Title: Not available

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 1-Propene, 2-methyl-, sulfurized, CASRN 68511-50-2

Remarks: Purity not provided

METHOD

Method/guideline followed: Modified OECD TG 403 

Test type: Acute Inhalation Toxicity

GLP compliant?: Yes

Year study performed: 1990

Test species: Sprague-Dawley rats (9 weeks old at initiation of study)

Number of animals/sex/dose: 10/sex/dose

Route of administration: Whole-body inhalation of vapor 

Vehicle: Not applicable

Doses/concentrations tested: 0.07 or 0.39 mg/L

Statistical methods used: Not specified

Test method/conditions remarks: Rats were exposed to 0.07 or 0.39 mg/L vapor for a single 

4-hour whole-body exposure. Sham control rats were placed in inhalation chambers in room air. 

One half of rats (5M, 5F) from each group were sacrificed 24 hour post-exposure; others 

maintained for 2 weeks recovery. Vapors from the methyl propene derivative were generated

from sample heated to 200°C in a counter current generator and delivered into the exposure 

chamber. Air samples were pulled through glass fiber filters to verify that particles were not 

present and animals were being exposed to pure vapor. Vapors were analyzed by GC to 

quantitate chamber concentrations using octane as a standard; qualitative analyses were 

performed by GC/MS. Animals were observed in the chambers and post exposure for clinical 

signs of toxicity. Body weights were taken and selected organs weighed at necropsy. 

Histopathology was conducted on liver, kidney, lungs, nasal turbinates, and tracheobronchial 

lymph nodes. 
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RESULTS

Endpoint value(s): LC50 > 0.39 mg/L

Results remarks: No mortality and minimal toxicity were observed. Abnormal clinical signs 

occurring during and immediately post-exposure included oral and ocular discharge, shallow 

respiration (some high-dose rats only), and decreased response to stimuli. No abnormal 

treatment-related clinical signs were observed from 1 hour post-exposure to the end of the 

recovery period. Group body weights and weights of liver, kidney, and lungs were unaffected by 

exposure. No treatment-related microscopic lesions were observed in the 5 organs examined. 

CONCLUSIONS

The LC50 > 0.39 mg/L was for a vapor generated from the starting material at 200°C, not from 

the whole methyl propene derivative. Vapor approximates conditions of workplace exposure. 

STUDY RELIABILITY

[2] Reliable with restrictions. Study performed with only two dose groups and is not a limit test. 

Physical and chemical properties provided with exception of structural formula and purity of 

starting material. 

REFERENCE

Reference information is not available (the underlying study contains confidential business 

information). This study was obtained from the Robust Summaries for the Alkyl Sulfides 

Category (submitted to EPA under the HPV Challenge Program by the Health, Environmental, 

and Regulatory Task Group of the American Chemistry Council’s Petroleum Additives Panel on 

May 19, 2005).
Final HPV3 Test Rule Data Adequacy Review (July 2014) CASRN 72162-15-3

32

Study 6: Acute inhalation toxicity study of CASRN 68515-88-8 in mice and guinea pigs 

Title: Not available

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Pentene, 2,4,4-trimethyl-, sulfurized, CASRN 68515-88-8

Remarks: Purity not provided

METHOD

Method/guideline followed: Consistent with EPA Health Effects Guideline OPPTS 870.1300

Test type: Acute Inhalation Toxicity

GLP compliant?: Yes

Year study performed: 1988

Test species: CD-1 strain mice, Hartley strain guinea pigs

Number of animals/sex/dose: 5/sex/dose

Route of administration: Aerosol inhalation (whole-body)

Vehicle: Mineral oil-based material, dosed as supplied

Doses/concentrations tested: 4.3 mg/L

Statistical methods used: Not specified

Test method/conditions remarks: Two groups of five mice/sex and five guinea pigs/sex were 

exposed for 4 hours to the test material (nominal concentration of 5 mg/L) as a liquid droplet 

aerosol generated by a Laskin nebulizer apparatus delivered into a plexi-glass chamber. Also, a 

control group of mice and guinea pigs was exposed to mineral oil in the same manner as the testmaterial-exposed group except that the test material was not administrated. The details of the 

whole-body exposure are consistent with those described in EPA Health Effects Guideline 

OPPTS 870.1300. The actual exposure concentration as measured by gravimetric analysis was 

4.3 mg/L. Particle size analyses were performed once/hour from the test material chamber using 

a cascade impactor. Animal observations for toxicological signs and mortality were recorded 

every 15 minutes during the exposure, and twice daily for the 14-day observation period. 

Individual weights were recorded on the day prior to exposure and on days 2, 3, 5, 8 and 14. At 

the conclusion of the observation period, the surviving animals were euthanized by 
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33

exsanguination under general anesthesia. All animals were subjected to gross necropsy (nasal 

passages, trachea, external surface, all orifices, the cranial cavity, the brain and spinal cord, and 

all viscera).

RESULTS

Endpoint value(s): LC50 (mice) > 4.3 mg/L; LC50 (guinea pigs) > 4.3 mg/L 

Results remarks: The mass median aerodynamic diameter for the studies was 1.6 microns with 

a geometric standard deviation of 2.1 (estimated percent of particles < 10 microns = 100%). One 

female guinea pig was euthanized on study day 7 because of a broken leg, an effect thought to be 

unrelated to the administration to the test material. All other animals survived the duration of the 

study. Observations noted during the test material exposure included reduced activity and 

matted coat. Signs exhibited by the test animals upon removal from the chamber and during the 

two-hour post-exposure observation period on day 1 included matted coat, yellow fur, yellow 

ano-gentital staining, and nasal discharge. The control groups were generally unremarkable 

during the exposure and immediately thereafter. During week 1, the test mice exhibited few 

signs other than matted coat. The test guinea pigs exhibited matted coat and ano-genital staining. 

During week 2, ano-genital staining was the only remarkable sign in the guinea pigs. No 

significant difference was noted between the test and control group weights for either species. 

No gross lesions that could be attributable to the test material were observed in any of the mice 

or guinea pigs. 

CONCLUSIONS

Ten of ten CD-1 mice and ten of ten Hartley guinea pigs received a single four-hour whole-body 

exposure to 4.3 mg/L test material as a respirable aerosol. All animals survived the exposure and 

the 14-day post-exposure observation period with the exception of a single guinea pig that was

euthanized for a broken limb. Signs of treatment included reduced activity and matted coat 

during the exposure. The treated animals were generally comparable to air-only control animals 

during the observation period. Body weight values and gross post mortem observations were 

generally unremarkable for differences between control and treated animals of either species. 

STUDY RELIABILITY

[1] Reliable without restrictions. 

REFERENCE

Reference information is not available (the underlying study contains confidential business 

information). This study was obtained from the Robust Summaries for the Alkyl Sulfides 

Category (submitted to EPA under the HPV Challenge Program by the Health, Environmental, 

and Regulatory Task Group of the American Chemistry Council’s Petroleum Additives Panel on 

May 19, 2005).
Final HPV3 Test Rule Data Adequacy Review (July 2014) CASRN 72162-15-3

34

Study 7: Acute inhalation toxicity study of CASRN 68515-88-8 in mice, rats, and guinea pigs

Title: Not available

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Pentene, 2,4,4-trimethyl-, sulfurized, CASRN 68515-88-8

Remarks: Purity not provided

METHOD

Method/guideline followed: Consistent with EPA Health Effects Guideline OPPTS 870.1300 

Test type: Acute Inhalation Toxicity

GLP compliant?: Yes

Year study performed: 1988

Test species: CD-1 COBS Swiss Albino mice, Sprague-Dawley CD rats, Hartley guinea pigs

Number of animals/sex/dose: 5/sex/dose

Route of administration: Aerosol inhalation (whole-body)

Vehicle: Mineral oil-based material, dosed as supplied

Doses/concentrations tested: 4.3 mg/L

Statistical methods used: Not specified

Test method/conditions remarks: Group of five mice/sex, five rats/sex, and five guinea 

pigs/sex were exposed for 4 hours to the test material as a liquid droplet aerosol generated by a 

Laskin nebulizer apparatus delivered into a plexi-glass chamber. Also, control groups of mice, 

rats and guinea pigs were exposed to mineral oil in the same manner as the test-material-exposed 

group except that the test material was not administrated. The details of the whole-body 

exposure are consistent with those described in EPA Health Effects Guideline OPPTS 870.1300. 

The actual exposure concentration as measured by gravimetric analysis was 4.3 mg/L. Particle 

size analyses were performed once/hour from the test material chamber using a cascade 

impactor. Animal observations for toxicological signs and mortality were recorded every 15 

minutes during the exposure, and twice daily for the 14-day observation period. Individual 

weights were recorded on the day prior to exposure and on days 2, 3, 5, 8, and 14. At the 

conclusion of the observation period, the surviving animals were euthanized by exsanguination 
Final HPV3 Test Rule Data Adequacy Review (July 2014) CASRN 72162-15-3

35

under general anesthesia. All animals were subjected to gross necropsy (nasal passages, trachea, 

external surface, all orifices, the cranial cavity, the brain and spinal cord, and all viscera).

RESULTS

Endpoint value(s): LC50 (mice) > 4.3 mg/L; LC50 (rat) < 4.3 mg/L; LC50 (guinea pigs) > 4.3 

mg/L

Results remarks: The test animals received an average analytical exposure concentration of 4.3 

mg/L test material with a nominal exposure concentration of 100 mg/L. Particle size distribution 

measurements showed an average mass median aerodynamic diameter of 3.8 microns with an 

average geometric standard deviation of 1.9 microns. Approximately 93 percent of the aerosol 

was 10 microns or less in size. 

Mortality and physical observations: Three female rats died within a day after exposure. A 

single male mouse and a single male guinea pig also died on test days 7 and 9, respectively. All 

other animals survived the duration of the study. Observations noted during exposure included 

nasal discharge, salivation, closed eyes and wet fur. Signs exhibited by the rats upon removal 

from the chamber and during the two-hour post-exposure observation period on day included 

numerous secretory responses, labored breathing, rales and wet fur. Also, several of the females 

showed tremors. One of the female rats died two hours after exposure. The mice and the guinea 

pigs were generally unremarkable except for contaminated fur. Two additional rats were found 

dead the morning after exposure. The surviving rats (both sexes) continued to show responses 

without a complete recovery during the 14-day post-exposure observation period, including nasal 

discharge, labored breathing, rales, and contaminated fur leading to alopecia. 

Body weight: Significant body weight losses were observed following exposure among all three 

species. The surviving mice and rats began to recover weight within a week after exposure. 

However, guinea pigs continued to lose weight throughout the first week and did not show a 

weight gain until the end of the second week. 

Gross post mortem observations: Discoloration of the lungs and nasal turbinates was noted 

among the spontaneously dying animals.

CONCLUSIONS

The LC50 values are as follows: LC50 (mice) > 4.3 mg/L; LC50 (rat) < 4.3 mg/L; LC50 (guinea 

pig) > 4.3 mg/L.

STUDY RELIABILITY

[2] Reliable with restrictions. Only one concentration was tested and mortality was observed in 

rats; a full study should have been conducted to determine the LC50 for rats.
Final HPV3 Test Rule Data Adequacy Review (July 2014) CASRN 72162-15-3

36

REFERENCE

Reference information is not available (the underlying study contains confidential business 

information). This study was obtained from the Robust Summaries for the Alkyl Sulfides 

Category (submitted to EPA under the HPV Challenge Program by the Health, Environmental, 

and Regulatory Task Group of the American Chemistry Council’s Petroleum Additives Panel on 

May 19, 2005).
Final HPV3 Test Rule Data Adequacy Review (July 2014) CASRN 72162-15-3

37

Study 8: Acute inhalation toxicity study of CASRN 68515-88-8 in rats 

Title: Not available

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Pentene, 2,4,4-trimethyl-, sulfurized, CASRN 68515-88-8

Remarks: Purity not provided

METHOD

Method/guideline followed: OECD TG 403

Test type: Acute Inhalation Toxicity

GLP compliant?: Yes

Year study performed: 1987

Test species: Sprague-Dawley Albino rats

Number of animals/sex/dose: 5/sex/dose

Route of administration: Aerosol inhalation (whole-body)

Vehicle: Mineral oil-based material dosed undiluted

Doses/concentrations tested: 1.5, 2.5, and 5.6 mg/L (measured)

Statistical methods used: The LC50 with 95% confidence intervals was computed using the 

method of Miller and Tainter (1944).

Test method/conditions remarks: Three groups of five rats/sex were exposed for 4 hours to 

the test material as a liquid droplet aerosol generated by a pressure spray apparatus delivered into 

a plexi-glass chamber. The details of the whole-body exposure are consistent with those 

described in OECD TG 403. The actual exposure concentrations as measured by gravimetric 

analysis were 1.5, 2.5, and 5.6 mg/L. Particle size analyses were performed twice/hour using a 

multi-stage cascade impactor. Animal observations for toxicological signs and mortality were 

recorded periodically during the exposure, and twice daily for the 14-day observation period.

Individual weights were recorded on the day prior to exposure and on days 4, 8, and 14. At the 

conclusion of the observation period, the surviving animals were euthanized using pentobarbital 

as an anesthetic followed by exsanguination. All animals were subjected to gross necropsy 

(external body surface and orifices, major visceral organs, body cavities and carcass). 
Final HPV3 Test Rule Data Adequacy Review (July 2014) CASRN 72162-15-3

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RESULTS

Endpoint value(s): LC50 (males) > 5.6 mg/L; LC50 (females) = 2.17 mg/L

Results remarks: The mass median aerodynamic diameter for the studies was 3.15 microns 

with a geometric standard deviation of 2.45 (estimated percent of particles < 12 microns = 

90.5%). Remarkable animal observations during the studies include alopecia (noted at all dose 

levels during second week of observation), ataxia (noted prior to the death of one female in the 

5.6 mg/L group), dark material around eye (noted in two animals/sex at the 5.6 mg/L dose), 

decreased activity (noted in all animals at the dose level of 2.5 and 5.6 mg/L; reversible by study 

day 5), respiratory irregularity (increased respiration noted in all groups during and immediately 

following exposure; reversible by study day 7), tremors (noted in one female during and 

immediately following exposure to 2.5 mg/L). No male deaths were recorded for any of the dose 

levels. Group mean body weights were decreased at day 4 among males exposed to 2.5 and 5.6 

mg/L. This effect was reversible by study observation day 8 and 14. Three of 5 females in the 

1.5 mg/L group died on day 2 following exposure. Four of 5 female rats exposed to 2.5 mg/L 

died on observation day 2. Three females in the high-dose group died on day 2 following 

exposure, with an additional death on day 6. Body weights decreased at day 4 in the surviving 

females, an effect that was reversible by days 8 and 14. No internal lesions or abnormalities 

were noted in any animal sacrificed at study termination. Pathological findings among females 

which died during the course of the observation period were observed in several organs, 

including the brain (prominent vascularization, and blood in the cranial cavity), nasal passages 

(reddening of the nasal passage, with the notation of clear fluid in the nasal passage), lungs 

(reddening of the lungs, with the observation of a ‘puffy’ lung in one female), and trachea (clear 

fluid noted in the trachea of one female). 

CONCLUSIONS

Following 4-hour whole-body exposure to a liquid droplet aerosol of the test material, the LC50

in male Sprague-Dawley rats is considered to be greater than 5.6 mg/L. The LC50 value in 

females was calculated to be 2.17 mg/L with upper and lower confidence limits of 3.69 and 0.64 

mg/L, respectively. 

STUDY RELIABILITY

[1] Reliable without restrictions. 

REFERENCE

Reference information is not available (the underlying study contains confidential business 

information). This study was obtained from the Robust Summaries for the Alkyl Sulfides 

Category (submitted to EPA under the HPV Challenge Program by the Health, Environmental, 

and Regulatory Task Group of the American Chemistry Council’s Petroleum Additives Panel on 

May 19, 2005).
Final HPV3 Test Rule Data Adequacy Review (July 2014) CASRN 72162-15-3

39

D-6. Repeated-Dose, Reproductive, and Developmental Toxicity

Summary of repeated-dose toxicity endpoint: Adequate

Summary of reproductive toxicity endpoint: Adequate

Summary of developmental toxicity endpoint: Adequate

Study 1: Combined repeated-dose oral toxicity study with the reproduction/developmental 

toxicity screening test in rats (McConnachie, 2014)

Title: A Combined Repeated Dose Toxicity Study with a Reproduction/Developmental Toxicity 

Screening of Sulfurated Decene by Oral Gavage in Rats

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 1-Decene, sulfurized, CASRN 72162-15-3; referred to as sulfurated decene or Mayco 

Base 1540 in the study report

Remarks: Purity: 100%; Dark brown liquid; Batch No. S31343. Certificate of analysis 

provided in the study report indicates a sulfur content of 38.1% for Batch No. S31343, but does 

not provide confirmation of the purity provided in the study report (100%).

METHOD

Method/guideline followed: OECD TG 422, “Combined Repeated Dose Toxicity Study with 

the Reproduction/Developmental Toxicity Screening Test”

Test type: Combined Repeated-Dose Toxicity Study with the Reproduction/Developmental 

Toxicity Screening Test

GLP compliant?: Yes, with the exception that concentrations in test formulations were not 

analytically verified.

Year study performed: 2013

Test species: Sprague-Dawley (Crl:CD(SD)) strain rats; obtained from Charles River UK Ltd., 

Margate, Kent, UK; age at initiation: ~11-12 wks (males) and ~10-11 wks (females); weight at 

initiation: 398-579 g (males) and 238-317 g (females).

Number of animals/sex/dose: Non-recovery groups: 10/sex/dose; Recovery group: 5 males at 

high dose only

Route of administration: Oral – gavage

Vehicle: Corn oil
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Exposure duration: Males: 30 days; Females: 43–47 days (throughout 14 days of pre-mating, 

mating, and gestation, and until postpartum/lactation day 4)

Doses/concentrations tested: Non-recovery groups: 0, 60, 250, and 1000 mg/kg-bw/day; 

Recovery group: 1000 mg/kg-bw/day

Statistical methods: “Unless otherwise stated, all statistical tests were two-sided and performed 

at the 5% significance level using in-house software. Males and females were analysed

separately.” [McConnachie 2014, p. 29]

“Pairwise comparisons were only performed against the control group (Group 1)... Body 

weight, food consumption, selected functional observational battery and motor activity data, 

hematology, coagulation and clinical chemistry were analyzed for homogeneity of variance using 

the ‘F-Max' test. If the group variances appeared homogeneous, a parametric ANOVA was used 

and pairwise comparisons were made using Fisher’s F protected LSD method via Student's t test 

i.e. pairwise comparisons were made only if the overall F-test was significant. If the variances 

were heterogeneous, log or square root transformations were used in an attempt to stabilize the 

variances. If the variances remained heterogeneous, then a Kruskal-Wallis non-parametric 

ANOVA was used and pairwise comparisons were made using chi squared protection (via z 

tests, the non-parametric equivalent of Student's t test).” [McConnachie 2014, p. 29]

“In circumstances where it was not possible to perform the F Max test due to zero standard 

deviation in at least one group, the non-parametric ANOVA results were reported.” 

[McConnachie 2014, p. 29] 

“Organ weights were analyzed using ANOVA as above and by analysis of covariance

(ANCOVA) using terminal kill body weight as covariate. In addition, organ weights as a 

percentage of terminal body weight were analyzed using ANOVA as above as an exploratory 

analysis.” [McConnachie 2014, p. 29]

“In circumstances where the variances in the ANCOVA remained heterogeneous following log 

or square root transformations, the data was subjected to a rank transformation prior to analysis. 

Where it was not possible to perform the F-Max test due to the small sample size (i.e. less than 3 

animals in any group), the untransformed parametric ANCOVA results were reported.” 

[McConnachie 2014, p. 29] 

Test method/conditions remarks: 

Range-Finding Test Procedure:

Dose levels in the definitive test were based on results from a 7-day range-finding toxicity test in 

rats.

1

 According to limited information provided in the definitive test study report, no adverse 

effects were seen in the range-finding test at a dose level up to 1000 mg/kg-bw/day.

Definitive Test Procedure:

 

1 The 7-day range-finding study is described in a separate study report (Charles River Study No. 496470), which 

was not available for review.
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Test Item Preparation: 

The test item was weighed into a container and the appropriate volume of vehicle was added. 

Formulations were magnetically stirred until visibly homogenous.

“The dosing formulations were prepared daily and dispensed immediately [up]on completion [of 

preparation] to the animal unit for dosing. The dosing formulations were stirred for at least 30 

minutes before dosing and continuously during dosing. Formulations were used within ca 1-4 

hours of preparation.” [McConnachie 2014, p. 15]

Analyses of Test Item Formulations: 

Test substance concentrations and stability of the dosing formulations were not determined in

samples collected for analysis because “an appropriate analytical method was not identified for 

the test item in corn oil”. [McConnachie 2014, p. 229] Attempts were made to validate a method 

for measurement of test item concentrations in the dosing formulations1using gas 

chromatography-flame ionization detection or mass spectroscopy detection; however, the test 

item could not be identified in the vehicle (corn oil) using either of these methods.

Dosing and Environmental Conditions:

Groups of sexually mature rats (10/sex/group), acclimated for ≥ 3 weeks, were administered the 

test item daily via gavage at dose levels of 0 (vehicle only), 60, 250, or 1000 mg/kg-bw/day at a 

constant dosing volume of 5 mL/kg-bw. An additional group (5 males), designated as a recovery 

group, was similarly administered 1000 mg/kg-bw/day. Exposure continued throughout the premating (14 days), mating, and post-mating periods. After the mating period, exposure continued 

in non-recovery animals until the day prior to euthanasia, for a total of 30 consecutive days of 

exposure for males and 43 to 47 consecutive days of exposure for females. Designated recovery 

males were held for an additional 14 days following dosing prior to being sacrificed. A control 

recovery group was not included.

“The volume to be administered to each animal was determined on each day by the weight of 

that animal as measured at the time of administration, except during late gestation; from Day 16 

until parturition is complete, the dose volume was determined by the weight of the animal on 

Day 16 of gestation.” [McConnachie 2014, p. 19]

“Where a female was found to be in the process of littering, or had recently littered, at the time 

of scheduled dose administration, the animal was not dosed on that day and dosing recommenced 

the following day.” [McConnachie 2014, p. 19]

Parental animals were housed in groups of 2 or 3 per sex prior to mating and in pairs (one male: 

one female) during mating. Animals were paired until evidence of mating (i.e., the presence of 

sperm in a vaginal lavage) was observed. After mating, males were returned their original cages 

and females were housed individually (or with their litters after parturition) throughout gestation 

and lactation. Non-recovery males were euthanized on day 31. Females were allowed to deliver 

and rear their pups until lactation day 5 to 7, when females and their pups were euthanized. 

 

1 Falconer D. (Study Director). 2014 Validation of methodologies for the formulation and analysis of sulfurated 

decene in oral (gavage) dosing formulations. Charles River, Edinburgh, UK. Study No. 430781. Sponsor: Dover 

Chemical Corporation, Dover, OH. [Validation study was provided in Appendix 3 of the study report.]
Final HPV3 Test Rule Data Adequacy Review (July 2014) CASRN 72162-15-3

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Mortality and Clinical Observations:

Animals were observed at least twice daily during dosing and recovery for mortality and clinical 

signs of toxicity. Detailed clinical observations were performed weekly during the dosing period 

outside of the cage.

Body Weight and Food/Water Consumption:

Individual body weights were recorded daily during the dosing and recovery periods and at the 

time of euthanasia. In the report, body weights were presented weekly for males throughout the 

study period; body weights for females are presented weekly during the pre-mating period, on 

gestation days 0, 7, 14, 16, and 20, and on lactation days 1 and 4. Food consumption was 

determined weekly for males and females during premating and during gestation days 0-7, 7-14, 

and 14-20 and lactation days 0-4 for mated females. Water intakes were monitored by visual 

inspection of water bottles on a regular basis throughout the study, but intakes were not 

quantitatively measured.

Ophthalmic Examinations:

The eyes were examined with an indirect ophthalmoscope during week 4 for males (nonrecovery and recovery) and prior to sacrifice for females. 

Neurological Assessments:

Detailed functional observations were performed cageside or in a standard arena once prior to the 

start of treatment (pretrial) and at weekly intervals thereafter. From week 6 onward, detailed 

functional observations were performed only on select females. Observations were performed

cageside for the following parameters: prostration, lethargy, writhing, circling, breathing 

abnormalities, gait abnormalities, tremor, fasciculation, convulsions, biting (of cage components 

or self-mutilating), vocalization, piloerection, ease of removal from the cage, body temperature, 

eye condition (pupillary function, miosis, mydriasis, exophthalmos, encrustation, and 

lacrimation), coat condition, salivation, and overall ease of handling. The following parameters 

were evaluated in a standardized arena: level of mobility, rearing, grooming,

urination/defecation, arousal, posture, tremor/convulsions, vocalization, piloerection, palpebral 

closure, gait abnormalities, stereotypy (excessive repetition of behaviors), and unusual behaviors. 

Full functional observations (performed prior to blood sampling) were performed on 5 select 

non-recovery males/group during weeks 4 to 5 and during lactation on the first 5 females/group 

that reared their litter until at least lactation day 3. In addition to the parameters listed above, full 

functional observations included reaction to sudden sound, reaction to touch, pain perception, 

landing foot splay, motor activity, grip strength, and observations for any other 

physical/functional abnormality not recorded in the previously mentioned functional screening 

tests.
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Hematology and Clinical Chemistry Analyses:

Blood samples for hematology and clinical chemistry evaluations were collected from 5 nonrecovery males during week 4, and on lactation day 4 (or later) from the first 5 females/group

that reared their litter until at least lactation day 3. Hematological parameters that were 

evaluated included hemoglobin, red blood cell count, hematocrit, mean corpuscular volume, 

mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, red blood cell 

distribution width, reticulocyte count, white blood cell count, differential leukocyte count 

(including counts for neutrophils, lymphocytes, monocytes, eosinophils, basophils), platelet 

count, large unstained cells, and other cells (where appropriate). Coagulation parameters 

determined from blood plasma included activated partial thromboplastin time, fibrinogen, and 

prothrombin time. 

The following clinical chemistry parameters were determined from plasma collected from each 

blood sample: urea, glucose, total protein, albumin, globulin, albumin/globulin ratio, sodium, 

potassium, chloride, calcium, inorganic phosphate, aspartate aminotransferase, alanine 

aminotransferase, alkaline phosphatase, creatinine, creatinine phosphokinase, cholesterol, total 

bilirubin, and lactate dehydrogenase. 

Gross Necropsy and Histopathology:

All animals (including non-recovery adults, their offspring, and recovery animals) were weighed 

and subjected to a full detailed external and internal necropsy. The following organs were

weighed for all non-recovery and recovery adult animals scheduled for sacrifice: adrenals, brain,

heart, kidneys, liver, lungs, spleen, thymus, thyroid glands with parathyroid, epididymides, 

prostate gland, ovaries, pituitary gland, testes, and uterus.

Histological examination was performed on tissues collected from 5 non-recovery animals/sex in 

the control and high-dose groups and on tissues from all recovery animals. The following tissues 

were examined microscopically: adrenals, aorta, brain, cecum, cervix, colon, duodenum, 

epididymides, eyes, femur, Harderian glands, heart, ileum, jejunum, kidneys, lacrimal glands, 

liver, lung, mandibular lymph node, mesenteric lymph node, mammary gland, esophagus, optic 

nerve, ovaries, oviducts, pancreas, parathyroid glands, pituitary gland, prostate gland, rectum, 

rib, sciatic nerve, seminal vesicle, skeletal muscle, skin and subcutis, spinal cord, spleen, 

sternum, stomach, testes, thymus, thyroid glands, tongue, trachea, ureters, urinary bladder, 

uterus, vagina, gut-associated lymphoid tissue (Peyer’s patch), mandibular salivary glands, and 

gross lesions/masses1.

Reproductive and Developmental Toxicity:

During mating, the following parameters were recorded: the numbers of males paired, numbers 

of siring males, numbers of females paired, number pregnant, and time to mate. Pregnant 

animals were observed daily and allowed to litter. Observed difficulties in littering or prolonged 

parturition were recorded. The following parameters were recorded: duration of gestation, 

 

1 Larynx, pharynx, and bone marrow samples were collected for histological examination according to “Text Table 

10” of the study report (p. 28). However, findings (either positive or negative) from histological examination of 

these tissues were not provided in the histopathology report (summary of findings in Tables 59 and 60 [pp. 126-

146]; individual findings in Appendices 54 and 55). Therefore, it is unknown whether or not these tissues were 

examined microscopically.
Final HPV3 Test Rule Data Adequacy Review (July 2014) CASRN 72162-15-3

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number producing a live litter, number of implantation sites per pregnancy, number of corpora 

lutea sites per pregnancy, total number of pups born per litter, number of live pups per litter 

(counted on lactation days 0, 1, and 4), number of dead pups born per litter, total number of male 

pups on lactation day 1, and total number of female pups lactation day 1. Calculated fertility, 

parturition, and offspring parameters included fertility indices for males and females, gestation 

index, birth index, live birth index, and viability index.

“The numbers of live and dead pups born in each litter ... [were] recorded as soon as possible 

after completion of parturition on Day 0 of lactation. The live pups were counted and examined 

daily for the presence of milk in the stomach and for any externally visible abnormalities. Each 

litter was weighed en masse (by sex) on Days 1 and 4 of lactation.” [McConnachie 2014, p. 23] 

During the lactation period, “[d]eficiencies in maternal care were recorded [as follows]: 

inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of 

pups, or apparently inadequate lactation or feeding.” [McConnachie 2014, p. 24] 

“... [Pups] found dead or killed were sexed, and then checked for the presence of milk in the 

stomach and for the presence of any externally visible abnormalities. All dead pups and any 

abnormal pups were, where practicable, examined or fixed in 10% formalin for optional further 

examination. Pups sacrificed along with dams were externally examined and checked for the 

presence of milk in the stomach.” [McConnachie 2014, p. 27]

At necropsy, reproductive organs of adult animals (non-recovery and recovery) were weighed 

and histologically examined, as described above. “The reproductive tract[s] of all females were 

examined for signs of implantation, [and] the number of any implantation sites was recorded. 

The total number of corpora lutea graviditatis was recorded for each female.” [McConnachie 

2014, p. 26]

RESULTS

Endpoint value(s): 

Systemic Toxicity:

LOAEL (male) = 1000 mg/kg-bw/day, based on reduced body weight gain

NOAEL (male) = 250 mg/kg-bw/day

NOAEL (female) = 1000 mg/kg-bw/day (highest dose tested)

Reproductive/Developmental Toxicity:

NOAEL = 1000 mg/kg-bw/day (highest dose tested)

Results remarks: 

Mortality and Clinical Observations:

Two parental females in the 60 mg/kg-bw/day group died on day 23 of gestation. No additional 

mortalities were observed in the parental non-recovery and recovery groups. Macroscopic 

findings noted in the two dead females at necropsy included spongy lungs, autolysis, and tissue 

discoloration. “Ulcerative inflammation in the esophagus was noted in both animals at 

histopathological examination, which is indicative of gavage injury, therefore this was 

considered to be the cause of death of these animals.” [McConnachie 2014, p. 31]
Final HPV3 Test Rule Data Adequacy Review (July 2014) CASRN 72162-15-3

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Clinical observations included dose-related “increased incidences of transient excess salivation, 

in both sexes, at all dose levels which was generally noted immediately post dose and was no 

longer evident 1 hours after dosing. Ploughing behavior (animal borrowing through bedding 

with its head) was also noted in one male and one female at 250 mg/kg/day and there were 

isolated incidences of pale eyes and piloerection in females at 1000 mg/kg/day.” [McConnachie 

2014, p. 31] “[H]owever due to the transient nature or low incidence of these observations, these 

were considered not to be adverse.” [McConnachie 2014, p. 35] “All other clinical signs were 

considered to be incidental and unrelated to treatment with Sulfurated Decene” [McConnachie

2014, p. 31]

Body Weight and Food/Water Consumption:

Non-recovery groups:

“In males dosed at 1000 mg/kg/day, reduced weight gain was noted from Day 15 onwards, and 

overall gain over the study period was also reduced (ca 15%), when compared to Controls and 

other treated groups.” [McConnachie 2014, p. 31] At the end of the exposure period, body 

weights of high-dose males were ~2% less than body weights of control males. No effects on 

body weight or body weight gain were noted in low- and mid-dose males. Mean body weights 

and body weight gains of dosed females were similar to those of controls during pre-mating, 

gestation, and lactation.

The reductions in body weight and body weight gain observed in male animals at the high dose 

did not reach statistical significance. However, given the magnitude of the reduction in body 

weight gain in males at 1000 mg/kg-bw/day (14.3%, relative to the control group, over the entire 

study period), this change is considered to be evidence of a possible effect of treatment. 

No effects on food consumption were observed in males or females. Results for water 

consumption were not provided.

Recovery group:

“Group mean [body] weight gain [in males] at ... [1000 mg/kg-bw/day] during the 2 week 

recovery period was also notably reduced in comparison to control weight gain over the final ca 

2 weeks of the main study period, which indicated that the reduced weight gain may not be fully 

recoverable two weeks after the cessation of test item administration.” [McConnachie 2014, p. 

35] 1

Ophthalmic Examinations:

No treatment-related changes were observed in the eyes.

 

1 A concurrent control group maintained during the recovery period was not included; therefore, it is unknown 

whether body weight gains in treated animals remained lower than controls during the 2-week recovery period.
Final HPV3 Test Rule Data Adequacy Review (July 2014) CASRN 72162-15-3

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Neurological Assessments: 

At 1000 mg/kg-bw/day, an increase in motor activity in one male animal resulted in a slight

increase in group mean values that did not reach statistical significance. This finding was not 

considered evidence of a treatment-related effect on motor activity. During detailed functional 

observations, a significant (p < 0.05) decrease in “sectors” was observed in males during weeks 2 

and 4 at both the low and high doses. During week 2, sectors were decreased in males by 50% at 

both 60 and 1000 mg/kg-bw/day. During week 4, sectors were decreased in males by 40% at

both 60 and 1000 mg/kg-bw/day. Due to the absence of a dose-related effect, the toxicological 

relevance of this change is not clear. A similar effect on measurements of sectors was not 

observed in females. No other changes in neurological parameters were observed. 

Hematology and Clinical Chemistry Analyses:

No treatment-related effects on hematology or clinical chemistry parameters were observed. 

Gross Necropsy and Histopathology:

Absolute ovary weight was significantly (p < 0.05) increased at 1000 mg/kg-bw/day (increase of 

10% relative to the control group). In addition, absolute pituitary gland weight was significantly 

increased in females at 60 (p < 0.01) and 250 (p < 0.05) mg/kg-bw/day (increase of 29% relative 

to control females at both doses). There were no treatment-related macroscopic or microscopic 

findings.

The ovary and pituitary gland weight changes were not considered to be evidence of a treatmentrelated effect in the absence of corroborating evidence of organ toxicity, such as histological 

findings, and the absence of a dose-related trend for pituitary gland weight changes. “As there 

were no patterns, trends or correlating data to indicate that these [changes in organ] weights were 

of any toxicological significance, these changes were considered to be incidental or due to 

differences in sexual maturity and unrelated to treatment with Sulfurated Decene.” 

[McConnachie 2014, p. 33]

Reproductive and Developmental Toxicity:

All females (10/group) became pregnant and fertility indices were 100% for all groups. As noted 

above, two females at the 60 mg/kg-bw/day dose level died on gestation day 23, presumably due 

to gavage trauma. One mid-dose (250 mg/kg-bw/day) female gave birth to a dead litter. No 

treatment-related effects were noted for mating, fertility, or duration of gestation. 

Slight reductions in the mean numbers of implantation sites and corpora lutea per pregnancy 

were observed in all treated groups (in the low-, mid-, and high-dose groups, respectively, the 

numbers of implantation sites were reduced by 16%, 7%, and 17% and the numbers of corpora 

lutea were reduced by 13%, 2%, and 11%); however, the changes did not reach statistical 

significance. The mean total numbers of pups born per litter were 16%, 13%, and 23% lower 

than the control group at 60, 250, and 1000 mg/kg-bw/day, respectively; however, these changes

also did not reach statistical significance. The mean number of live pups per litter on lactation 

days 0, 1, or 4 was lower in the treated groups than the control group, although the changes did 

not reach statistical significance; a dose-related trend for this parameter was evident on lactation 

day 0, but not on lactation days 1 or 4. At doses of 60, 250, and 1000 mg/kg-bw/day, decreases

in the mean numbers of live pups per litter (relative to the control group) were 19%, 23%, and 
Final HPV3 Test Rule Data Adequacy Review (July 2014) CASRN 72162-15-3

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25% on lactation day 0; 38%, 30%, and 25% on lactation day 1; and 42%, 35%, and 24% on 

lactation day 4, respectively. 

“The number of implant[ation] sites, corpora lutea, [and] total and live pups born were reduced 

in treated groups when compared with Controls, however the reductions did not follow a dose

related pattern and values for each parameter were within the historical [control] ranges for this 

type of study [conducted] at [the laboratory].” [McConnachie 2014, p. 33] Therefore, changes in 

these parameters were not considered to be related to treatment.

The calculated birth index and live birth index were similar among all groups. However, a high 

incidence of pup death was observed during lactation days 0 to 4 in all groups, including the 

control group. In the 0, 60, 250, and 1000 mg/kg-bw/day groups, 6/10, 4/81, 7/92, and 5/10 

dams, respectively, lost 2 or more pups during lactation days 0 to 4. For this period, the mean 

viability indices were 48%, 62%, 46%, and 59% for the 0, 60, 250, and 1000 mg/kg-bw/day

groups, respectively. In addition, a relatively high incidence of total litter loss was observed 

among all groups, including the control group. The percentages of females with total litter loss3

during lactation days 0 to 4 at 0, 60, 250, and 1000 mg/kg-bw/day were 50% (5/10), 13% (1/8), 

44% (4/9), and 40% (4/10), respectively. The study author did not provide an explanation for the 

unusually high incidence of pup death and total litter loss observed among all groups, including 

the controls. Laboratory historical control data for animals of the same species, strain, age, and 

body weight, with exposure to an identical vehicle (corn oil), were not available for review.

“Intergroup differences [in pup survival indices] did not follow a dose related pattern or were 

due to a large number of litter losses across the study and could therefore not be attributed to 

treatment with Sulfurated Decene.” [McConnachie 2014, p. 33]

There were no treatment-related clinical signs or macroscopic abnormalities observed in pups. 

No effects on pup body weights were observed. In all, no statistically significant effects of 

treatment were observed on reproductive and developmental parameters and no dose-related 

trends in these parameters were apparent.

CONCLUSIONS

Systemic Toxicity:

The LOAEL for systemic toxicity in males is 1000 mg/kg-bw/day, based reduced body weight 

gain. The NOAEL for systemic toxicity in males is 250 mg/kg-bw/day.

The NOAEL for systemic toxicity in females is 1000 mg/kg-bw/day, based on no adverse effects 

at the highest dose tested.

 

1 Two females died at this dose on gestation day 23; therefore, both females were excluded from this ratio.

2 One female at this dose gave birth to only dead fetuses; therefore, this female was excluded from this ratio.

3 Calculated as: (number of dams with no live pups remaining on lactation day 4) / (number of dams with live pups 

on lactation day 0) x 100%.
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Reproductive/Developmental Toxicity:

The NOAEL for reproductive toxicity and developmental toxicity is 1000 mg/kg-bw/day, based 

on no adverse effects at the highest dose tested.

STUDY RELIABILITY

[2] Reliable with restrictions. Concentrations of the test substance in the dosing formulation

were not analytically verified and stability of the test substance in the vehicle (corn oil) was not 

determined due to the inability of the laboratory to develop a validated analytical method. A 

concurrent recovery control group was not included in the study. The study author did not 

provide an explanation for the high incidences of pup mortality and total litter loss observed 

during lactation among all dose groups, including the control group. Deviations from OECD TG 

422 included the following: clinical chemistry measurements did not include bile acids; ovaries, 

testes, epididymides, and accessory sex organs were only examined microscopically in 5 

animals/sex/dose, rather than in all animals; and bone marrow samples were not examined 

microscopically.

REFERENCES

McConnachie, K. (Study Director). 2014. A combined repeated dose toxicity study with a 

reproduction/developmental toxicity screening of sulfurated decene by oral gavage in rats. 

Charles River Laboratories, Edinburgh, UK. Study No. 496486. Sponsor: Dover Chemical 

Corporation, Dover, OH. 

Text in quotes is taken directly from McConnachie (2014).
Final HPV3 Test Rule Data Adequacy Review (July 2014) CASRN 72162-15-3

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Study 2: Repeated-dose dermal toxicity study of CASRN 68511-50-2 in rats

Title: Not available

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 1-Propene, 2-methyl-, sulfurized CASRN 68511-50-2

Remarks: Purity not provided

METHOD

Method/guideline followed: Comparable to OPPTS 870.3250

Test type: Thirteen-week dermal subchronic toxicity study

GLP compliant?: Yes

Year study performed: 1989

Test species: Sprague-Dawley (Tac:N[SD]fBR)

Number of animals/sex/dose: 10/sex/dose

Route of administration: Dermal 

Vehicle: Mineral oil (solvent-refined naphthenic base stock), density 0.88 g/mL

Exposure duration: 5 days/week, 13 weeks

Doses/concentrations tested: Part 1: 500 and 2000 mg/kg/day undiluted test material; 500 

mg/kg/day diluted 50% w/v in mineral oil base stock; Part 2: 500, 250, 100, 50, and 10 

mg/kg/day diluted in mineral oil base stock 

Statistical methods used: Analysis of Variance followed by multiple range tests; serum 

chemistry and hematology data were evaluated using the F test for ANOVA and StudentNewman-Keuls multiple comparison test.

Test method/conditions remarks: 

Age at initiation: 7 weeks old following 2 weeks acclimation.

Study was designed to identify inherent toxicity of test material and to determine whether 

dilution in a mineral oil carrier would alter toxicity. Methyl propene derivative was applied to 

the clipped backs of groups of 20 Sprague-Dawley rats (10M, 10F) 5 days per week for 13 weeks 
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at dose levels of 500 or 2000 mg/kg/day undiluted or at dose levels of 500, 250, 100, 50, or 10 

mg/kg/day diluted in mineral oil on the same schedule. Rats were fitted with Elizabethan collars 

to minimize ingestion of test material, which was left uncovered on the skin. One vehicle and 2 

untreated shaved control groups were included in the study. Assessments for toxic response 

included: daily clinical observations; weekly skin irritation scoring; weekly body weights;

terminal organ weights; hematology, serum chemistry, and urinalysis at weeks 5 and 13; and

gross necropsy evaluations, sperm morphology, and histopathology at study termination. 

Endpoint value(s): LOAEL = 100 mg/kg/day based on increased production of white blood 

cells in spleen and bone marrow; NOAEL = 50 mg/kg/day

Results remarks: Male rats treated with methyl propene derivative for 13 weeks at dose levels 

≥ 250 mg/kg/day gained less weight (15% less at study termination) than controls. Female 

weights were unaffected. At doses ≥ 250 mg/kg/day, both sexes had decreased levels of red 

blood cells and increased levels of neutrophils in circulation, increased spleen size and increased 

pigment and red pulp in the spleen. At doses ≥ 100 mg/kg/day, there was increased production 

of WBC in spleen and bone marrow. Mean liver-to-body weights were increased in male rats at 

dose levels ≥ 250 mg/kg and in female rats at ≥ 500 mg/kg/day. Male rats treated with undiluted 

test material at 500 or 2000 mg/kg/day had increased kidney weights correlated with dose-related 

increase in hyaline droplet formation indicative of light hydrocarbon nephropathy. Undiluted 

test material and dilutions at 25% (500 mg/kg, 250 mg/kg in Part 2) induced moderate to strong 

reaction in the skin, characterized by erythema, edema, increased thickness, and stiffness; these 

effects were more severe in the 500 mg/kg (diluted 50% w/v) group. Microscopically, 

hyperkeratosis, hyperplasia of sebaceous gland, increased mitosis in epidermis, and dermal 

abscesses were observed. Virtually no irritation was observed in the vehicle control group or in 

dose groups of 100, 50, or 10 mg/kg/day where dilutions were made at 10% or 5% w/v. No 

effects on sperm motility or morphology were observed in rats treated with 2000 mg/kg/day. 

The relative weight increases in livers of higher dose animals of both sexes had no microscopic 

correlate and is considered an adaptive response to treatment. The increase in kidney weight and 

hyaline droplet formation in male rats is indicative of light hydrocarbon nephropathy, a condition 

considered by EPA to be specific to male rats and not predictive of comparable toxicity in 

humans. Although many changes in hematology parameters can be associated with infections 

which can occur with severe skin irritation, increased dose-related neutrophil production at ≥ 250 

mg/kg/day was observed in animals with minimal skin irritation and can be considered a direct 

effect of the test material. 

CONCLUSIONS

The NOAEL for systemic toxicity is 50 mg/kg/day based on increased production of white blood 

cells in spleen and bone marrow at 100 mg/kg/day. The minimally irritating concentration of 

methyl propene derivative diluted in mineral oil base stock is 10%.

STUDY RELIABILITY

[1] Reliable without restrictions. 
Final HPV3 Test Rule Data Adequacy Review (July 2014) CASRN 72162-15-3

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REFERENCES

Reference information is not available (the underlying study contains confidential business 

information). This study was obtained from the Robust Summaries for the Alkyl Sulfides 

Category (submitted to EPA under the HPV Challenge Program by the Health, Environmental, 

and Regulatory Task Group of the American Chemistry Council’s Petroleum Additives Panel on 

May 19, 2005).
Final HPV3 Test Rule Data Adequacy Review (July 2014) CASRN 72162-15-3

52

Study 3: Repeated-dose dermal toxicity study of CASRN 68511-50-2 in rabbits (28 days)

Title: Not available

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 1-Propene, 2-methyl-, sulfurized, CASRN 68511-50-2

Remarks: Purity not provided

METHOD

Method/guideline followed: Reference for study design: Federal Register, Volume 43, Number 

163

Test type: 28-Day Subchronic Dermal Toxicity Study

GLP compliant?: Yes

Year study performed: 1982

Test species: Albino rabbits

Number of animals/sex/dose: 6/sex/dose

Route of administration: Dermal

Vehicle: None

Exposure duration: 4 weeks

Doses/concentrations tested: Group 1: 200 mg/kg/day undiluted test material; Group 2: 2000 

mg/kg/day undiluted test material 

Statistical methods used: All data was submitted to analysis of variance (method: Statistical 

Analysis System, SAS Statistical Institute 1979) followed by evaluation using the NewmanKeuls method for all significant dose differences. 

Test method/conditions remarks: Age at initiation of treatment: Not specified. Treatment 

began following 1 week acclimation. Number of animals tested: 36 animals (6M, 6F/group): 2 

treatment groups, 1 untreated control group. Study was designed to evaluate the subchronic 

toxicity of the test material when applied dermally. Methyl propene derivative was applied to 

the shaved dorsal trunk area (approximately 10% of the body surface) of 2 groups of 12 albino 

rabbits (6M, 6F) 5 days per week for 4 weeks at dose levels of 200 or 2000 mg/kg/day of 
Final HPV3 Test Rule Data Adequacy Review (July 2014) CASRN 72162-15-3

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undiluted test material on the same test schedule. Half of the animals in each group were 

abraded once per week throughout the study. The abrasion penetrated the stratum corneum, but 

did not cause bleeding. The treated skin was occluded for at least 6 hours daily and the trunk of 

each animal covered with an impervious material. One untreated shaved control group of 6 

animals (3 intact, 3 abraded) was included in the study. Assessments for local and systemic 

effects included: clinical observations; skin irritation scoring 5 days per week; body weights (at 

pretest, every 3 to 4 days during testing, termination); hematology, serum chemistry, and 

urinalysis at pretest and termination; and gross necropsy evaluations at study termination. 

Endpoint value(s): LOAEL = 2000 mg/kg/day based on weight loss in males and increased 

monocytes; NOAEL = 200 mg/kg/day 

Results remarks: One male rabbit death occurred at the higher dose level. Body weight gains

were observed in control (0.5 to 1.0 kg) and lower dose group (0.2 to 1.0 kg). A trend of weight 

loss and change in food consumption among the high-dose males was observed in the latter half 

of the study. Weight gain was observed in 5 of 6 high-dose females. Treatment with the test 

material caused severe skin irritation at both doses. Abrasion of skin increased the degree of 

irritation at the low-dose level. No irritation was observed in the control group. Urinalysis 

values were normal for all groups. The low-dose group showed an increase in chloride and a 

decrease in albumin. The high-dose group showed decreased alkaline phosphatase and an 

increase in chloride and globulin. Hematology showed no trends in the control and low-dose 

groups while monocyte determinations were significantly different (increased) in the high-dose 

group. Gross and histopathological examination of tissues did not reveal any pattern of changes 

attributable to dermal contact with the test material. At autopsy, one animal in the control group 

was found to be female instead of male and one animal in the low-dose group was found to be 

male instead of female. Statistical evaluation including and excluding these two animals showed 

no significant differences. 

CONCLUSIONS

LOAEL = 2000 mg/kg/day; NOAEL = 200 mg/kg/day. No minimally irritating concentration 

was identified by this study. 

STUDY RELIABILITY

[2] Reliable with restrictions. Animal ages were not included in the report. An uneven sex 

distribution was used. Clinical behavior determinations beyond that of morbidity were not 

included in the report. Only two doses were tested.

REFERENCES

Reference information is not available (the underlying study contains confidential business 

information). This study was obtained from the Robust Summaries for the Alkyl Sulfides 

Category (submitted to EPA under the HPV Challenge Program by the Health, Environmental, 

and Regulatory Task Group of the American Chemistry Council’s Petroleum Additives Panel on 

May 19, 2005).
Final HPV3 Test Rule Data Adequacy Review (July 2014) CASRN 72162-15-3

54

Study 4: Repeated-dose dermal toxicity study of CASRN 68511-50-2 in rabbits (21 days)

Title: Not available

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 1-Propene, 2-methyl-, sulfurized, CASRN 68511-50-2

Remarks: Purity not provided

METHOD

Method/guideline followed: None cited

Test type: 21-Day Repeated Dose Dermal Toxicity Study

GLP compliant?: No

Year study performed: 1979

Test species: New Zealand White rabbit

Number of animals/sex/dose: 3 treatment groups (7M/3F, 8M/2F, 6M/4F), 1 untreated control 

(5M/5F)

Route of administration: Dermal 

Vehicle: None

Exposure duration: 21 days

Doses/concentrations tested: 140, 560, and 2240 mg/kg/day undiluted test material 

Statistical methods used: Not available

Test method/conditions remarks: Age of animals at initiation: Not specified. Exposure was 

initiated following at least 2 weeks acclimation. Study was designed to evaluate local and 

systemic effects of test material when applied dermally. Methyl propene derivative was applied 

to the shaved backs and sides (approximately 10% of the body surface) of 3 groups of 10 New 

Zealand White rabbits 5 days per week for 3 weeks at dose levels of 140, 560, or 2240 

mg/kg/day of undiluted test material on the same test schedule. The animals were fitted with 

plastic collars to inhibit ingestion of the test material, which was left uncovered on the skin and 

not removed prior to the next dose. One untreated shaved control group of 10 animals was 

included in the study. Assessments for local and systemic effects included: twice daily (morning 
Final HPV3 Test Rule Data Adequacy Review (July 2014) CASRN 72162-15-3

55

and afternoon) clinical observations; skin irritation scoring 5 days per week; weekly body 

weights; hematology, serum chemistry, and urinalysis at pretest and termination; and gross 

necropsy evaluations at study termination. 

Endpoint value(s): NOAEL = 2240 mg/kg/day based on no systemic effects at the highest dose 

tested

Results remarks: All rabbits survived the duration of the test. Body weight changes were 

within expected ranges and comparable for all groups. Rabbits in all groups had lethargy, ptosis, 

G.I. disturbances, nasal and ocular discharges, and respiratory distress, all more often in the 

second and third weeks with no discernible pattern of response. Skin responses included slight 

to moderate erythema and very slight to slight edema during Week 1 for all treated groups. 

During Week 2, responses in all treated groups were moderate to severe erythema with 

additional signs of cracked skin, bleeding, and discoloration. Edema was slight at the lowest 

dose and slight to moderate at the higher doses. During Week 3, all treated groups had severe 

erythema with cracked and bleeding skin, eschar, and discoloration and edema was slight to 

moderate. No irritation was observed in the control group. Urinalysis values were normal in all 

groups. Several individual and isolated hematological and serum chemistry values were out of 

expected range, but with no discernible treatment related changes in the mean values for all 

groups. At necropsy, sporadic occurrences of dark lungs and liver, red and bloated intestines, 

pale kidney, or small or gray spleen were noted with no relationship to treatment. Epithelial 

hyperplasia of the treated skin was observed in all rabbits with the treated groups exhibiting 

slightly more severe grades of hyperplasia than the control group. The rabbits were grouped by 

sex at the start of the study. At necropsy, six errors in sexing were discovered which resulted in 

uneven sex distribution within the groups. Since there were no apparent effect differences 

between the sexes, the study is not considered to be compromised. With no discernable pattern 

of response in both test and control groups, observed clinical signs are considered to be related to 

handling. The occurrence of hyperplasia in all groups suggests a relationship to clipping rather 

to test material administration. However, in-life dermal observations revealed severe erythema 

responses in all treated rabbits and none in the sham-treated control group. 

CONCLUSIONS

The NOAEL is 2240 mg/kg/day based on no systemic effects at the highest dose tested. No 

minimally irritating concentration was identified by this study. 

STUDY RELIABILITY

[2] Reliable with restrictions. Animal age and organ weight data were not included in the report. 

The test site was not occluded following test material application. An uneven sex ratio was used.

REFERENCES

Reference information is not available (the underlying study contains confidential business 

information). This study was obtained from the Robust Summaries for the Alkyl Sulfides 

Category (submitted to EPA under the HPV Challenge Program by the Health, Environmental, 
Final HPV3 Test Rule Data Adequacy Review (July 2014) CASRN 72162-15-3

56

and Regulatory Task Group of the American Chemistry Council’s Petroleum Additives Panel on 

May 19, 2005).
Final HPV3 Test Rule Data Adequacy Review (July 2014) CASRN 72162-15-3

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Study 5: Repeated-dose inhalation toxicity study of CASRN 68515-88-8 in rats

Title: Not available

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Pentene, 2,4,4-trimethyl-, sulfurized, CASRN 68515-88-8

Remarks: Purity not provided

METHOD

Method/guideline followed: OECD TG 412

Test type: 4-Week Inhalation Toxicity Study in Rats

GLP compliant?: Yes

Year study performed: 1989

Test species: Sprague-Dawley CD rats (7 weeks old at initiation of treatment)

Number of animals/sex/dose: 10/sex/dose for low- and mid-dose levels; 15 male and 20 female 

rats for the control and high-dose level

Route of administration: Aerosol inhalation (whole-body)

Vehicle: None

Exposure duration: 4 weeks followed by a 3-week recovery period

Doses/concentrations tested: 0, 15, 50, and 160 mg/m3

Statistical methods used: Body weight, food consumption, hematology and clinical chemistry 

parameters, organ weights, and organ/body weight ratios were analyzed. Mean values of all dose 

groups were compared to control at each time interval. Tests included parametric ANOVA with 

a Dunnett’s post-hoc test, non-parametric Kruskal-Wallis and Dunn’s rank sum test, Bartlett’s 

test for equal variances, and Student’s t-test.

Test method/conditions remarks: The rats were exposed for 6 hours/day, 5 days/week for 4 

weeks to the test material (target concentrations = 15, 50, and 150 mg/m3) as a liquid droplet 

aerosol generated by an air atomizing nozzle apparatus delivered into a plexi-glass chamber.

Control rats were exposed in the same manner as the test-material-exposed group except that

mineral oil only was administered at a level of 150 mg/m3. The details of the whole-body 
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exposure are consistent with those described in OECD TG 412. The actual exposure 

concentrations, as measured by gravimetric analysis, were 15, 50, and 160 mg/m3. Particle size 

analyses were performed once/week from the test material chamber using a cascade impactor. 

Animal observations for toxicological signs and mortality were recorded twice daily, once in the 

morning and once in the afternoon. Individual weights were recorded twice pre-test and then 

weekly during the exposure and recovery periods, and at termination. At the conclusion of the 

observation period, the surviving animals were euthanized with carbon dioxide. Animals were 

fasted prior to sacrifice. Five rats/sex were subjected to post-exposure blood analysis (routine 

hematology and clinical chemistry parameters) on test day 1 for the control and high-dose 

groups, at termination on 5 rats/sex for all dose groups, and on 5 rats/sex from the control and

high-dose group after three weeks of recovery. Complete gross post mortem examinations were 

performed on all animals (nasal passages, trachea, external surface, all orifices, the cranial 

cavity, the brain and spinal cord, and all viscera). Nine major organs were weighed to obtain 

organ/body weight calculations, 42 individual organs and/or tissues were preserved, and 10 

major organs and/or tissues were examined for histopathology. 

Endpoint value(s): NOAEL (male) = 160 mg/m3based on no relevant effects at the highest 

dose tested; LOAEL (female) = 160 mg/m3based on increased hemoglobin levels; NOAEL 

(females) = 50 mg/m3

Results remarks: The mass median aerodynamic diameter for the studies ranged from 1.9 to 

2.6 microns with a geometric standard deviation ranging from 1.8 to 2.2. These data indicated 

that the aerosol was of a respirable size in the rat, with at least 96% of the particles 10 microns or 

less in diameter. 

Mortality: One high-dose female had convulsive behavior following the third day of exposure, 

and was found dead the next morning. The cause of death was unclear. There were no other 

unscheduled deaths in the study. 

Physical observations: The animals were unremarkable during the exposure period. Weekly 

detailed observations included an increased incidence of nasal discharge or dried red material on 

the facial area among the high-dose animals. However, these findings were not temporally 

consistent nor were they apparent in the lowest two doses of test material. No significant 

respiratory sounds were noted. 

Body weights: Although there were no significant differences seen between control and treated 

groups, there was a trend toward lower body weight gains during the exposure period of the 

study at all dose levels in the males and with the two highest dose levels in the females. During 

the three-week recovery period, the high-dose animals did not regain the difference in body 

weight compared to the controls. 

Hematology: The only significant difference from control values was increased hemoglobin 

concentration in the high-dose females sacrificed after 4 weeks of exposure. 
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59

Clinical chemistry: There were several statistically significant differences from the control 

values at both the post-exposure and post-recovery time intervals. However, these differences 

did not correlate with dose, with sex, with potential target organs, or with sacrifice interval. 

Terminal organ and body weights: Following 4 weeks of exposure to test material, increases in 

kidney weights were seen in the males at all three dose levels, and were statistically significant in 

the higher two levels. This effect was considered to be related to renal effects seen 

microscopically in males (see below). This difference in weight abated following 3 weeks of 

recovery. Following 4 weeks of exposure, statistically significant increases were seen in highdose liver weights and liver/body ratio in both sexes. These differences abated following 3 

weeks of recovery. Spleen and adrenal weights increased compared to controls in the high-dose 

groups of both sexes. Post-recovery increases in testes, heart, lung, and spleen weights were 

recorded. These effects were not accompanied by pathologic microscopic findings, and 

therefore, the biological significance was considered equivocal. A few visible gross changes, 

such as discolored lungs, were noticed in the sacrificed animals. Microscopically, treatmentrelated effects were seen in the kidneys in the males in a dose-related profile. Findings included 

globular casts at the corticomedulary junction, the cortex and medulla, as well as hyaline droplets 

in the proximal convoluted tubule cells. These responses were seen in males in all treatment 

groups following 4 weeks of exposure, and in the high-dose group after 3 weeks of recovery.

The nephropathy in males is consistent with the alpha 2μ-globulin-mediated mechanism that is 

male rat-specific and is not considered by EPA to be predictive of comparable toxicity in 

humans. All other microscopic tissue alterations observed in other organs were considered 

incidental findings.

CONCLUSIONS

The LOAEL for females is 160 mg/m3based on increased hemoglobin levels in females. The 

NOAEL is 50 mg/m3in females. The NOAEL for males is 160 mg/m3based on no human 

health-related effects at the highest dose tested.

STUDY RELIABILITY

[1] Reliable without restrictions. 

REFERENCES

Reference information is not available (the underlying study contains confidential business 

information). This study was obtained from the Robust Summaries for the Alkyl Sulfides 

Category (submitted to EPA under the HPV Challenge Program by the Health, Environmental, 

and Regulatory Task Group of the American Chemistry Council’s Petroleum Additives Panel on 

May 19, 2005).
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60

Study 6: Repeated-dose dermal toxicity study of CASRN 68515-88-8 in rats 

Title: Not available

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Pentene, 2,4,4-trimethyl-, sulfurized, CASRN 68515-88-8

Remarks: Purity not provided

METHOD

Method/guideline followed: Consistent with OECD TG 410 and OPPTS 870.3200

Test type: 28-Day Dermal Toxicity Study in Rats

GLP compliant?: Yes

Year study performed: 1988

Test species: Sprague-Dawley CD rat (21 days old at initiation of treatment)

Number of animals/sex/dose: 5 males/dose

Route of administration: Dermal

Vehicle: None

Exposure duration: 5 days/week, 4 weeks

Doses/concentrations tested: 0 or 1000 mg/kg/day

Statistical methods used: Continuous data including body weight, body weight gain, and food 

consumption were analyzed by analysis of variance.

Test method/conditions remarks: Rats were administered the test substance at 0 or 1000 

mg/kg/day topically on shaved, unabraded skin under semi-occluded conditions for 6 hours/day, 

5 days/week for 4 weeks (20 applications). Controls rats received topical application of mineral 

oil. Following the exposure period, the test material was removed with mineral oil. 

Endpoint value(s): NOAEL = 1000 mg/kg/day based on no systemic toxicity at the highest 

dose tested
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Results remarks: All animals survived throughout the study and physical examinations were 

generally unremarkable. No difference between the test material-treated animals and control 

animals was noted for the parameters of body weight, body weight gain, or food consumption. 

Detail gross pathological examination of external and internal features of the animals revealed no 

remarkable findings with the exception of weak-moderate irritation responses at the site of test 

material application. These responses were characterized by erythema, eschar, and flaking of the 

skin that persisted over the majority of the 28-day treatment period.

CONCLUSIONS

All animals survived throughout the study and physical examinations were generally 

unremarkable. The NOAEL is 1000 mg/kg/day (only dose tested).

STUDY RELIABILITY

[2] Reliable with restrictions. Data for clinical chemistry, hematology, and histopathology are 

not presented.

REFERENCES

Reference information is not available (the underlying study contains confidential business 

information). This study was obtained from the Robust Summaries for the Alkyl Sulfides 

Category (submitted to EPA under the HPV Challenge Program by the Health, Environmental, 

and Regulatory Task Group of the American Chemistry Council’s Petroleum Additives Panel on 

May 19, 2005).
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62

D-7. Genetic Toxicity – Gene Mutation

Summary of endpoint: Adequate

Study 1: Mutagenicity study of CASRN 68511-50-2 in Salmonella typhimurium 

Title: Not available

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 1-Propene, 2-methyl- sulfurized, CASRN 68511-50-2

Remarks: Purity not provided

METHOD

Method/guideline followed: Consistent with guidelines outlined in OECD TG 471

Test type: Bacterial Reverse Mutation Assay

GLP compliant?: No

Year study performed: 1978

Test species: Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538, and TA98

Metabolic activation: With and without

Concentrations tested: 0, 0.01, 0.05, 0.1, 0.5, 1.0 μL/plate

Statistical methods used: Determination of mean ± S.D. of replicate plate counts

Test method/conditions remarks: The vehicle was DMSO. All stock and working solutions 

were stored at 4°C in glass screw-capped bottles. All sterility controls were negative for 

bacterial growth. Vehicle was tested as negative control. Activation consisted of adult male 

Sprague-Dawley rat liver S-9 fraction, induced with Aroclor 1254 (100 μl/plate).

RESULTS

Genotoxicity result: Not mutagenic

Cytotoxic concentration (if applicable): Not specified
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Results remarks: For all strains and dose levels with and without metabolic activation, the 

criteria for a positive mutagen (at least 3 times the number of colonies as the controls for 

spontaneous reversion) were not met. Positive controls (9-aminoacridine and 2-nitrofluorene 

without activation and 9-aminoacridine, 2-nitrofluorene, aflatoxin, and 6-aminochrysene with 

activation) had at least 3 times the number of colonies as the control. 

CONCLUSIONS

The test chemical does not induce gene mutations in bacteria either with or without metabolic 

activation.

STUDY RELIABILITY

[2] Reliable with restrictions. Cytotoxicity was not noted and the assay was conducted using 

very low concentrations. 

REFERENCE

Reference information is not available. This study was obtained from the Robust Summaries for 

the Alkyl Sulfides Category (submitted to EPA under the HPV Challenge Program by the 

Health, Environmental, and Regulatory Task Group of the American Chemistry Council’s 

Petroleum Additives Panel on May 19, 2005).
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64

Study 2: Mutagenicity study of CASRN 68515-88-8 in Salmonella typhimurium

Title: Not available

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Pentene, 2,4,4-trimethyl-, sulfurized CASRN 68515-88-8

Remarks: 97% purity

METHOD

Method/guideline followed: Consistent with guidelines outlined in OECD TG 471

Test type: Bacterial Reverse Mutation Test

GLP compliant?: Yes

Year study performed: 1982

Test species: Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538

Metabolic activation: With and without

Concentrations tested: 0, 0.01, 0.03, 0.1, 0.3, and 1 μL/plate 

Statistical methods used: The mean number of his+revertants/plate for three replicate assay 

plates was calculated for each concentration and strain.

Test method/conditions remarks: No significant deviations from guideline protocols. The 

vehicle was DMSO.

RESULTS

Genotoxicity result: Negative with and without metabolic activation.

Cytotoxic concentration (if applicable): Not observed

Results remarks: The test material was tested without metabolic activation at 1, 0.3, 0.1, 0.03, 

and 0.01 μL of test material/plate and found to be non-mutagenic to the bacterial strains tested. 

The number of revertant colonies as a result of treatment with the test material did not differ 

significantly from the number produced by the DMSO vehicle control. The test material was not 

toxic to any strain at any concentration. The positive controls, sodium azide, 2-nitrofluorene, 

and 9-aminoacridine at concentrations ranging from 2.5-100 μg/plate produced more than a 10-
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65

fold greater incidence of his+ revertants/plate with the bacterial strains used in this study. The

test material was also tested in the presence of an S9 microsomal fraction from Aroclor 1254-

treated rat livers. The concentrations tested (1, 0.3, 0.1, 0.03, and 0.01 μL of test material/plate) 

in the activated system did not induce significant detectable mutagenic events with the bacterial 

strains used. The positive metabolic activated control, 2-anthramine (2.5 μg/plate) produced 

positive mutagenic responses in the bacterial strains used in this study.

CONCLUSIONS

The test material did not produce significant mutation in any of the Salmonella strains in the 

quantitative mutagenesis assay, either in the presence or absence of metabolic activation. Thus, 

under the conditions of the assay employed, the test material was determined to be nonmutagenic in the Salmonella/microsome mutagenesis assay. 

STUDY RELIABILITY

[2] Reliable with restrictions. Cytotoxicity was not observed and the assay was conducted using 

very low concentrations.

REFERENCE

Reference information is not available (the underlying study contains confidential business 

information). This study was obtained from the Robust Summaries for the Alkyl Sulfides 

Category (submitted to EPA under the HPV Challenge Program by the Health, Environmental, 

and Regulatory Task Group of the American Chemistry Council’s Petroleum Additives Panel on 

May 19, 2005).
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66

Study 3: Mutagenicity study of CASRN 67762-55-4 in Salmonella typhimurium 

Title: Not available

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Alkenes, C15-18 alpha-, sulfurized, CASRN 67762-55-4

Remarks: Purity not provided

METHOD

Method/guideline followed: Designed to be in compliance with microbial mutagenicity testing 

as set forth by OECD 1981, EPA 1982, FDA 1993

Test type: Bacterial Reverse Mutation Test

GLP compliant?: Yes (except analyses were not performed to verify the homogeneity, stability 

or accuracy of the test/control article preparation )

Year study performed: 1996

Test species: Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102; 

Escherichia coli strain WP2 uvrA

Metabolic activation: With and without

Concentrations tested: Pre-screen, duplicate cultures: 50.0, 167, 500, 1670, and 5000 μg/plate, 

plus control; Triplicate cultures: 50.0, 167, 500, 1670, 5000, and 10,000 μg/plate 

Statistical methods used: Statistical analyses are performed using the program developed by 

Snee and Irr (1981), with significance established at the 95% confidence limit.

Test method/conditions remarks: Test article was first evaluated in a pre-screen using both 

liquid preincubation and plate incorporation treatment conditions. Duplicate cultures of strains 

TA1537, TA100, and WP2 uvrA were treated with article at doses of 50.0, 167, 500, 1670, and 

5000 μg/plate, as well as the solvent control, in the absence of S9. The test article was found to 

be incompletely soluble (droplets were observed) at all doses. The article was next evaluated 

using both treatment conditions. Based upon the results of the pre-screen, the article was 

evaluated in triplicate cultures in strains TA1535, TA1537, TA98, TA100, TA102, and WP2 

uvrA in the presence and absence of S9 at doses of 50.0, 167, 500, 1670, 5000, and 10,000 

μg/plate. Six doses of the article were evaluated in the event of unacceptable toxicity and/or 

insolubility at the highest dose levels evaluated in the mutation assay. The S9 mixture included 

6% (v/v) Aroclor 1254-induced male Sprague-Dawley rat liver homogenate with the appropriate 
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67

buffer and cofactors. The test article was again found to be incompletely soluble at all doses, 

under both treatment conditions.

 

RESULTS

Genotoxicity result: Negative with and without metabolic activation

Cytotoxic concentration (if applicable): Not specified

Results remarks: In the pre-screen, results indicated that the article was not toxic. In the 

following study, normal growth was observed in all tester strains at all doses evaluated with and 

without S9. Revertant frequencies for all doses of article in all tester strains, with and without S9 

under both treatment conditions approximated or were less than those observed in the concurrent 

negative control cultures. All positive and negative controls were within acceptable ranges. 

CONCLUSIONS

The results were negative in this study, using liquid pre-incubation and plate incorporation 

treatments.

STUDY RELIABILITY

[2] Reliable with restrictions. No analyses were supplied to verify the homogeneity, stability, or 

accuracy of test article preparation. The test article was found to be incompletely soluble 

(droplets were observed) at all doses, under both treatment conditions. 

REFERENCE

Reference information is not available (the underlying study contains confidential business 

information). This study was obtained from the Robust Summaries for the Alkyl Sulfides 

Category (submitted to EPA under the HPV Challenge Program by the Health, Environmental, 

and Regulatory Task Group of the American Chemistry Council’s Petroleum Additives Panel on 

May 19, 2005).
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68

D-8. Genetic Toxicity – Chromosomal Aberrations

Summary of endpoint: Adequate

Study 1: Micronucleus assay of CASRN 68511-50-2 in mice 

Title: Not available

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 1-Propene, 2-methyl, sulfurized-, CASRN 68511-50-2

Remarks: Purity not provided

METHOD

Method/guideline followed: OECD TG 474

Test type: Mammalian Erythrocyte Micronucleus Test (in vivo)

GLP compliant?: Yes

Year study performed: 1989

Test species: B6C3F1 mice

Number of animals/sex/dose: 15/sex/dose (5/sex/dose/sample interval)

Route of administration: Intraperitoneal

Vehicle: Hydroxypropyl methylcellulose (Methocel K4M Premium - Dow Chemical)

Exposure duration: Single dose

Doses tested: 3500 mg/kg bw

Statistical methods used: Normal test for equality of proportion (one-tailed). Because of 

multiplicity of comparisons, a Dunnett adjustment was made. 

Test method/conditions remarks: Young male and female mice were treated with a single 

intraperitoneal injection of 3.5 g/kg test material, 50 mg/kg cyclophosphamide in saline, or 

methylcellulose vehicle alone. Dose had been determined in a preliminary toxicity test to 

identified MTD for this study. Animals were sacrificed and femurs removed at 24, 48, or 72 

hours post-dosing (5M, 5F per interval) for test material and negative control, and at 24 hours 
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post-dosing only for cyclophosphamide. Bone marrow smears were prepared and immature red 

blood cells (polychromatic erythrocytes, PCEs) and mature red blood cells (normochromatic 

erythrocytes, NCEs) were evaluated for toxicity and the presence of micronuclei. Slides were 

stained with acridine orange and scored under a fluorescence microscope. Slides from all dose 

groups were sorted by a computerized random number system and the cytogeneticist was 

unaware of what dose group any individual slide was from. The ratio of PCE or NCE per the 

first 1000 erythrocytes counted was calculated to determine cytotoxicity, if any.

RESULTS

Genotoxicity result: Negative

Cytotoxic dose: Not observed

Results remarks: In the preliminary toxicity test (2M, 2F/group) all mice died at 5.0 g/kg and 

all survived at 3.5 g/kg with no cytotoxicity in bone marrow cells 24 hours after injection. Data 

from the full study demonstrate that the frequency of mironucleated PCEs in femoral bone 

marrow for males and females treated with the test material was not significantly elevated (p <

0.05) when compared to negative controls for groups sampled at 24, 48, or 72 hours 

postinjection. Results from both sexes combined demonstrate the same results. 

Cyclophosphamide, the positive control material, did induce statistically significant increases in 

micronucleated PCEs in all animals demonstrating that a valid study was performed.

CONCLUSIONS

Methyl propene derivative administered via i.p. injection at 3.5 g/kg body weight did not induce 

the formation of micronuclei in PCEs in male or female mice at any time interval and is not 

considered clastogenic in this test system. 

STUDY RELIABILITY

[1] Reliable without restrictions. 

REFERENCE

Reference information is not available (the underlying study contains confidential business 

information). This study was obtained from the Robust Summaries for the Alkyl Sulfides 

Category (submitted to EPA under the HPV Challenge Program by the Health, Environmental, 

and Regulatory Task Group of the American Chemistry Council’s Petroleum Additives Panel on 

May 19, 2005).
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70

Study 2: Micronucleus assay of CASRN 68511-50-2 in rats 

Title: Not available

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 1-Propene, 2-methyl-, sulfurized, CASRN 68511-50-2

Remarks: Purity not provided

METHOD

Method/guideline followed: Modified OPPTS 870.5395

Test type: Mammalian Erythrocyte Micronucleus Test (in vivo)

GLP compliant?: Yes

Year study performed: 1987

Test species: Sprague-Dawley (Tac:N[SD]fBR) rats

Number of animals/sex/dose: 5/sex/dose

Route of administration: Dermal

Vehicle: Mineral oil (solvent refined naphthenic base stock); density 0.88 g/L

Exposure duration: 5 days/week for 13 weeks

Doses tested: 500 or 2000 mg/kg/day undiluted test material; 500 mg/kg/day diluted 50% w/v 

in mineral oil base stock

Statistical methods used: SAS ANOVA and ANOVA F test; Tukey’s Studentized Range test 

and Scheffe’s test; SAS General Linear Model, a studentized linear regression analysis to 

determine dose responsiveness. Statistical analyses compared test values to negative control 

data; a significant increase in micronuclei is an indicator of clastogenic activity by the test 

material

Test method/conditions remarks: 

Age at initiation: 7 weeks old following 2 weeks acclimation.
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Methyl propene derivative was applied to the clipped backs of groups of 20 Sprague-Dawley rats 

(10M, 10F) 5 days per week for 13 weeks at dose levels of 0, 500, or 2000 mg/kg/day undiluted 

or 500 mg/kg diluted (50% w/v) in mineral oil base stock. Rats were fitted with Elizabethan

collars to minimize ingestion of test material, which was left uncovered on the skin. At 

termination of the 13 week subchronic study, approximately 24 hours after the final dermal 

administration, bone marrow was harvested from femurs of the first 5 rats/sex/group necropsied. 

Three bone marrow slides were prepared for each animal. Slides were stained with acridine 

orange and scored under a fluorescence microscope. All slides were randomized by a computergenerated random numbers table so that the cytogeneticist was unaware of what dose group any 

individual slide was from. Immature red blood cells (polychromatic erythrocytes, PCE) and 

mature red blood cells (normochromatic erythrocytes, NCE) were evaluated for toxicity and the 

presence of micronuclei. The ratio of PCE to NCE per the first 1000 erythrocytes counted was 

calculated to determine cytotoxicity if any. At least 1000 PCE and 1000 NCE were scored for 

the presence of micronuclei.

RESULTS

Genotoxicity result: Negative

Cytotoxic dose: No data

Results remarks: Methyl propene derivative undiluted (500 or 2000 mg/kg/day) and methyl 

propene derivative (500 mg/kg/day) 50% w/v in mineral oil base stock were not cytototoxic to 

red blood cell formation. These test materials did not induce any statistically significant increase 

in the formation of micronucleated PCEs or NCEs in bone marrow red blood cells of male or 

female rats exposed dermally for 13 weeks.

CONCLUSIONS

Methyl propene derivative does not cause chromosome damage in rats following regular and 

prolonged dermal exposure in this test system. NOEL= 2000 mg/kg/day 

STUDY RELIABILITY

[2] Reliable with restrictions. Study does not include a positive control. Samples were collected 

only once approximately 24 hours after the final dose. Information on test material composition, 

purity, and stability are not part of this report, but the reader is referred to the adjunct 13-week 

subchronic report.

REFERENCE

Reference information is not available (the underlying study contains confidential business 

information). This study was obtained from the Robust Summaries for the Alkyl Sulfides 

Category (submitted to EPA under the HPV Challenge Program by the Health, Environmental, 

and Regulatory Task Group of the American Chemistry Council’s Petroleum Additives Panel on 

May 19, 2005).
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72

Study 3: Micronucleus assay of CASRN 68515-88-8 in mice 

Title: Not available

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Pentene, 2,4,4-trimethyl-, sulfurized, CASRN 68515-88-8

Remarks: 97% purity

METHOD

Method/guideline followed: OECD TG 474

Test type: Mammalian Erythrocyte Micronucleus Test (in vivo)

GLP compliant?: Yes

Year study performed: 1988

Test species: B6C3F1 mice

Number of animals/sex/dose: 5/sex/dose

Route of administration: Oral gavage

Vehicle: Not indicated

Exposure duration: Single dose

Doses tested: 5000 mg/kg

Statistical methods used: Group mean body weights, total polychromatic erythrocytes (PCEs), 

normochromatic erythrocytes (NMEs), PCEs with micronuclei, and NMEs with micronuclei 

were compared. For each animal, a minimum of 1000 PCEs were counted for the presence of 

micronucleated PCEs. The frequency of micronucleated cells per animals was expressed as the 

number of micronucleated PCEs per 1000 PCEs counted. The ration of PCEs/NMEs was also 

recorded. The data were analyzed for statistical significance on a binomial distribution, at a level 

of significance of 0.05, and using the table of Kastenbaum and Bowman (Mutation Res. 9:527-

549, 1970). 

Test method/conditions remarks: 

Number of animals per dose: 5/sex/group.
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Control groups and treatment: 5/sex negative control (mineral oil); 5/sex positive control 

(cyclophosphamide, 50 mg/kg intraperitoneal injection).

Mice were approximately 12 weeks old and 17-31 grams at study initiation. Animals were 

observed daily and body weights were recorded after 18, 24, and 48 hours. Test material and 

negative control groups were sacrificed after 18, 24, and 48 hours, whereas the positive control 

group was terminated after 24 hours. 

RESULTS

Genotoxicity result: Negative

Cytotoxic dose: No data

Results remarks: The frequency of PCEs with micronuclei ranged from 1.0 to 5.9/1000 PCEs 

in negative control mice with groups means of 2.6, 3.0, and 2.4 PCEs for the three time points. 

These averages and group means were within the expected range based on published data on the 

performing laboratory historical controls. In contrast, male animals dosed with 

cyclophosphamide had 9.0 to 24.0 micronucleated PCEs/1000 PCEs, with a mean of 14.5 for the 

group. The average frequencies of micronucleated PCEs obtained from male animals receiving 

the test material after the three time periods were 5.1, 3.0, and 5.7/1000 PCEs. These group 

means were not significantly higher than the negative control values. The mean PCE/NME 

ratios in negative male group for the three time periods were 0.60, 0.60, and 0.69, respectively. 

The test material was not cytotoxic since the PCE/NME ratio at the three time points was 0.60, 

0.59, and 0.66. The mean frequency of micronucleated PCEs/1000 PCEs for female mice was 

1.9, 2.1, and 2.9, respectively. The average micronucleated PCEs value for the 

cyclophosphamide treated females was 20.5. Female mice treated with the test material were 

found to have mean micronucleated PCEs values of 1.1, 2.0, and 1.2 at the three time points, 

respectively. A comparison of the PCE/NME ratio between the negative control and test 

material treated female mice did not vary significantly. 

CONCLUSIONS

The subject material was tested for its genotoxicity using mouse in vivo micronucleus screening 

assay in bone marrow. There was no significant increase in micronucleated PCEs in animals 

exposed to the test substance. Thus, the test material was negative in this assay. 

STUDY RELIABILITY

[1] Reliable without restrictions. 

REFERENCE

Reference information is not available (the underlying study contains confidential business 

information). This study was obtained from the Robust Summaries for the Alkyl Sulfides 

Category (submitted to EPA under the HPV Challenge Program by the Health, Environmental, 
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74

and Regulatory Task Group of the American Chemistry Council’s Petroleum Additives Panel on 

May 19, 2005).
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75

E. DATA ADEQUACY SUMMARY TABLE

Endpoint Study Information 

Available

Acceptable Data Gap

Aquatic Effects

Acute Toxicity to Fish Yes Yes No

Acute Toxicity to Aquatic Invertebrates Yes Yes No

Chronic Toxicity to Aquatic 

Invertebrates

Yes Yes No

Toxicity to Aquatic Plants 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 Toxicity Yes Yes No

Developmental Toxicity Yes Yes No