SRC TR-12-034

HPV3 TEST PLAN AND DATA ADEQUACY REVIEW

1-Decene, sulfurized, CASRN 72162-15-3

Prepared by:

Teresa Manyin, Christopher Janson, William L. Richards, and Gary Diamond

Chemical, Biological and Environmental Center

SRC, Inc.

7502 Round Pond Road

North Syracuse, NY 13212

Contract No. EP-W-09-027

Task FG007.2.009.006

Submitted to:

OCSPP/OPPT/RAD (7403M)

U.S. Environmental Protection Agency

1200 Pennsylvania Avenue

Washington, D.C. 20460

Debra Milligan, Project Officer

Meena Sonawane, Work Assignment Manager

David Brooks, Alternate Work Assignment Manager

March 14, 2012
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TABLE OF CONTENTS

A. INTRODUCTION .................................................................................................................... 1

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

C. ROBUST SUMMARIES .......................................................................................................... 4

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

C-2. Acute Toxicity to Daphnia............................................................................................... 10

C-3. Chronic Toxicity to Daphnia ........................................................................................... 13

C-4. Toxicity to Plants (Algae) ................................................................................................ 14

C-5. Acute Toxicity.................................................................................................................. 17

C-6. Repeated-Dose Toxicity................................................................................................... 34

C-7. Genetic Toxicity – Bacterial Reverse Mutation............................................................... 47

C-8. Genetic Toxicity – Chromosomal Aberrations................................................................ 53

C-9. Reproductive Toxicity...................................................................................................... 60

C-10. Developmental Toxicity................................................................................................. 61

D. DATA ADEQUACY SUMMARY TABLE........................................................................... 62
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HPV3 Test Plan and Data Adequacy Review: 1-Decene, sulfurized, CASRN 72162-15-3

A. INTRODUCTION

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

Chemicals” (76 FR 65385, October 21, 2011), requires the following toxicity tests for 1-decene, 

sulfurized (CASRN 72162-15-3):

C1 – Aquatic Toxicity

D – Mammalian Acute Toxicity

E1 – Bacterial Reverse Mutation 

E2 – Chromosomal Damage (Aberration or Micronucleus Test)

F1 – Mammalian Repeated Dose/Reproduction/Developmental Toxicity

For the C1 testing requirement, the test rule requires the use of Test Group 1 if log Kow < 4.2, 

whereas Test Group 2 is required if log Kow ≥ 4.2. Given that the estimated log Kow of 1-decene, 

sulfurized is > 6.0, the use of Test Group 2 is required for this chemical substance. Test Group 2 

consists of Chronic Toxicity to Daphnia and Toxicity to Plants (Algae).

To address the required endpoints, Dover Chemical Corporation (referred to as Dover hereafter) 

submitted a test plan,1

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

which utilizes data from members of the Alkyl Sulfides HPV Category in 

an analog approach. The submitter proposes the use of data for the following three analogs for 

some of the required toxicity endpoints:

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

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

The proposed analog chemicals were classified as the “Alkyl Polysulfides” Subcategory in 

EPA’s Hazard Characterization of the Alkyl Sulfides Category.2

EPA accepts the use of the proposed analogs because they are structurally similar to the test rule 

chemical substance3 with similar functional groups and all four chemicals share similar 

physicochemical characteristics,4

 1 Letter dated February 20, 2012.

including high partition coefficient (log Kow), low water 

2 Screening-Level Hazard Characterization: Alkyl Sulfides Category, dated September 2009; available at 

http://www.epa.gov/chemrtk/hpvis/hazchar/Category_Alkyl%20Sulfides_Sept2009.pdf. 3 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 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 at 

http://www.epa.gov/hpv/pubs/summaries/alkylsul/c12549tp.pdf). 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. 4 Values estimated using EPISuite (p. 4 of Dover’s test plan).
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solubility, low vapor pressure and high boiling point. Additionally, the molecular weights of the 

analogs and test rule chemical are generally similar.

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

No experimental toxicity data are available for the test rule chemical. Dover proposes a readacross (“bridging”) strategy using analog data, as well as modeled data, to satisfy the test-rulerequired endpoints. For aquatic toxicity endpoints, Dover suggests that testing is “not applicable 

based upon physicochemical properties of the substance, primarily low water solubility which 

makes certain tests not feasible”. Dover agrees to conduct a test for only one of the toxicity 

endpoints: acute oral toxicity.

A summary of EPA’s response/assessment for each endpoint follows:

1. Chronic Toxicity to Daphnia

No experimental data are available for the test rule chemical or analogs. Although Dover 

provides ECOSAR-estimated chronic daphnid toxicity values for “plausible structures of 

lower molecular weight decyl sulfides”, modeled data are not acceptable as a substitute 

for measured data. Furthermore, low water solubility does not preclude the conduct of a 

chronic daphnid test. The test rule guideline, ASTM E 1193-97 (Reapproved 2004) 

provides guidance for use of test substances that are slightly soluble in water. Therefore, 

the testing requirement for chronic toxicity to Daphnia is maintained.

2. Toxicity to Plants (Algae)

Adequate data are available from the analog CASRN 68511-50-2 to satisfy this testing 

requirement.

3. Acute Oral Toxicity (Rat)

Adequate data are available from the analogs CASRNs 68511-50-2 and 68515-88-8 to 

satisfy this testing requirement. Although Dover proposes to conduct an acute oral 

toxicity test on CASRN 72162-15-3, such testing is unnecessary because adequate analog 

data are available for this endpoint.

4. Bacterial Reverse Mutation

Adequate data are available from the analogs CASRNs 68511-50-2, 68515-88-8, and 

67762-55-4 to satisfy this testing requirement.

5. Chromosomal Aberration

Adequate data are available from the analogs CASRNs 68511-50-2 and 68515-88-8 to 

satisfy this testing requirement. Data for the C12-C16 alkyl sulfide substance were not 
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considered for satisfaction of the endpoint because no justification was provided for use 

of this analog.

6. Repeated Dose/Reproduction/Developmental

Adequate data are available from dermal studies of the analogs CASRNs 68511-50-2 and

68515-88-8 and an inhalation study of the analog CASRN 68515-88-8 to satisfy the 

repeated dose testing requirement. EPA comments on the initial test plan for the Alkyl 

Sulfide Category5 stated that “based on the use of these materials (lubricant additives)”... 

“the most appropriate routes of exposure in terms of relevance to humans appear to be 

either the inhalation or dermal routes.” No analog data are available for the 

reproduction/developmental toxicity endpoint. The data from mammalian toxicity tests 

(e.g., repeated-dose toxicity tests) cannot be considered to constitute a substitute or 

alternative for reproduction/developmental toxicity testing. In general, the potential for 

reproduction/developmental toxicity is not inherently apparent from other types of 

mammalian toxicity testing. Therefore, the testing requirement for 

reproduction/developmental toxicity testing is maintained. Given that adequate data are 

available for the repeated dose toxicity endpoint, the submitter may satisfy the F1 testing 

requirement by conducting 40 CFR 799.9355 (Reproduction/Developmental Toxicity 

Screening Test).

Toxicity data presented below for the analog chemicals 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, 2005.6 Each available toxicity study is summarized in 

Section C. Using standard testing guidelines and professional judgment as guides, each study 

was assigned a Klimisch reliability code. A table summarizing the data adequacy for each 

toxicity endpoint is provided on the last page of this document (Section D). 

 5 EPA comments on the initial test plan for the Alkyl Sulfides Category are available at 

http://www.epa.gov/oppt/chemrtk/pubs/summaries/alkylsul/c12549ct.pdf

6 The final submission of robust summaries for the Alkyl Sulfide Category is available at 

http://www.epa.gov/hpv/pubs/summaries/alkylsul/c12549rr2.pdf.
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C. ROBUST SUMMARIES

C-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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C-2. Acute Toxicity to Daphnia

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-1

m-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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C-3. Chronic Toxicity to Daphnia

Summary of endpoint: Not adequate

No studies were available for this endpoint.
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C-4. Toxicity to Plants (Algae)

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 
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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/m2

sec. 

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. 
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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).
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17

C-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.
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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 Plan and Data Adequacy Review CASRN 72162-15-3

19

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.
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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 Plan and Data Adequacy Review CASRN 72162-15-3

21

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. 
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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 Plan and Data Adequacy Review 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 Plan and Data Adequacy Review CASRN 72162-15-3

25

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 Plan and Data Adequacy Review CASRN 72162-15-3

27

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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28

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 Plan and Data Adequacy Review CASRN 72162-15-3

29

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 Plan and Data Adequacy Review CASRN 72162-15-3

30

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.
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31

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 Plan and Data Adequacy Review CASRN 72162-15-3

32

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). 
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33

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).
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34

C-6. Repeated-Dose Toxicity

Summary of endpoint: Adequate

Study 1: 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.
Final HPV3 Test Plan and Data Adequacy Review CASRN 72162-15-3

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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 

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%.
Final HPV3 Test Plan and Data Adequacy Review CASRN 72162-15-3

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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).
Final HPV3 Test Plan and Data Adequacy Review CASRN 72162-15-3

37

Study 2: 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 Plan and Data Adequacy Review 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 Plan and Data Adequacy Review CASRN 72162-15-3

39

Study 3: 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 
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40

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 Plan and Data Adequacy Review CASRN 72162-15-3

41

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

May 19, 2005).
Final HPV3 Test Plan and Data Adequacy Review CASRN 72162-15-3

42

Study 4: 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/m3 based on no relevant effects at the highest 

dose tested; LOAEL (female) = 160 mg/m3 based 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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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/m3 based on increased hemoglobin levels in females. The 

NOAEL is 50 mg/m3 in females. The NOAEL for males is 160 mg/m3 based 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).
Final HPV3 Test Plan and Data Adequacy Review CASRN 72162-15-3

45

Study 5: 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).
Final HPV3 Test Plan and Data Adequacy Review CASRN 72162-15-3

47

C-7. Genetic Toxicity – Bacterial Reverse 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

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 
Final HPV3 Test Plan and Data Adequacy Review CASRN 72162-15-3

48

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).
Final HPV3 Test Plan and Data Adequacy Review CASRN 72162-15-3

49

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-

fold greater incidence of his+ revertants/plate with the bacterial strains used in this study. The 
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50

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).
Final HPV3 Test Plan and Data Adequacy Review CASRN 72162-15-3

51

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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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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C-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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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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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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59

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

May 19, 2005).
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C-9. Reproductive Toxicity

Summary of endpoint: Not adequate

No studies were available for this endpoint.
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C-10. Developmental Toxicity 

Summary of endpoint: Not adequate

No studies were available for this endpoint.
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D. DATA ADEQUACY SUMMARY TABLE

Endpoint Study Information 

Available

Acceptable Data Gap

Aquatic Effects

Acute Toxicity to Fish Yes Yes No

Acute Toxicity to Daphnia Yes Yes No

Chronic Toxicity to Daphnia No — Yes

Toxicity to Plants (Algae) 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 No — Yes

Developmental Toxicity No — Yes