SRC TR

- 1 2

- 034

HPV 3 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 R ound Pond Road North Syracuse, NY 13212

Contract No. EP

- W

- 09

- 027

Task FG007. 2 .009.00 6

Submitted to:

OCSPP/OPPT/RAD (7403M) U.S. Environmental Protection Agency 1200 Pennsylvania Avenue

Washington, D.C. 20460

Debra Milligan, Project Officer

Meena Sona wane, Work Assignment Manager David Brooks, Alternate Work Assignment Manager

March 14 , 201 2Final HPV3 Test Plan and Data Adequacy Review CASRN 72162

- 15

- 3

i 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

................................ ................................ ........... 62Final HPV 3 Test Plan and Data Adequacy Review CASRN 72162

- 15

- 3

1 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 followin g 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/Developme ntal Toxicity

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

ow < 4.2, whereas Test Group 2 is required if log K

ow ≥ 4.2. Given that the estimated log K

ow of 1

- decene, sulfurized is > 6.0, the use of Test Group 2 i s 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 util izes 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 substance 3 with similar functional groups and all four chemicals share similar physicochemical characteristics, 4

1 Letter dated February 20, 2012. including high partition coefficien t (log K

ow ), low water

2 Screening

- Le vel 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 Abstr act Service nomenclature

convention s , both the 1

- decene feedstock and the test rule chemical, 1

- decene, sulfurized (CASRN 72162

- 15

- 3)

, are

non

- branched. O f the three chemicals that Dover proposes as analogs of the test rule chemical, o nly 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 Ad ditives 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 pla n)

.Final HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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2 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 read

across (“bridging”) strategy using analog data, as well as modeled data, to satisfy the test

- rule

required endpoints. For aquatic toxicity endpoints, Dover suggests that testi ng 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 struc tures 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 (Reappr oved 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 CASR N 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 notFinal HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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- 3

3 considered for satisfaction of the e ndpoint because no justification was provided for use of this analog.

6. Repeated Dose/Reproduction/Developmental

A dequate 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 t o satisfy the repeated dose testing requirement. EPA comments on the initial test plan for the Alkyl Sulfide Category 5 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 consider ed 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 test ing 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 Reg ulatory 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 summar izing 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 fin al submission of robust summaries for the Alkyl Sulfide Category is available at http://www.epa.gov/hpv/pubs/summaries/alkylsul/c12549rr2.pdf

.Final HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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4 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 Tox icity 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; t otal organic carbon (TOC) measurements o f 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 rem arks: 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 ; p retreatment : none, fish held for a minimum of 14 days before testing. No feeding

occurred during the test.Final HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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5 Test System: Individual WAFs were prepared for each test level and renewed daily. A measured weight of test material was added to a measure d 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. Followi ng 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 substanc e 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 trea tment, 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 hardne ss of 176 mg/L as CaCO 3

. 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. Alka linity was not reported. Dissolved oxygen : 7.1 to 8.7 mg/L ; pH : 7.8 to 8.5 ; condu ctivity : 570 to 650

μ mhos/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 LL 50 s: Statistical analysis of survival data was not warranted.

Test Substance: No undissolved test mater ial was seen on the surface of the test vessels during the entire aquatic toxicity test.

Reference Substance: No

RESULTS

Endpoint value(s): 96

- h LL 50 > 1000 mg/L (nominal); 96

- h L OEC = 10 00 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 th e 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 wat er 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 micronFinal HPV 3 Test Plan and Data Adequacy Review CASRN 72162

- 15

- 3

6 filter prior to TOC analysis, that the test vessels were covered, or that the continuous te mperature 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 t he 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 i n 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 toxi city

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. I t 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 t he Water Accommodated Fractions (WAFs) of CMA 613

to t he 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 und er 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 HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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7 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 variegat u s

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; t otal 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 (rang e = 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 ;

p retreatment

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

Test System: Individual WS Fs 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 Telfon

c oated 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 fromFinal HPV 3 Test Plan and Data Adequacy Review CASRN 72162

- 15

- 3

8 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 solubl e 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 th rough 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 1 0 ,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), respective ly

.

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

Calculation of LL 50 s: 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 t oxicity test.

Reference Substance: Sodium lauryl sulfate (SLS). The 96

- h LC 50 was 1.2 mg/L. No

information was provided on method of calculation.

RESULTS

Endpoint value(s): Nominal concentrations: 96

- h LL 50 > 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 t o 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 th at the exposure concentration in the

W S F 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. )Final HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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9 CONCLUSIONS

No mortality o r 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 Summari es 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 HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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10 C

- 2

. Acute Toxicity to Daphnia

Sum mary 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) meas urements.

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, 3 00

, 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 oCFinal HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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11 Dilution water: Filtered well water collected at Hampton, New Hampshire and adjusted to the appropriate hardness 168 to 172 mg/L as CaCO 3

. The water was passed through activated carbon, a particle filter, and then an ultraviolet sterilizer and s tored 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 ph otoperiod was maintained with cool

- whit e 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 μ mhos/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

tes t 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 actu al 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 EC 50 > 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 co ncentration of 100 0 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 conc entrations tested. Ninety

five to 100% survival occurred at all test concentrations. No sublethal effects were noted during

the test.Final HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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12 STUDY RELIABILITY

[1 ] Reliable with out restrictions.

REFERENCE

Ward, T.J. 1993

. Acute Toxicity of the Water Acc ommodated 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, Environment al, and Regulatory Task Group of the American Chemistry Council’s Petroleum Additives Panel on May 19, 2005)

.Final HPV 3 Test Plan and Data Adequacy Review CASRN 72162

- 15

- 3

13 C

- 3. Chronic Toxicity to Daphnia

Summary of endpoint: Not adequate

No studies were available for this endpoint.Final HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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14 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

- Propen e, 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) measuremen ts 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 prepar ed 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 spe ed 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 surfa ce or bottom, aFinal HPV 3 Test Plan and Data Adequacy Review CASRN 72162

- 15

- 3

15 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 treat ment 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. C ool

- white fluorescent lights provided a light intensity of 47 to 50 μ Ein/m 2 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 susp ended 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 cont rol 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 EL 50 s and NOELRs: Moving avera ge and probit methods (Stephan, 1983) were

used to calculate EC 50 s (i.e., EL 50 s). A parametric one

- way analysis of variance (ANOVA) and

Dunnett’s test were used to calculate the no

- observed effect concentration (i.e., EL 0 s) when data were normally distrib uted 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: N o

RESULTS

Endpoint value(s): Based on nominal loading rates: 72

- hour EL 50 (cell density) = 26 (21

- 32) mg/L; 72

- hour NOELR (cell density) = 5.0 mg/L; 96

- hour EL 50 (cell density) = 34 (29

- 39) mg/L; 96

- hour NOELR (cell density) = 10 mg/L ; 72

- hour EL 50 (g rowth rate) > 100 mg/L; 72

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

- hour EL 50 (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 p H of the sterile media at the start of the test was 7.0 rather than 7.5. These deviations did not compromise the study.Final HPV 3 Test Plan and Data Adequacy Review CASRN 72162

- 15

- 3

16 72

- hour EL 50 for cell density was determined by moving average method. 96

- hour EL 50 for cell density was determined by Probit method . 96

- hour NOELR s 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 exposur e, reductions in cell density and growth rate were observed at a loading rate > 10 mg/L.

STUDY RELIABILITY

[1] Reliable without restrictions.

REFERENCE

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

the Alkyl Sulfides Category (submitted to EPA under the HPV Challenge Program by the Health, Environmental, and Regulatory Task Group of the American Chemistry Council’s Petroleum Additives Panel on May 19, 2005)

.Final HPV 3 Test Plan and Data Adequacy Review CASRN 72162

- 15

- 3

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

Th is 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 (LD 50 )

GLP compliant?: Pre

- GLP

Year study performed: 1975

Test species: Sherman/Wistar rat (young adult)

Number of animals/sex/do se: 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

m L /kg. Animals observed for 14 days post

- dosing for signs of toxicity or mortality

. Body weights were not taken; gross necropsies and hist opathology were not performed

.Final HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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18 RESULTS

Endpoint value(s): LD 50 = 5.7 mL /kg; 19/2 0 confidence limits = 4

- 8 mL /kg

Results remarks: No deaths were observed at 2.0 or 4.0 m L /kg

. A t 8 m L /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 m L /kg. No data were presented on parameters other than mortality.

CONCLUSIONS

The oral LD 50 is 5.7 mL /kg (~5700 mg/kg) for male rats.

STUDY RELIABILITY

[2] Reliable with restrictions. D etai ls 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 bus iness 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 A dditives Panel on May 19, 2005)

.Final HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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- 3

19 Study 2 : Acute oral toxicity study of CASRN 68515

- 88

- 8 in rats

Title: Not available

Th is study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Pentene, 2,4,4,

- trimethyl, s ulfurized

, CASRN 68515

- 88

- 8

Remarks: Purity not provided

METHOD

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

Test type: Acute Oral Toxicity

GLP compliant?: Y es

Year study performed: 1987

Test species: Albino r ats 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/co nditions remarks: The animals were fasted overnight before dosing. The sample was administered as supplied at a limit dose of 5.0 g/kg via o ral gavage with a syringe

and Nelaton catheter. Following administration, th e 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 wer e euthanized by carbon dioxide at the conclusion of the observation period. Gross autopsies were performed on all animals that died during the observation period and on all survivors after day 15.Final HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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20 RESULTS

Endpoint value(s): LD 50 > 5.0 g/kg

Results re marks: 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 administratio n. 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 LD 50 is greater than 5.0 g/kg for male and female rats.

STUDY RELIABILITY

[1] Reliable with out r estrictions.

REFERENCE

Reference information is not available (the underlying study contains con fidential business information) . This study was obtained from the Robust Summaries for the Alkyl Sulfides Category (submitted to EPA under the HPV Challeng e Program by the Health, Environmental, and Regulatory Task Group of the American Chemistry Council’s Petroleum Additives Panel on May 19, 2005)

.Final HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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- 3

21 Study 3 : Acute dermal toxicity study of CASRN 68515

- 88

- 8 in rabbits

Title: Not available

Th is 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

- ho ur 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 freque ntly on the day of treatment. Thereafter, all surviving rabbits were examined for outward signs of toxicity on c e per day, for

the entire 14

- day observation period. Individual weights were recorded on the day of dosage, weekly thereafter and prior to sacr ifice. The surviving animals were euthanized at the conclusion of the observation period. Gross autopsies were performed on all animals after 14 days.Final HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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22 RESULTS

Endpoint value(s): LD 50 > 2.0 g/kg

Results remarks: One rabbit died on day 14 of the stud y. 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 d ay 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

mort ality. 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 LD 50 greater than 2.0 g/kg.

STUDY RELIABILITY

[1] Reliable with out 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 Categor y (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 HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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23 Study 4 : Acute dermal toxicity study of CASRN 67762

- 55

- 4 in rabbi ts

Title: Not available

Th is 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: N ot 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 t ested: 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 dorsa l skin of each animal. The patches were removed after 24

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

RESULTS

Endpoint value(s): LD 50 > 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.Final HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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24 CONCLUSIONS

The dermal L D 50 is greater tha n 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 S ulfides 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 HPV 3 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

Th is study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: 1

- Propene, 2

- methyl

- , sulfurized, CASRN 68511

- 50

- 2

Remarks: Purity not provided

METHOD

Method/guideli ne 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 adm inistration: 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 gro up were sacrificed 24 hour post

- exposure; others maintained for 2 weeks recovery. Vapors from the methyl propene derivati ve 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 vapo r. 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.Final HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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26 RESULTS

Endpoint value(s): LC 50 > 0.39 mg/L

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

occurr ing 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 obser ved 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 L C 50 > 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 on ly 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 busi ness 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 Ad ditives Panel on May 19, 2005)

.Final HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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27 Study 6 : Acute inhalation toxicity study of CASRN 6851 5

- 88

- 8 in mice and guinea pigs

Title: Not available

Th is study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Pente ne, 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 per formed: 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 t ested: 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 ge nerated 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 test

material

- exposed group except that the test material was not administrate d. 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 recorde d 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 byFinal HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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

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

RESULTS

Endpoint value(s): LC 50 (mice) > 4.3 mg/L; LC 50 (guinea pigs) > 4.3 mg/L

Results remarks: The mass median aerodynamic diameter for the stu dies 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 t he 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 mic e 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

- h our 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 treatme nt 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 f or 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 stud y 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 HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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29 Study 7 : Acute inhalation toxicity study of CASRN 6851 5

- 88

- 8 in mice , rats
, and guinea pigs

Title: Not available

Th is study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Penten e, 2,4,4

- trimethyl

- , s ulfurized

, 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 sp ecies: 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/concentrati ons 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

Laski n 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 deta ils 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 th e 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 exsanguinationFinal HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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

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

RESULTS

Endpoint value(s): LC 50 (mice) > 4.3 mg/L; LC 50 (rat) < 4.3 mg/L; LC 50 (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 de viation of 1.9 microns. Approximately 93 percent of the aerosol

was 10 microns or less in size.

Mortality and physical observations: Three female rats die d 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 c h amber 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 die d two hours after exposure. The mice and the guinea pig s 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 we ight 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 w as noted among the spontaneously dying animals.

CONCLUSIONS

The LC 50 values are as follows: LC 50 (mice) > 4.3 mg/L; LC 50 (rat) < 4.3 mg/L ; LC 50 (guinea pig) > 4.3 mg/L.

STUDY RELIABILITY

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

rats ; a full study should have been conducted to determine t he LC 50 for rats.Final HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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- 3

31 REFERENCE

Reference information is not available (the underlying study contains confidential business information). This study was obtained fr om 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 HPV 3 Test Plan and Data Adequacy Review CASRN 72162

- 15

- 3

32 Study 8 : Ac ute inhalation toxicity study of CASRN 6851 5

- 88

- 8 in rats

Title: Not available

Th is 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 a dministration: 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 LC 50 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. T he 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

- s tage 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 visc eral organs, body cavities and carcass).Final HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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33 RESULTS

Endpoint value(s): LC 50 (males) > 5. 6 mg/L; LC 50 (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 animal s /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 dur ing 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 da y 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. T hree females in the high

- dose group died on day 2 following exposure, with an addition al 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 n oted 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 LC 50 in male Sprague

- Dawley rats is considered to be greater than 5.6 mg/L. The LC 50 value in females was calculated to be 2.17 mg/L with upper and lo wer 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 obtain ed 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 HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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34 C

- 6 . Re peated

- Dose Toxicity

Summary of endpoint: Adequate

Study 1: Repeated

- dose dermal toxicity study of CASRN 68511

- 50

- 2 in rats

Title: Not available

Th is 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: C omparable 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

Do ses/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 ; s erum chemistry and hematology data were evaluated using the F test for ANOVA and Student

Newman

- Keuls multiple comparison test.

Test method/conditions remarks: Age at initiation: 7 weeks old following 2 w eeks acclimation

.Final HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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35 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

- Dawle y 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 Elizabeth an collars to minimize ing estion 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 produ ction 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. Fe male 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/da y, 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

re action 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 epider mis

, 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 formati on 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 NO AEL 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 HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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36 STUDY RELIABILITY

[1] Reliable without restrictions.

REFERENCE S

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 t he American Chemistry Council’s Petroleum Additives Panel on May 19, 2005)

.Final HPV 3 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

Th is study is: Adequate Not Ad equate

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 Subchron ic 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 teste d: 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 b y evaluation using the Newman

Keuls 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, 6 F/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 bo dy 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 ofFinal HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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38 undiluted test material on the same test schedule. Half of the animals in each group were abraded once per week throughout the stu dy. 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 in tact, 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 te sting, termination) ; hematology , serum ch emistry

, and urinalysis at pretest and termination ; and gross necropsy evaluations at study termination.

Endpoint value(s): LOAEL = 200 0 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 ha lf 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 i n 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. Hematolo gy 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 tw o animals showed no significant differences.

CONCLUSIONS

LOAEL = 200 0 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 incl uded in the report. An u neven 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 con tains 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 HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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- 3

39 Study 3 : Repeated

- dose dermal toxicity study of CASRN 68511

- 50

- 2 in rabbits (21 days)

Title: Not available

Th is study is: Adequate Not Adequate

TEST SUBSTAN CE

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 designe d 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 (morningFinal HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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40 and afternoon) clinical observations ; skin irritation scoring 5 days per week ; weekly bo dy 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 mod erate 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 s kin, 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 trea tment. 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

, s ix 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 r evealed 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 b y 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.

REFERENCE S

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, Environment al,Final HPV 3 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 HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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- 3

42 Study 4 : Repeated

- dose inhalation toxicity study of CASRN 6851 5

- 88

- 8 in rats

Title: Not available

Th is 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

- do se 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/m 3

Statistical methods used: Body weight, food con sumption, 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 te st, 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 concentrat ions = 15, 50, and 150 mg/m 3 ) 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/m 3

. The details of the whole

- bodyFinal HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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43 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/m 3

. 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 p re

- 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 subj ected 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 ob tain 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/m 3 based on no relevant effects at the highest

dose te sted ; LOAEL (female) = 160 mg/m 3 based on increased hemoglobin levels; NOAEL (females) = 50 mg/m 3

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. Th e s e 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 fou nd 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 inciden ce 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 not ed.

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 d ose 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

conc entration in the high

- dose females sacrificed after 4 weeks of exposure.Final HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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44 Clinical chemistry: There were several statistically significant differences from the control values at both the post

- exposure and post

- recovery time intervals. However, these dif ferences 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 do se 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 week s of exposure, statistically significant increases were seen in high

dose 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 teste s , 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 vi sible gross changes, such as discolored lungs, were noticed in the sacrificed animals. Microscopically, treatment

related effects were seen in the kidneys in the males in a dose

- related profile. Findings included globular casts at the corticomedulary jun ction, 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 nephro pathy 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 con sidered incidental findings.

CONCLUSIONS

The LOAEL for females is 160 mg/m 3 based on increased hemoglobin levels in females. The

NOAEL is 50 mg/m 3 in females. The NOAEL for males is 160 mg/m 3 based on no human

health

- related effects at the highest dose tested.

STUDY RELIABILITY

[1] Reliable without restrictions.

REFERENCE S

Reference information is not available (the underlying study contains confidential business information). This study was obtained from the Robust Summaries for the Alkyl Sulfid es 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 HPV 3 Test Plan and Data Adequacy Review CASRN 72162

- 15

- 3

45 Study 5 : Repeated

- dose dermal toxicity study of CASRN 68 51 5

- 88

- 8 in rats

Title: Not available

Th is study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Pentene, 2,4,4

- trimethyl

- , sulfurized

, CASRN 68515

- 88

- 8

Remarks: Purity not provided

METHOD

Method/guid eline 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 a nimals/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 ga in

, 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/w eek 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 th e highest

dose testedFinal HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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46 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 weig ht, 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 applica tion. 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

unrema rkable. 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.

REFERENCE S

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 HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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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

Th is 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 outl ined in OECD TG 471

Test type: Bacterial Reverse Mutation Assay

G LP compliant?: No

Year study performed: 1978

Test species: Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538, and TA98

Metabolic activation: With and without

Concentration s tested: 0, 0.01, 0.05, 0.1, 0.5, 1.0 μ L /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

- c apped bottles

. All sterility controls were negative for bacterial growth

. Vehicle was tested as negative control

. Activation consisted of a dult male Sprague

- Dawley rat liver S

- 9 fraction, induced with Aroclor 1254 ( 100 μ l/plate ).

RESULTS

Genotoxicit y 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 forFinal HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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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 a s 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 l ow 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 HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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49 Study 2 : Mutagenicity study of CASRN 6851 5

- 88

- 8 in Salmonella typhimurium

Title: Not available

Th is study is: Adequate Not A dequate

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

G LP 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 met hods 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/pl ate 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 n ot 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 bacteri al strains used in this study. TheFinal HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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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 non

mutagenic 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 informati on 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 Re gulatory Task Group of the American Chemistry Council’s Petroleum Additives Panel on May 19, 2005).Final HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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51 Study 3 : Mutagenicity study of CASRN 67762

- 55

- 4 in Salmonella typhimurium

Title: Not available

Th is 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 b y OECD 1981, EPA 1982, FDA 1993

Test type: Bacterial Reverse Mutation Test

G LP 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 lim it.

Test method/conditions remarks: Test article was first evaluated in a pre

- screen using both liquid preincubation and plate incorporation treatment conditions. Duplicate cultur es of strains TA1537, TA100, an d WP2 uvrA were treated with article at do ses 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 conditi ons. 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 appropriateFinal HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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- 3

52 bu ff er 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 frequen cies 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 r anges.

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 bu siness 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 HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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- 3

53 C

- 8 . Genetic Toxicity

– Chromosomal Aberrations

Summary of endpoint: Adequate

Study 1: Micronucleus assay of CASRN 68511

- 50

- 2 in mice

Title: Not available

Th is 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 com pliant?: 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: H ydroxypropyl methylcellulose (Methocel K4M Premium

- Dow C hemical )

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 rema rks: 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 M TD 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 hoursFinal HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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- 3

54 post

- dosing only for cyclophosphamide. Bone marrow smears were prepared and imm ature 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 m icroscope. 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 b one 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 P CEs 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 inter val and is not considered clastogenic in this test system.

STUDY RELIABILITY

[1 ] Reliable with out restrictions.

REFERENCE

Reference information is not available (the underlying study contains confidential business information). This study was obtai ned 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 HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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- 3

55 Study 2: Micronucleus assay of CASRN 68511

- 50

- 2 in rats

Title: Not available

Th is 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 acc limation

.Final HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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56 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 stoc k. Rats were fitted with Elizabeth an 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 harve sted 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 computer

generated ra ndom 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 toxicit y 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 te st 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 approxim ately 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 th e American Chemistry Council’s Petroleum Additives Panel on May 19, 2005).Final HPV 3 Test Plan and Data Adequacy Review CASRN 72162

- 15

- 3

57 Study 3: Micronucleus assay of CASRN 6851 5

- 88

- 8 in mice

Title: Not available

Th is 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

Tes t 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 polychr omatic erythrocytes (PCEs), normochromatic erythrocytes (NMEs), PCEs with micronuclei, and NMEs with micronuclei were compared. For each animal, a minimu m 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

.Final HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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58 Control groups and treatment: 5/sex negative control (mineral oil); 5/sex positive control (cyclop hosphamide, 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 contra st, 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 perio ds 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 w as 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 cyclophosph amide 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 with out restrictions.

REFERENCE

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

in formation). 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 HPV 3 Test Plan and Data Adequacy Review CASRN 72162

- 15

- 3

59 and Regulatory Task Group of the American Chemistry Council’s Petroleum Additives Panel on May 19, 2005).Final HPV 3 Test Plan and Data Adequacy Review CASRN 72162

- 15

- 3

60 C

- 9 . Reproductive Toxicity

Summary of endpoint: Not adequate

No studies were available for this endpoint.Final HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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- 3

61 C

- 1 0 . Developmental Toxicity

Summary of endpoint: Not adequate

No studies were available for this endpoint.Final HPV 3 Test Plan and Data Adequacy Review CASRN 72162

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62 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

H ealth 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