1 

### Substance Group: Group 3: Alkaryl Sulfonates

Summary prepared by: Petroleum Additives Panel Health & Environmental Research Task Group
October 20052 

### 1.0 Physico-chemical Data Category: Alkaryl Sulfonates

1.1 Melting and Boiling Points

CAS # Parameter Value*

o 

### C melting point 349.84
61789-86-4 o 

### C boiling point 935.88

o 

### C melting point 349.84
61790-48-5 o 

### C boiling point 935.88

o 

### C melting point 309.31
68608-26-4 o 

### C boiling point 707.03

o 

### C melting point 349.84
68783-96-0 o 

### C boiling point 935.88

o 

### C melting point 349.84 Analog of
70024-69-0 o 

### C boiling point 935.88

o 

### C melting point 208.45
71549-79-6 o 

### C boiling point 506.34

o 

### C melting point 349.84
71786-47-5 o 

### C boiling point 935.88

o 

### C melting point 347.25
78330-12-8 o 

### C boiling point 788.26

o 

### C melting point 349.84
115733-09-0 o 

### C boiling point 935.88

o 

### C melting point 208.45
115829-36-2 o 

### C boiling point 506.34

*These data were modeled by an HERTG member company representative. The reliability code that should accompany the robust summarie s prepared using these data is: (2) Valid with restrictions. The selection of this code is based on the data being modeled rather than measured. The use of these data should always be accompanied by the caveat that they were modele d using a structure based modeling program (see reference) and that the values selected are based on a structure that is repr esentative of the CAS#.

The reference for the model is: MPBPWIN (v1.31) In: Meylan W. and P. Howard. 1999. EPIWIN Modeling Program, Syracuse Research Corporation. Environmental Science Center, 6225 Running Ridge Road, North Syracuse, NY, 13212-2510,
USA.3 

### 2.0 Environmental Fate and Pathways Category: Alkaryl Sulfonates

2.1 Water Solubility Robust Summary 3 – WS - 1

CAS No. CAS# 115733-09-0 Test Substance Name C14-24 alkaryl calcium salt derivative Remarks For more information on this chemical, see Section 2.0
“General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category. Method/Guideline Flask Method, Method A6 of Commission Directive
92/69/EEC and OECD Method 105 GLP (Y/N) Yes
Year 2004 Remarks for Test Conditions The solubility in water of a substance is specified by the
saturation mass concentration of the substance in water at a given temperature. A preliminary test was conducted by diluting 0.2645 g of the test material to 600 mL with glass double-distilled water. After shaking at 30 o C for 17.5 hours and standing at 20 o C for 3 hours, the solution was centrifuged at 13,500 rpm for 20 minutes and analyzed.

Based on the preliminary results, mixtures of double distilled
water and test substance were added to each of three conical flasks. The flasks were shaken for approximately 24, 48 and 72 hours (one flask/time period) at 100 rpm and 30°C. The flasks were then allowed to stand for approximately 24 hours at 20°C. The contents of each flask was then centrifuged at 13,500 rpm for 20 minutes and transferred to measuring cylinders. The pH of each solution was measured. The concentration of the test material in the sample solutions was determined in duplicate with duplicate injections of each sample by an LCMS method with direct flow injection. Results Prior to centrifuging the samples were clear and colorless with excess test material floating on the surface. Following centrifugation the supernatants were clear, colorless and free
from excess test material.

The water solubility of the test material was determined to be
4.79 x 10 -4 g/l at 20.0 + 0.5°C.

The linearity of the detector response in respect to concentration was acceptable over a range of concentrations up to 11.900 mg/L with a correlation coefficient of 0.962. Recovery analysis of the sample procedure was assessed and proved adequate for the test. Value (g/L) at temperature °C 4.79 x 10 -4 g/l at 20.0 + 0.5°C4 Conclusions The solubility of the test material in double distilled water
was found to be 4.79 x 10 -4 g/l at 20.0 + 0.5°C. Data Quality Reliable without restriction (Klimisch Code) References Determination of Water Solubility SafePharm Laboratories Project No.: 1666/013 (27 Jul 2004)5 

### Robust Summary 3 - WS - 2

CAS No. CAS# 68608-26-4 Test Substance Name Sulfonic acids, petroleum, sodium salts Remarks For more information on this chemical, see Section 2.0
“General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category. Method/Guideline Flask Method, Method A6 of Commission Directive
92/69/EEC and OECD Method 105 GLP (Y/N) Yes
Year 2004 Remarks for Test Conditions The solubility in water of a substance is specified by the
saturation mass concentration of the substance in water at a given temperature. A preliminary test was conducted by diluting 0.1305 g of the test material to 600 mL with glass double-distilled water. After shaking at 30 o C for 3 hours and standing at 20 o C for 3 hours, the solution was centrifuged at 13,500 rpm for 20 minutes and analyzed.

Based on the preliminary results and subsequent definitive test results, mixtures of double distilled water and test
substance were added to each of nine conical flasks. The flasks were shaken for approximately 24, 48, 72, 96, 120 and 144 hours at 30°C (Flasks 1-6) and for approximately 24, 28 and 72 hrs at 30°C (Flasks 7-9). After standing at 20°C for not less than 24 hours, the contents of each flask were centrifuged and/or filtered as follows: Flasks 1-6 were sampled, centrifuged at 13500 rpm for 20 minutes and the supernatant removed by pipettes. Flasks 7 to 9 were sampled and filtered through 0.2 um filters. The concentration of the test material in the sample solutions was determined in duplicate with duplicate injections of each sample by an
HPLC method with Mass Selective detection. Results Prior to sampling the samples contained excess test material and were clear and colorless. After sampling and treatment the samples were clear, colorless and visually free from
excess test material.

The water solubility of the test material was shown to
increase with time shaken at 30°C. It appeared that complete saturation of the test samples had not occurred. However the
test material contained mineral oil and this could have acted
as a co-solvent. There was also the possibility that with time shaken at 30°C, gradual emulsification of the test samples occurred causing an apparent increase in solubility. While the test samples were clear and colorless it is possible that micro-dispersions could have been present and undetected. Filtration of the samples had no effect on solubility. Based6 on these considerations it was determined that the water solubility should be expressed as an upper limit value (based on the sample taken at 144 hrs at 30°C) and that the results
should be considered as an overestimate of the water solubility. This was concluded since the experimental value was found to be higher than would be expected based onchemical structure and computer estimation. The computerbased estimate (WSKOWWIN, version 1.41 USEPA, 2000) was calculated to be 0.0144 mg/L at 25°C. This was based on a molecular weight of 432.64 for the total test material with approximately 330 of the molecular weight contributed from alkyl/aromatic groups which did not take into account any mineral oil or impurities in the test material.

The water solubility of the test material was determined to be less than or equal to 6.38 x 10 -3 g/l at 20.0 + 0.5°C.

The linearity of the detector response in respect to concentration was acceptable over a range of concentrations of 0 to 50 mg/L with a correlation coefficient of 0.998. Recovery analysis of the sample procedure was assessed and proved adequate for the test. Value (g/L) at temperature °C Less than or equal to 6.38 x 10 -3 g/l at 20.0 + 0.5°C Conclusions The solubility of the test material in double distilled water was found to be less than or equal to 6.38 x 10 -3 g/l at 20.0

+ 0.5°C. Data Quality Reliable with restriction (Klimisch Code). Restriction due to the fact that the water solubility was expressed as an upper limit value and that it was judged that the results should be considered as an overestimate of the water solubility. References Determination of Water Solubility SafePharm Laboratories Project No.: 1666/014 (04 Oct 2004)7 

### Robust Summary 3 – WS - 3

CAS No. CAS# 61790-48-5 Test Substance Name Sulfonic acids, petroleum, barium salts Remarks For more information on this chemical, see Section 2.0
“General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category. Method/Guideline Flask Method, Method A6 of Commission Directive
92/69/EEC and OECD Method 105 GLP (Y/N) Yes
Year 2004 Remarks for Test Conditions The solubility in water of a substance is specified by the
saturation mass concentration of the substance in water at a given temperature. A preliminary test was conducted by diluting 0.1325 g of the test material to 250 mL with glass double-distilled water. After shaking at 30 o C for 3 hours and standing at 20 o C for 20 hours, the solution was centrifuged at 13,500 rpm for 20 minutes and analyzed.

Based on the preliminary results, mixtures of double distilled
water and test substance were added to each of three conical flasks. The flasks were shaken for approximately 24, 48 and 72 hours (one flask/time period) at 100 rpm and 30°C. The flasks were then allowed to stand for approximately 24 hours at 20°C. The pH of each solution was measured. The concentration of the test material in the sample solutions was determined in duplicate with duplicate injections of each sample by an LCMS method with direct flow injection. Results The samples were clear and colorless and free of excess test material. Prior to treatment the samples were clear and
colorless with excess test material present at the bottom of the flasks. The linearity of the det ector response in respect to concentration was acceptable over a range of concentrations up to 20 mg/L with a correlation coefficient of 0.998. Recovery analysis of the sample procedure was assessed and proved adequate for the test.

The water solubility of the test material was determined to be
less than 1.03 x 10 -3 g/l at 20.0 + 0.5°C. A limit value was provided since it was evident from sample spectra that the chromatography peak did not relate to the test material itself but rather to low molecular weight, water soluble fractions or impurities from the test material.

Recovery analysis of the sample procedure were assessed and proved adequate for the test. Value (g/L) at temperature °C <1.03 x 10 -3 g/l at 20.0 + 0.5°C. Conclusions The solubility of the test material in double distilled water8 was found to be < 1.03 x 10 -3 g/l at 20.0 + 0.5°C. Data Quality Reliable without restriction (Klimisch Code) References Determination of Water Solubility SafePharm Laboratories Project No.: 1666/012 (10 Sep 2004)9 

### Robust Summary 3 – WS – 4

CAS No. CAS# 78330-12-8

Test Substance Name Benzenesulfonic aci d,C15-C30 alkyl. derivs., sodium salts Remarks For more information on this chemical, see Section 2.0
“General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category. Method/Guideline Flask Method, Method A6 of Commission Directive
92/69/EEC and OECD Method 105 GLP (Y/N) Yes
Year 2004 Remarks for Test Conditions The solubility in water of a substance is specified by the
saturation mass concentration of the substance in water at a given temperature. A preliminary test was conducted by diluting 0.0098 g of the test material to 250 mL with glass double-distilled water. After shaking at 30 o C for 3 hours and standing at 20 o C for 19 hours, the solution was centrifuged at 40,000 g for 20 minutes and analyzed.

Based on the preliminary results, mixtures of double distilled
water and test substance were added to each of three conical flasks. The flasks were shaken for approximately 24, 48 and 72 hours (one flask/time period) at 100 rpm and 30°C. The flasks were then allowed to stand for approximately 24 hours at 20°C. The contents of each flask was filtered through 0.2 μm filters and transferred to measuring cylinders. The pH
of each solution was measured. The concentration of the test material in the sample solutions was determined in duplicate with duplicate injections of each sample by an LCMS method with direct flow injection. Results The filtered samples were clear and colorless and free of
excess test material. Prior to filtration the samples were slightly hazy with excess test material dispersed and suspended in solution. Filtration recoveries showed that approximately 30% of the test material was either lost during analysis or absorbed by the filters. Therefore a recovery correction factor was used at the 10 mg/L nominal
concentration level.

The water solubility of the test material was determined to be
3.82 x 10 -2 g/l at 20.0 + 0.5°C.

Recovery analysis of the sample procedure were assessed and proved adequate for the test. Value (g/L) at temperature °C 3.82 x 10 -2 g/l at 20.0 + 0.5°C Conclusions The solubility of the test material in double distilled water
was found to be 3.82 x 10 -2 g/l at 20.0 + 0.5°C. Data Quality Reliable without restriction (Klimisch Code)10 References Determination of Water Solubility SafePharm Laboratories Project No.: 1666/030 (10 Sep 2004)11 

### Robust Summary 3 – WS - 5

CAS No. CAS# 61789-86-4 Test Substance Name Sulfonic acids, petroleum, calcium salts Remarks For more information on this chemical, see Section 2.0
“General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category. Method/Guideline Flask Method, Method A6 of Commission Directive
92/69/EEC and OECD Method 105 GLP (Y/N) Yes
Year 2004 Remarks for Test Conditions The solubility in water of a substance is specified by the
saturation mass concentration of the substance in water at a given temperature. A preliminary test was conducted by diluting 0.0511 g of the test material to 250 mL with glass double-distilled water. After shaking at 30 o C for 3 hours and standing at 20 o C for 18 hours, the solution was centrifuged at 13,500 rpm for 20 minutes and analyzed.

Based on the preliminary results, mixtures of double distilled
water and test substance were added to each of three sealed conical flasks. The flasks were shaken for approximately 24, 48 and 72 hours (one flask/time period) at 100 rpm and 30°C. The flasks were then allowed to stand for approximately 24
hours at 20°C. The contents of each flask was then centrifuged at 13,500 rpm for 20 minutes and transferred to measuring cylinders. The pH of each solution was measured. The concentration of the test material in the sample solutions was determined in duplicate with duplicate injections of each sample by an LC-MS method with direct flow injection. Results Prior to centrifuging the samples were clear and colorless with excess test material floating on the surface. Following centrifugation the supernatants were clear, colorless and free
from excess test material.

There was a spread of results for all samples ranging from approximately 2 x 10 -4 to 6 x 10 -4 g/L. Although this was a relatively wide range in percenta ge terms it is not in terms of concentration. Excluding one or two data points does not alter the overall result significantly. Given the method of analysis and the difficult nature of the test substance (low water soluble reaction product containing mineral oil) the
mean concentration was consid ered an appropriate expression of the test materials water solubility.

The water solubility of the test material was determined to be
4.02 x 10 -4 g/L at 20.0 + 0.5°C.12 The linearity of the detector response in respect to concentration was acceptable over a range of concentrations up to 10 mg/L with a correlation coefficient of 0.999. Recovery analysis of the sample procedure was assessed and proved adequate for the test. Value (g/L) at temperature °C 4.02 x 10 -4 g/L at 20.0 + 0.5°C Conclusions The solubility of the test material in double distilled water
was found to be 4.02 x 10 -4 g/L at 20.0 + 0.5°C. Data Quality Reliable without restriction (Klimisch Code) References Determination of Water Solubility SafePharm Laboratories Project No.: 1666/015 (10 Sep 2004)13 

### 2.2 Biodegradation

Robust Summary 3-Biodeg-1

Test Substance

CAS # 61789-86-4 Chemical Name Sulfonic acids, petroleum, calcium salts

Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline followed OECD 301F Test Type (aerobic/anaerobic) Aerobic GLP (Y/N) Y Year (Study Performed) 1996 Contact time (units) 28 days. Inoculum Activated sludge from domest ic wastewater treatment plant.

Remarks for test conditions Inoculum : The supernatant from the homogenized activated sludge was used as inoculum. The inoculum was pre-adapted to the test material for 14 days during which the test substance was added incrementally at concentrations equivalent to 4, 8, and 8 mg carbon/L on days 0, 7, and 12, respectively. The targeted microbial level in the test mixture was 10,000 to 1,000,000 cells/mL.

Concentration of test chemical : Test substance concentration was approximately 100 mg/L, giving at least 50 to 100 mg ThOD per L test medium. No organic solvents were used to facilitate the dispersion of the test material. The test substance was weighed onto a teflon coupon
and introduced into the medium.

Temp of incubation : 23 + 1oC

Dosing procedure : A measured volume of the inoculated mineral medium containing approximately 100 mg/L test substance was continuously stirred in a closed system for 28 days.

Sampling frequency : The oxygen uptake was monitored continuously and recorded every 4 hours throughout the test.

Controls : Yes (blank and positive controls per guideline); abiotic and toxicity checks were not included. Sodium benzoate was used as the positive control.

Analytical method : Oxygen uptake was measured using a BI-1000 electrolytic respirometer system.14 Method of calculating measured concentrations : N/A

Other: The inoculum was pre-adapted to the test substance for 14 days.

Results Degradation % after time 8.6% after 28 days

Kinetic (for sample, positive and negative controls) Reference (sodium benzoate) – >60% (3d) Test substance – 9.0% (28d) Breakdown Products (Y/N) If yes describe breakdown products N

Remarks
Conclusions 8.6% in 28 days. The reference s ubstance, sodium be nzoate, reached a level of 88.8% in the same test period. Data Quality (1) Reliable without restriction

References This robust summary was prepared from an unpublished study by an individual member company of the HERTG (the underlying study contains confidential business information).

Other Updated: 9-6-0015 

### Robust Summary 3-Biodeg-2

Test Substance

CAS # 71486-79-8 Chemical Name Benzensulfonic acid, mono-C15-30-branched alkl and di-C11-13- branched and linear alkylderivs., calcium salts, overbased Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline followed OECD 301F Test Type (aerobic/anaerobic) Aerobic GLP (Y/N) Y Year (Study Performed) 1997 Contact time (units) 28 days Inoculum Activated sludge from domest ic wastewater treatment plant.

Remarks for test conditions Inoculum : The supernatant from the homogenized activated sludge was used as inoculum. The inoculum was pre-adapted to the test material for 14 days during which the test substance was added incrementally at concentrations equivalent to 4, 4, and 8 mg carbon/L on days 0, 7, and 12, respectively. The targeted microbial level in the test mixture was 10,000 to 1,000,000 cells/mL.

Concentration of test chemical : Test substance concentration was approximately 100 mg/L, giving at least 50 to 100 mg ThOD per L test medium. No organic solvents were used to facilitate the dispersion of the test material. The test substance was weighed onto a teflon coupon
and introduced into the medium.

Temp of incubation : 23 + 1oC

Dosing procedure : A measured volume of the inoculated mineral medium containing approximately 100 mg/L test substance was continuously stirred in a closed system for 28 days.

Sampling frequency : The oxygen uptake was monitored continuously and recorded every 4 hours throughout the test.

Controls : Yes (blank and positive controls per guideline); abiotic and toxicity checks were not included. Sodium benzoate was used as the positive control.

Analytical method : Oxygen uptake was measured using a BI-1000 electrolytic respirometer system.

Method of calculating measured concentrations : N/A16 Other: The inoculum was pre-adapted to the test substance for 14 days.

Results Degradation % after time 8.6% after 28 days. Kinetics (for sample, positive and negative controls) Positive control substance (sodi um benzoate): >60% (3d) Test substance: 8.6%% (28d) Breakdown Products (Y/N) If yes describe breakdown products N

Remarks
Conclusions 8.6% in 28 days. Data Quality (1) Reliable without restriction

References This robust summary was prepared from an unpublished study by an individual member company of the HERTG (the underlying study contains confidential business information).

Other Updated: 9-6-0017 Robust Summary 3- Biodeg-3
Test Substance CAS # Analog of 71786-47-5 Chemical Name Magnesium long chain alkaryl sulfonate Remarks This substance is an analog for the group of substances referred to as alkaryl magnesium salt derivative , in HERTG’s Final Submission for the Alkaryl Sulfonates Category. It is chemically similar to members of this CAS number, but does not belong to this CAS number. For more information on the chemicals, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.
Method Method/Guideline Followed OECD 301B, Ready Biodegradability, Modified Sturm Test Test Type (aerobic/anaerobic) Aerobic GLP (Y/N) Y Year (study performed) 1995 Contact time (units) 28 days Inoculum (source) Domestic activated sewage sludge
Remarks For Test Conditions Inoculum: Activated sewage sludge from domestic WWTP prepared per test guideline. Inoculum was not acclimated.

Replicates: Triplicates for test substance, positive control material, and
control blank.

Temperature of incubation: 20 – 23 °C

Dosing procedure: Neat test chemical was gravametrically determined on glass cover slips, which were then added to culture medium in test
vessels.

Sampling: Days 2, 4, 7, 10, 14, 17, 21, 24, 29 (after acidification on day 28)

Concentration of test substance: Loading into each of 3 test vessels were 19.9, 20.1, and 20.0 mg C/L.

Controls: Blank and positive contro ls used per guideline; toxicity control not used. Positive control was benzoic acid (Na salt) added to each control vessel at a loading of 20.2 mg C/L.

Analytical method: Titration of residual Ba(OH)2 (0.05 N initially) in trapping solution, using 0.1N HCl.

Method of calculating biodegradati on values: Percent biodegradation calculated as percent ratio of cumulative net carbon dioxide to theoretical carbon dioxide as determined from elemental analysis of
test material.18 Results Degradation % After Time Test substance: 1.5% in 28 days Positive control substance: 89.2% in 28 days Kinetics (for sample, positive and negative controls) Positive control t 1/2 : <10 days

Breakdown Products (Y/N) (if yes describe breakdown products) N

Conclusions 1.5% in 28 days Data Quality (1) Reliable without restriction References This robust summary was prepared from an unpublished study by an individual member company of the HERTG (the underlying study contains confidential business information).
Other Updated: 9-6-0019 

### Robust Summary 3- Biodeg-4
Test Substance

CAS # Analog of 70024-69-0 Chemical Name Benzenesulfonic acid, mono-C16-C24-alkyl derivatives, calcium salts

Remarks This substance is an analog fo r the group of substances in HERTG’s Final Submission for the Alkaryl Sulfonates Category. It is chemically similar to members of this CAS number, but does not belong to this CAS number. For more information on this chemical, see Section 2.0
“General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.
Method
Method/Guideline followed Closed bottle test according to OECD Guideline No. 301D, EEC Directive 79/831 and EEC Directive 67/548 Annex V C.6 as published
in 84/499/EEC. Test Type (aerobic/anaerobic) Aerobic GLP (Y/N) Y Year (Study Performed) 1989 Contact time (units) 28 days Inoculum Domestic activated sewage sludge

Remarks for test conditions Inoculum: Activated sludge bacteria from dom estic sewage treatment plant used at about 1 drop sludge filtrate inoculum/L basal medium.

Concentration of test chemical: 2 mg/L. Inoculum was not preacclimated to test substance. Two replicates run per treatment.

Temperature of incubation: 20 ± 1 °C

Dosing procedure: Test chemical was added onto Whatman GFA filter paper that were then placed inside test vessels immediately before
culture medium was added to the vessels.

Sampling: Days 0, 5, 15, and 28 after inoculation.

Controls: Yes (blank and positive contro ls used per guideline); toxicity
control not used. Standard Control Substances: Sodium benzoate and Aniline tested at 2 mg/L.

Analytical method: Chemical oxygen demand (COD) of the test
substance and standard control subs tances determined using the Hach semi-micro sample digestion methods followed by direct reading of the CODs using a Hach DR/2 Spectrophotometer. During the biodegradability test dissolved oxygen concentrations for each test medium were determined in duplicate using a Yellow Springs BOD
Probe.20 Remarks for test conditions,
cont’d Inoculum: Sludge from domestic WWTP used at 10 mL/L basal
medium

Conc of test chemical: Test chemical added directly to test vessels at 13.3 mg C/L (28.6 mg/L CAS# 68511-50-2). No preacclimation was
used.

Temp of incubation: 23 – 24 °C Dosing procedure: Neat test chemical added by micropipettor to culture medium in vessels immediately prior to addition of sewage and
soil inocula

Sampling: Days 1, 3, 6, 10, 14, 21, 29 (after acidification on d 28)

Controls: Yes (blank and positive contro ls used per guideline); toxicity control not used. Positive Control was Benzoic acid (Na salt) at 20 mg
C/L

Analytical method: Titration of residual Ba(OH)2 in trapping solution, using HCl

Method of calculating measured c oncentrations: The oxygen depletion values for the test substance and standard substances at each sampling time are corrected by means of the blank values and expressed as a percentage of the theoretical oxygen demand or chemical oxygen demand determined by the Hach semi -micro sample digestion method.

Other:

• Biodegradation of the Standard Control Substances: Sodium
benzoate and Aniline attained 97% and 61% degradation within 28 days. Because both standard substances achieved greater or equal to 60% degradation the test was deemed valid.

• Two replicates were run per treatment; values are average of replicates. The % biodegradation value reported is slightly inflated by the use of zero titration volume rather than negative volume when corrected for blanks; however, comparison of titration volumes for the test chemical and blank show them to be very sim ilar, so inhibition of inoculum is not suspected.
Results Degradation % after time Test Substance degraded 8.0% by day 28. Kinetic (for sample, positive and negative controls) None given

Breakdown Products (Y/N) If yes describe breakdown products NA21 Conclusions 8.0% Not Readily biodegradable; biodegradation was essentially zero Data Quality (1) Reliable without restriction References Douglas, M.T. (1989) The Ready Biodegradability of Analog of CAS# 70024-69-0 in a Closed bottle Test System, Huntington Research Centre, Ltd., Study #30/891706.
Other Updated: 9-6-0022 

### Robust Summary 3-Biodeg-5
Test Substance CAS # Analog for 68783-96-0 Chemical Name Calcium alkaryl sulfonate Remarks This substance is an analog for the group of substances referred to as petroleum derived calcium salt, overbased , in HERTG’s Test Plan for the Alkaryl Sulfonates Category. It is chemically similar to members of this CAS number, but does not belong to this CAS number. For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.
Method Method/Guideline Followed OECD 301B, Ready Biodegradability, Modified Sturm Test Test Type (aerobic/anaerobic) Aerobic GLP (Y/N) Y Year (study performed) 1995 Contact time (units) 28 days Inoculum (source) Domestic activated sewage sludge
Remarks For Test Conditions Inoculum: Activated sewage sludge from domestic WWTP prepared per test guideline. Inoculum was not acclimated.

Replicates: Triplicates for test substance, positive control material, and
control blank.

Temperature of incubation: 20 – 23 °C

Dosing procedure: Neat test chemical was gravametrically determined on glass cover slips, which were then added to culture medium in test
vessels.

Sampling: Days 1, 3, 5, 7, 10, 13, 17, 20, 24, 29 (after acidification on day 28)

Concentration of test substance: Loadings into 3 test vessels were 19.8, 20.1, and 19.4 mg C/L.

Controls: Blank and positive contro ls used per guideline; toxicity control not used. Positive control was benzoic acid (Na salt) added to each control vessel at a loading of 21.2 mg C/L.

Analytical method: Titration of residual Ba(OH)2 (0.05 N initially) in trapping solution, using 0.1N HCl.

Method of calculating biodegradati on values: Percent biodegradation calculated as percent ratio of cumulative net carbon dioxide to theoretical carbon dioxide as determined from elemental analysis of
test material.23 Results Degradation % After Time Test substance: 9.1% in 28 days Positive control substance: 86.1% in 28 days Kinetics (for sample, positive and negative controls) Positive control t 1/2 : <10 days

Breakdown Products (Y/N) (if yes describe breakdown products) N

Conclusions 9.1% in 28 days Data Quality (1) Reliable without restriction References This robust summary was prepared from an unpublished study by an individual member company of the HERTG (the underlying study contains confidential business information).
Other Updated: 9-6-0024 

### 3. Ecotoxicity Category: Alkaryl Sulfonates

AQUATIC ORGANISMS

3.1 Acute Toxicity to Fish

Robust Summary 3-Fish -1

Test Substance
CAS # 61789-86-4 Chemical Name Sulfonic acids, petroleum, Calcium salts Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed Test protocol followed OECD Guideline for Testing of Chemicals #203, Fish Acute Toxicity Test (1984). Test Type Static renewal test; a one level screening test GLP (Y/N) Y Year (Study Performed) 1986 Species/Strain Sheepshead minnow ( Cyprinodon variegatus ) Analytical Monitoring Total organic carbon (T OC) measurements of each freshly prepared test solution and control and after 24-h on test just before daily
renewal with fresh test solution. Exposure Period (unit) 96 hours Statistical methods Statistical analysis of su rvival data not warranted because there was no mortality in the study.
Remarks field for test conditions (fill as applicable) Test Organisms: source – a commercial supplier in New Hampshire, age – 14 days old, total length – 12 mm average (range 10 to 15 mm; n =30), wet weight – 0.040 g average (range 0.004 to 0.11 g; n = 30). Loading – 0.080 g biomass/L, Pretreatment – none, fish held for a minimum of 7 days before testing. No feeding during the test.

Test System: Individual WAFs (individual water accommodated fractions) 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 accomplished using a Telfon coated magnetic stir bar. Mixing speed adjusted such that a vortex formed between 30 to 50% of the distance to the bottom. Following the mixing period, the test solution was allowed to stand for 2 hours25 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 t est substance with the lower end approximately midway between bottom and surface. The siphoned water phase, designated WAF, 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 per treatment, 10 fish per replicate (20 per treatment). Test vessels were loosely covered to reduce entry of dust.

Dilution Water: Natural seawater collected from Cape Cod Canal, Bourne, Massachusetts. The water was filtered through 0.5-micron polypropylene core filter and activated carbon, then stored for 1 to 4 days prior to use while being constantly aerated. During storage the water had a salinity of 32 to 34 ppt and pH of 7.7 to 7.8. During the test: dissolved oxygen – 6.3 mg/L to above 100% saturation (7.2 mg/L), pH – 7.8 to 8.1, salinity – 32 to 34, temperature – 22 to 23 C. Mean measured TOC levels in the control and 10,000 mg/L WAF test level were 4.1 mg/L (range 2.3 to 7.9) and 10.2 mg/L (range 6.4 to 15.0), respectively

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

Test Findings: No mortality or signs of toxicity were noted in the 10,000 WAF test level and the control throughout the entire test.

Calculation of LL 50 s: Statistical analysis of survival data not
warranted.

Test Substance: No undissolved test material was report on the surface of the test vessels during the entire aquatic toxicity test.

Reference Substance: Sodium lauryl sulfate (SLS). The 96-h LC 50 was 1.2 mg/L. No information provided on method of calculation.

Results Nominal concentrations: 96-h LL 50 >10,000 mg/L. This is equivalent
to 96-h LL 0 = 10,000 mg/L (no mortality or toxic signs noted).
Remarks Measured concentration: n/a

Unit: mg/L

Statistical results: Statistical analysis of survival data not warranted.

Other:

• Test results reported in original study as “lethal concentrations”26 are reported in this summary as “lethal loading”, because test results are based on WAF loading rates.

• Control response was satisfactory.
Conclusions No mortality or signs of toxicity were noted in the 10,000 WAF test level and the control throughout the entire test. Data Quality (1) Reliable without restriction References Nicholson, R.B. (1986) Acute toxicity of CMA Test Material Code 504 to Sheepshead Minnow, Cyprinodon variegatus. Springborn Bionomics Study #10823-0186-6100-500-504, Report #BW-86-04-
1983.
Other Updated: 9-6-0027 Robust Summary 3-Fish - 2
Test Substance CAS # analog for Analog of CAS # 70024-69-0 (material tested = CAS #70024-71-4) Chemical Name analog to benzenesulfonic acid, mono-C16–C24 alkyl derivatives, calcium salts, overbased
Remarks The tested substance is an anal og for the group of substances referred to as C16-C24 alkyl calcium salt overbased derivative in the HERTG’s Final Submission for Alkaryl Sulfonates Category. It is chemically similar to members of this CAS number, but does not belong to this CAS number. For more information on this chemical, see Section 2.0
“General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed Test protocol followed OECD Guideline for Testing of Chemicals #203, Fish Acute Toxicity Test (1984). Test Type Static renewal test; a one level screening test GLP (Y/N) Y Year (Study Performed) 1986 Species/Strain Sheepshead minnow ( Cyprinodon variegatus ) Analytical Monitoring Total organic carbon (T OC) measurements of each freshly prepared test solution and control and after 24-h on test just before daily
renewal with fresh test solution. Exposure Period (unit) 96 hours Statistical methods Statistical analysis of su rvival data not warranted because there was no mortality in the study.
Remarks field for test conditions (fill as applicable) Test Organisms: source – a commercial supplier in New Hampshire, age – 10 to 15 days old, total length – 11 mm average (range 10 to 13 mm; n =30), wet weight – 0.028 g average (range 0.02 to 0.05 g; n = 30). Loading – 0.056 g biomass/L, Pretreatment – none, fish held for a minimum of 6 days before testing. No feeding during the test.

Test System: Individual water accommodated fractions (WAFs) 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 accomplished using a Telfon coated magnetic stir bar. Mixing speed adjusted such that a vortex formed between 30 to 50% of the distance to the bottom. Following the mixing period, the test solution
were allowed to stand for 2 hours be fore the water phase was removed. To avoid removing test material from the surface or bottom, a siphon was placed in the mixing vessel prior to addition of water and test substance with the lower end approximately midway between bottom and surface. The siphoned water phase, designated water accommodated fraction (WAF), was used for the aquatic toxicity test.28 About 90% of the test solution in each test vessel was renewed daily after 24, 48, and 72 hours. Two 5-L replicates per treatment, 10 fish per replicate (20 per treatment). Test vessels were loosely covered to reduce entry of dust. Dilution Water: Natural seawater collected from Cape Cod Canal, Bourne, Massachusetts. The water was filtered through 0.5-micron polypropylene core filter and activated carbon, then stored for 1 to 4 days prior to use while being constantly aerated. During storage the water had a salinity of 32 to 34 ppt and pH of 7.7 to 7.8. During the test: dissolved oxygen – 5.5 mg/L to above 100% saturation (7.2 mg/L), pH – 7.9 to 8.1, salinity – 32 to 34, temperature – 22 to 23 C. Mean measured TOC levels in the control and 10,000 mg/L WAF test level were 3.0 mg/L (range 1.2 to 4. 4) and 6.4 mg/L (range 5.0 to 7.9), respectively.

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

Test Findings: No mortality or signs of toxicity were noted in the 10,000 WAF test level and the control throughout the entire test.

Calculation of LL 50 s: Statistical analysis of survival data not warranted because there was no mortality in the study.

Test Substance: No undissolved test material was report on the surface of the test vessels during the entire aquatic toxicity test.

Reference Substance: Sodium lauryl sulfate (SLS). The 96-h LC 50 was 1.2 mg/L. No information provided on method of calculation.

Results Nominal concentrations: 96-h LL 50 >10,000 mg/L. This is equivalent
to 96-h LL 0 = 10,000 mg/L (no mortality or toxic signs noted).
Remarks Measured concentration: n/a

Unit: mg/L

Statistical results: Statistical analysis of survival data not warranted.

Other:

• Test results reported in original study as “lethal concentrations” are reported in this summary as “lethal loading”, because test results are based on WAF loading rates.

• Control response was satisfactory.29 Conclusions No mortality or signs of toxicity were noted in the 10,000 WAF test level and the control throughout the entire test. Data Quality (1) Reliable without restriction References Nicholson, R.B. (1986) Acute toxicity of CMA Test Material Code 514 to Sheepshead Minnow, Cyprinodon variegatus. Springborn Bionomics Study #10823-0186-6100-500-514, Report #BW-86-04-
1993.
Other Updated: 9-6-0030 Robust Summary 3-Fish - 3
Test Substance
CAS # 71486-79-8 Chemical Name Benzenesulfonic acid, mo no-C15-30-branched alkyl and di-C11-13- branched and linear alkyl deri vatives, calcium salts, overbased Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed Test protocol followed US EPA Toxic Substances Control Act Test Guideline #797.1400 (1985), OECD Guideline for Testing of Chemicals #203, Fish Acute Toxicity Test (1984).

Test Type Static renewal test
GLP (Y/N) Y Year (Study Performed) 1983 Species/Strain Fathead minnow ( Pimephales promelas ) Analytical Monitoring Total organic carbon (TOC) measurements of initial (0-h) test solutions and after one day on test ( 24-h) before renewal of fresh test solutions. Water samples were passed through 0.45-micron filter prior to TOC analysis using EPA Method 415.1 Exposure Period (unit) 96 hours Statistical methods Statistical analysis of su rvival data not warranted because there was no mortality in the study.
Remarks field for test conditions (fill as applicable) Test Organisms: Acquired from Aquatic Research Organisms, Hampton, New Hampshire, age: juve nile, total length: 29 mm average (longest fish not more than twice the shortest fish), wet weight: 0.2 g average (no range reported). Loading: <0.5 g biomass/L, Pretreatment: none, fish held for a minimum of 14 days before testing. No feeding during the test.

Test System: Individual water accommodated fractions (WAFs) were prepared for each test level and renewed daily. A measured weight of test material was added to a measured volume of dilution water (30-L) in a glass vessel and stirred for 24 hours. Stirring accomplished using a Telfon coated magnetic stir bar. Mi xing speed adjusted such that a
vortex formed between 30 to 50% of the distance to the bottom. Following the mixing period, the test solutions were allowed to stand for 1 hour before the water phase was removed. To avoid removing test material from the surface or bottom, a siphon was placed in the mixing vessel prior to addition of water and test substance with the lower end 1-2 inches off the bottom. The siphoned water phase (i.e., WAF) was used in the aquatic toxicity test. About 80% of the solution in each test level was renewed daily after 24, 48, and 72 hours. Two 15-L replicates per treatment, 10 fish per replicate (20 per treatment).31 Test vessels loosely covered to reduce entry of dust.

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

Light: 16-h light per day using c ool-white fluorescent lights with an intensity of 20 uEin/m 2

.

Test Temperature: 21.4 to 22.8 C.

Water Chemistry: Dissolved oxygen: 7.3 – 8.6 mg/L, pH: 7.4 - 8.1, conductivity: 860 – 910 umhos/cm. Alkalinity not reported.

Element: Mortality

Test Levels: Control, 100, 300, & 1,000 mg/L WAF loading rates. No
undissolved test material was seen on the surface of the test vessels during the entire aquatic toxicity test.

Test Findings: No mortality or signs of toxicity was observed in all treatments and the control throughout the entire test. No undissolved
test material was seen on the su rface of the test vessels during the entire aquatic toxicity test.

Analytical Monitoring: TOC levels were between 2.3 - 3.0 mg/L in the control, 2.8 - 3.2 mg/L at 100 mg/L loading, between 2.6 - 3.2 mg/L at 300 mg/L loading and 2.6 - 3.3 mg/L at the 1,000 mg/L loading. TOC
levels were not considered to be indicative of actual test material
concentrations and results are th erefore based on nominal loading

rates.

Reference Substance: No
Results Nominal concentrations: 96-h LL 50 >1,000 mg/L. This is equivalent
to 96-h LL 0 = 1,000 mg/L (no mortality or toxic signs noted).
Remarks Measured concentration: n/a

Unit: mg/L

LC50, LC0, LL50 or LL0 at 48, 72, 96-hours: LL 50 and LL 0 reported32 as LC 50 and NOEC, respectively, although test results were based on WAF loading rate.

Statistical results: Statistical analysis of survival data not warranted.

Other:

• Test results reported in original study as “lethal concentrations” are reported in this summary as “lethal loading”, because test results are based on WAF loading rates.

• Control response was satisfactory.
Conclusions No mortality or signs of toxicity were observed in any of the treatments (100, 300, and 1,000 mg/L WAF loading rates) or in the control throughout the entire test. Data Quality (1) Reliable without restriction References Ward, T.J. (1993) Acute Toxicity of The Water Accommodated Fractions (WAFs) of CMA 605 to The Fathead Minnow, Pimephales promelas. T.R. Wilbury Study #9176-CMA/ESI-605.
Other Updated: 9-6-0033 Robust Summary 3-Fish - 4
Test Substance
CAS # 71786-47-5 Chemical Name Benzenesulfonic acid, mono and dialkyl derivatives, magnesium salts Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed Test protocol followed US EPA Toxic Substances Control Act Test Guideline #797.1400 (1985), OECD Guideline for Testing of Chemicals #203, Fish Acute Toxicity Test (1984). Test Type Static renewal test
GLP (Y/N) Y Year (Study Performed) 1983 Species/Strain Fathead minnow ( Pimephales promelas ) Analytical Monitoring Total organic carbon (TOC) measurements of initial (0-h) test solutions and after one day on test ( 24-h) before renewal of fresh test solutions. Water samples were passed through 0.45-micron filter prior to TOC analysis using EPA Method 415.1 Exposure Period (unit) 96 hours Statistical methods Statistical analysis of su rvival data not warranted because there was no mortality in this study.
Remarks field for test conditions (fill as applicable) Test Organisms: Acquired from Aquatic Research Organisms, Hampton, New Hampshire, age: juvenile, total length: 38.4 mm average (longest fish not more than twice the shortest fish), wet weight: 0.5 g average (no range reported). Loading: <0.5 g biomass/L, Pretreatment: none, fish held for a minimum of 14 days before testing. No feeding during the test.

Test System: Individual water accommodated fractions (WAFs) were prepared for each test level and renewed daily. A measured weight of test material was added to a measured volume of dilution water (30-L) in a glass vessel and stirred for 24 hours. Stirring accomplished using a Telfon coated magnetic stir bar. Mi xing speed adjusted such that a
vortex formed between 30 to 50% of the distance to the bottom. Following the mixing period, the test solutions were allowed to stand for 1 hour before the water phase was removed. To avoid removing test material from the surface or bottom, a siphon was placed in the mixing vessel prior to addition of water and test substance with the lower end 1-2 inches off the bottom. The siphoned water phase (i.e., WAF) was used in the aquatic toxicity test. About 80% of the solution in each test level was renewed daily after 24, 48, and 72 hours. Two 15-L replicates per treatment, 10 fish per replicate (20 per treatment). Test vessels loosely covered to reduce entry of dust.34 Dilution Water: Filtered well water collected at Hampton, New Hampshire and adjusted to the appropriate hardness 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.

Light: 16-h light per day using c ool-white fluorescent lights with an intensity of 20 uEin/m 2

.

Test Temperature: 21.6 to 22.8 C.

Water Chemistry: Dissolved oxygen: 6.9 – 8.3 mg/L, pH: 7.0 - 7.9, conductivity: 870 – 890 umhos/cm. Alkalinity not reported.

Element: Mortality

Test Levels: Control, 100, 300, & 1,000 mg/L WAF loading rates. No
undissolved test material was seen on the surface of the test vessels during the entire aquatic toxicity test.

Test Findings: No mortality or signs of toxicity was observed in all treatments and the control throughout the entire test.

Calculation of LL 50 s: Statistical analysis of survival data not
warranted.

Analytical Monitoring: TOC levels were between 2.8 - 3.2 mg/L in the control, 3.3 - 3.8 mg/L at 100 mg/L loading, between 3.1 - 4.0 mg/L at 300 mg/L loading and 3.2 - 4.4 mg/L at the 1,000 mg/L loading. TOC
levels were not considered to be indicative of actual test material
concentrations and results are th erefore based on nominal loading

rates.

Reference Substance: No
Results Nominal concentrations: 96-h LL 50 >1,000 mg/L. This is equivalent
to 96-h LL 0 = 1,000 mg/L (no mortality or toxic signs noted).
Remarks Measured concentration: n/a

Unit: mg/L

Statistical results: Statistical analysis of survival data not warranted.35 Other:

• Test results reported in original study as “lethal concentrations” are reported in this summary as “lethal loading”, because test results are based on WAF loading rates.

• Control response was satisfactory.
Conclusions No mortality or signs of toxicity were observed in any of the treatments (100, 300, and 1,000 mg/L WAF loading rates) or in the control throughout the entire test. Data Quality (1) Reliable without restriction References Ward, T.J. (1993) Acute Toxicity of The Water Accommodated Fractions (WAFs) of CMA 609 to The Fathead Minnow, Pimephales promelas. T.R. Wilbury Study #9176-CMA/ESI-609.
Other Updated: 9-6-0036 Robust Summary 3-Fish - 5
Test Substance
CAS # 71786-47-5 Chemical Name Benzenesulfonic acid, mono and dialkyl derivatives, Magnesium salts Remarks This substance is referred to as alkaryl magnesium salt derivative in the HERTG’s Test Plan for Alkaryl Sulfonates Category. For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed Test protocol followed OECD Guideline for Testing of Chemicals #203, Fish Acute Toxicity Test (1984). Test Type static renewal test; a one level screening test GLP (Y/N) Y Year (Study Performed) 1986 Species/Strain Sheepshead minnow ( Cyprinodon variegatus ) Analytical Monitoring Total organic carbon (T OC) measurements of each freshly prepared test solution and control and after 24-h on test just before daily
renewal with fresh test solution. Exposure Period (unit) 96 hours Statistical methods Statistical analysis of su rvival data not warranted because there was no mortality in this study.
Remarks field for test conditions (fill as applicable) Test Organisms: source – a commercial supplier in New Hampshire, age – 17 to 22 days old, total length – 11 mm average (range 10 to 13 mm; n =30), wet weight – 0.028 g average (range 0.02 to 0.05 g; n = 30). Loading - 0.056 g biomass/L, Pretreatment – none, fish held for a minimum of 20 days before testing. No feeding during the test.

Test System: Individual water accommodated fractions (WAFs) 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 accomplished using a Telfon coated magnetic stir bar. Mixing speed adjusted such that a vortex formed between 30 to 50% of the distance to the bottom. Following the mixing period, the test solution
were allowed to stand for 2 hours be fore the water phase was removed. To avoid removing test material from the surface or bottom, a siphon was placed in the mixing vessel prior to addition of water and test substance with the lower end approximately midway between bottom and surface. The siphoned water phase, designated WAF 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 per treatment, 10 fish per replicate (20 per treatment). Test vessels were loosely covered to reduce entry of dust.37 Dilution Water: Natural seawater collected from Cape Cod Canal, Bourne, Massachusetts. The water was filtered through 0.5-micron polypropylene core filter and activated carbon, then stored for 1 to 4 days prior to use while being constantly aerated. During storage the water had a salinity of 32 to 34 ppt and pH of 7.7 to 7.8. During the test: dissolved oxygen – 4.9 mg/L to above 100% saturation (7.4 mg/L), pH – 7.6 to 8.1, salinity – 32 ppt, temperature – 22 to 23 C. Mean measured TOC levels in the control and 1,000 mg/L WAF test level were 5.0 mg/L (range 2.6 to 7. 0) and 3.6 mg/L (range 1.0 to 7.5), respectively.

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

Test Findings: No mortality or signs of toxicity were noted in the 10,000 WAF test level and the control throughout the entire test.

Analytical Monitoring: ). Mean measured TOC in the 10,000 mg/L WAF test level was 5.0 mg/L compared to 3.6 mg/L in the control.

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

Reference Substance: Sodium lauryl sulfate (SLS). The 96-h LC 50 was 1.2 mg/L. No information provided on method of calculation.

Results Nominal concentrations: 96-h LL 50 >10,000 mg/L. This is equivalent
to 96-h LL 0 = 10,000 mg/L (no mortality or toxic signs noted).
Remarks Measured concentration: n/a

Unit: mg/L

Statistical results: Statistical analysis of survival data not warranted because there was no mortality in this study.

Other:

• Test results reported in original study as “lethal concentrations” are reported in this summary as “lethal loading”, because test results are based on WAF loading rates.

• Control response was satisfactory.38 Conclusions No mortality or signs of toxicity were noted in the 10,000 WAF test level and the control throughout the entire test. Data Quality (1) Reliable without restriction References Nicholson, R.B. (1986) Acute toxicity of CMA Test Material Code 523 to Sheepshead Minnow, Cyprinodon variegatus. Springborn Bionomics Study #10823-0186-6100-500-523, Report #BW-86-04-
1986.
Other Updated: 9-6-0039 

### Robust Summary 3-Fish - 6
Test Substance
CAS # 115733-09-0 Chemical Name Benzenesulfonic acid, C14-C24 branched & linear alkyl derivatives. Calcium
salts Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed OECD Guideline for Testing of Chem icals #203 Fish Acute Toxicity Test

Test Type Semi-Static acute toxicity test (renewal) GLP (Y/N) Y Year (Study Performed) 2004 Species/Strain Oncorhynchus mykiss Analytical Monitoring Not performed. Exposure Period (unit) 96 hours Statistical methods An estimate of the lethal loading rates (LL 50 ) was determined by inspection.

Remarks field for test conditions (fill as applicable) Fingerlings were obtained from a commercial breeder and were acclimated for 13 days. The fish had a mean length of 4.7 cm and a mean weight of 1.54 g at
the end of the definitive test. Individual test concentrations were prepared for each test level. A measured
volume of test material was added to a measured volume of dilution water and continuously stirred for 24 hours. Fo llowing settling for 1 hour the aqueous phase or WAF (water accommodated fraction) was removed by mid-depth siphoning. A sealed 96 hours semi-static test was carried out with daily renewal of the test WAF’s. Duplicate 20-liter glass exposure vessels were filed with the WAF. A separate chamber served as the control. Ten fish were placed in each chamber
and the chambers were c overed to reduce evaporation. The chambers were aerated. The fish were not fed during the study.

The fish were observed for toxicity at 3, 24, 48, 72 and 96 hours. At 24, 48
and 72 hours the fish were transferred to fresh WAFs or control water.

Dissolved oxygen, water temperature a nd pH were determined throughout the study. Vortex depth was recorded at the start and end of each mixing period.40 Test Concentrations (Nominal) 100mg/L (Water Accommodated Fraction-WAF) test concentration was selected based on a range-finding study.
Results The 96-hour LL 50 (loading level likely to cause 50% mortality) was >100 mg/L WAF. The No Observed Effect Loading rate was 100 mg/L. Remarks In the range find study no mortality or sublethal effects were observed at 10 or 100 mg/L. During the main study, no toxicity or mortality was observed at 100 mg/L (WAF). The LL 50 s (loading levels likely to cause 50% mortality)
were as follows:
Time (hours) LL 50 (mg/L) 95% Confidence Limits (mg/L)
3 >100 -
6 >100 -
24 >100 -
48 >100 -
72 >100 -
96 >100 - Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test material to be present. Water chemistry: Temperature: 12.7-14.3 o C; Dissolved Oxygen: 10.0-10.3 mg/L; pH: 7.7-8.2.
Conclusions The 96 hour LL 50 (loading level likely to cause 50% mortality) was >100 mg/L WAF. The No Observed Effect Loading rate was 100 mg/L. Data Quality Reliable with restriction (Klimisch Code). Restriction due to the lack of analytical confirmation of test concentrations. References Acute Toxicity to Rainbow Trout SafePharm Study Number: 1666/066 (03 Mar 2005)
Other Updated: 3/22/200541 

### Robust Summary 3-Fish - 7
Test Substance
CAS # 68608-26-4 Chemical Name Sulfonic acids, petroleum, sodium salts Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed OECD 203

Test Type Acute Toxicity to Fish (Water Accommodated Fraction-WAF) GLP (Y/N) Y Year (Study Performed) 2005 Species/Strain Rainbow Trout ( Oncorhynchus mykiss ) Fingerlings were obtained from a commercial breeder. Fish Size Average length 5.5 cm and a mean weight of 1.76 g, loading rate of 0.88 g body weight/L Number of Fish Range Find: 3/concentration Definitive Study: 20/concentration (10/replicate) Analytical Monitoring No

Nominal Test Substance
Concentration Levels Range Find Study A: 0, 10, 100 mg/L WAF Range Find Study B: 0, 32, 320 mg/L WAF Range Find Study C: 0, 10, 100 mg/L WAF Definitive Study: 0, 100 mg/L WAF
Test Concentration Preparation Individual test concentrations were prepared for each test level. A
measured volume of test material was added to a measured volume of
dilution water and continuo usly stirred for 24 hours. Following settling for 1 hour the aqueous phase or WAF (water accommodated fraction) was removed by mid-depth siphoning. Exposure Period 96 hours Exposure Conditions Static-renewal test conditions. The test prep arations were renewed daily.
Vehicle None Statistical Analysis LL 50 values determined by inspection. Dose Rangefinding Study Yes Test Chambers Covered, 20-liter glass aquaria containing the test solution Diluent Water Dechlorinated, softened water Diluent Water Chemistry Hardness 100 mg/l as CaCO 3 pH 7.6-8.5. Diluent Water Chemistry During 96 Hour Exposure
Period. Dissolved Oxygen: 8.3-9.6 mg/L pH: 7.5-8.3

Photoperiod 16 hours of light, 8 hours of dark Temperature Range Approximately 14 O C during holding period
14.5-14.9 O C during exposure period42 Remarks field for test
conditions All organisms were observed for mortality and the number of individuals exhibiting clinical signs of toxicity or abnormal behavior at 3, 6, 24, 48, 72, and 96 hours after initiation of test material exposure.43 Results At the start of the mixing period, the 100 mg/L loading rate was clear, with an oily slick at the surface. Microscopic examination of the WAF indicated a significant amount of dispersed test material present in the
water column. The WAF was therefore filtered through glass wool prior to use. Microscopic examination post filtration showed no particles or micro dispersions present.

Cumulative mortality data was as follows:
Cumulative mortality (%) Concentration mg/L (WAF) Number of
Trout 24 Hours 96 Hours

Range Find A and B
0 3 0 0
10 3 0 0
32 3 0 0
100 3 0 33.3
320 3 0 33.3

Range Find C
0 3 0 0
10 3 0 0
100 3 0 0

Definitive Study
100 20 (10/replicate) 0 0

Time (hours) Lethal Loading
Rate 50 (mg/L) Method of Determination

3 >100 Inspection 6 >100 Inspection 24 >100 Inspection 48 >100 Inspection 72 >100 Inspection 96 >100 Inspection

The 96-hour Lethal Loading rate (LL 50 ) was >100 mg/L (WAF). The No Observed Effect Level, based on mortality and the absence of any sub lethal effects of exposure was 100 mg/L.
Conclusions Under the conditions of this study th e 96-hour Lethal Loading rate (LL 50 ) was >100 mg/L (WAF). The 96-hour no observed effect level was 100 mg/L (WAF). Data Quality Reliable without restriction (Klimisch Code) References Acute Toxicity to Rainbow Trout SafePharm Laboratories Project No.: 1666/062 (21 Jul 2005)44 Other Updated: 8/15/200545 

### Robust Summary 3-Fish - 8
Test Substance
CAS # 61789-86-4 Chemical Name Sulfonic acids, petroleum, calcium salts Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed OECD 203

Test Type Acute Toxicity to Fish (Water Accommodated Fraction-WAF) GLP (Y/N) Y Year (Study Performed) 2005 Species/Strain Rainbow Trout ( Oncorhynchus mykiss ) Fingerlings were obtained from a commercial breeder. Fish Size Average length 4.6 cm and a mean weight of 1.26 g, loading rate of 0.63 g body weight/L Number of Fish Range Find: 3/concentration Definitive Study 20/concentration (10/replicate) Analytical Monitoring No

Nominal Test Substance
Concentration Levels Range Find Study: 0, 10, 100 mg/L WAF Definitive Study: 0, 100 mg/L WAF
Test Concentration Preparation Individual test concentrations were prepared for each test level. A
measured volume of test material was added to a measured volume of
dilution water and continuo usly stirred for 24 hours. Following settling for 1 hour the aqueous phase or WAF (water accommodated fraction) was removed by mid-depth siphoning. Exposure Period 96 hours Exposure Conditions Static-renewal test conditions. The test prep arations were renewed daily.
Vehicle None Statistical Analysis LL 50 values determined by inspection. Dose Range finding Study Yes Test Chambers Covered, 20-liter glass aquaria containing the test solution Diluent Water Dechlorinated, softened water Diluent Water Chemistry Hardness 100 mg/l as CaCO 3 pH 7.6-8.5. Diluent Water Chemistry During 96 Hour Exposure
Period. Dissolved Oxygen: 9.2-9.9 mg/L pH: 7.5-8.0

Photoperiod 16 hours of light, 8 hours of dark Temperature Range Approximately 14 O C during holding period
13.6-14.4 O C during exposure period
Remarks field for test
conditions All organisms were observed for mortality and the number of individuals exhibiting clinical signs of toxicity or abnormal behavior at 3, 6, 24, 48,46 72, and 96 hours after initiation of test material exposure.
Results At the start of the mixing period, the 100 mg/L loading rate was clear, with an oily slick at the surface. Microscopic examination of the WAF indicated a slight amount of dispersed test material present in the water column. The WAF was therefore filtered through glass wool prior to use. Microscopic examination post filtration showed no particles or micro dispersions present.

Cumulative mortality data was as follows:
Cumulative mortality (%) Concentration mg/L (WAF) Number of
Trout 24 Hours 96 Hours

Range Find
10 3 0 0
100 3 0 0

Definitive Study
100 20 (10/replicate) 0 0

Time (hours) Lethal Loading
Rate 50 (mg/L) Method of Determination

3 >100 Inspection 6 >100 Inspection 24 >100 Inspection 48 >100 Inspection 72 >100 Inspection 96 >100 Inspection

The 96-hour Lethal Loading rate (LL 50 ) was >100 mg/L (WAF). The No Observed Effect Level, based on mortality and the absence of any sub lethal effects of exposure was 100 mg/L.
Conclusions Under the conditions of this study th e 96-hour Lethal Loading rate (LL 50 ) was >100 mg/L (WAF). The 96-hour no observed effect level was 100 mg/L (WAF). Data Quality Reliable without restriction (Klimisch Code) References Acute Toxicity to Rainbow Trout SafePharm Laboratories Project No.: 1666/065 (5 Aug 2005)
Other Updated: 8/15/200547 

### Robust Summary 3-Fish - 9
Test Substance
CAS # 61790-48-5 Chemical Name Sulfonic acids, petroleum, barium salts Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed OECD 203

Test Type Acute Toxicity to Fish (Water Accommodated Fraction-WAF) GLP (Y/N) Y Year (Study Performed) 2005 Species/Strain Rainbow Trout ( Oncorhynchus mykiss ) Fingerlings were obtained from a commercial breeder. Fish Size Average length 4.2 cm and a mean weight of 0.88 g, loading rate of 0.44 g body weight/L Number of Fish Range Find: 3/concentration Definitive Study: 20/concentration (10/replicate) Analytical Monitoring No

Nominal Test Substance
Concentration Levels Range Find Study: 0, 10, 100 mg/L WAF Definitive Study: 0, 100 mg/L WAF
Test Concentration Preparation Individual test concentrations were prepared for each test level. A
measured volume of test material was added to a measured volume of
dilution water and continuo usly stirred for 24 hours. Following settling for 1 hour the aqueous phase or WAF (water accommodated fraction) was removed by mid-depth siphoning. Exposure Period 96 hours Exposure Conditions Static-renewal test conditions. The test prep arations were renewed daily.
Vehicle None Statistical Analysis LL 50 values determined by inspection. Dose Range finding Study Yes Test Chambers Covered, 20-liter glass aquaria containing the test solution Diluent Water Dechlorinated, softened water Diluent Water Chemistry Hardness 100 mg/l as CaCO 3 pH 7.6-8.5. Diluent Water Chemistry During 96 Hour Exposure
Period. Dissolved Oxygen: 9.1-9.9 mg/L pH: 7.6-8.2

Photoperiod 16 hours of light, 8 hours of dark Temperature Range Approximately 14 O C during holding period
13.9-14.5 O C during exposure period
Remarks field for test
conditions All organisms were observed for mortality and the number of individuals exhibiting clinical signs of toxicity or abnormal behavior at 3, 6, 24, 48,48 72, and 96 hours after initiation of test material exposure.
Results At the start of the mixing period, the 100 mg/L loading rate was clear, with brown oily globules at the surface. Microscopic examination of the WAF indicated small globules of test material were present in the water column. The WAF was therefore filtered through glass wool prior to use. Microscopic examination post filtration showed no globules present.

Cumulative mortality data was as follows:
Cumulative mortality (%) Concentration mg/L (WAF) Number of
Trout 24 Hours 96 Hours

Range Find
10 3 0 0
100 3 0 0

Definitive Study
100 20 (10/replicate) 0 0

Time (hours) Lethal Loading
Rate 50 (mg/L) Method of Determination

3 >100 Inspection 6 >100 Inspection 24 >100 Inspection 48 >100 Inspection 72 >100 Inspection 96 >100 Inspection

The 96-hour Lethal Loading rate (LL 50 ) was >100 mg/L (WAF). The No Observed Effect Level, based on mortality and the absence of any sub lethal effects of exposure was 100 mg/L.
Conclusions Under the conditions of this study th e 96-hour Lethal Loading rate (LL 50 ) was >100 mg/L (WAF). The 96-hour no observed effect level was 100 mg/L (WAF). Data Quality Reliable without restriction (Klimisch Code) References Acute Toxicity to Rainbow Trout SafePharm Laboratories Project No.: 1666/067 (19 Jul 2005)
Other Updated: 8/15/200549 

### 3.2 Acute Toxicity to Aquatic Invertebrates (e.g. Daphnia)

Robust Summary 3-DAPH - 1
Test Substance
CAS # 115733-09-0 Chemical Name Benzenesulfonic acid, C14- C24 branched and linear alkyl derivatives,
calcium salts Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed Test protocol followed US EPA Toxic Substances Control Act Test Guideline #797.1300 (1985, 1987), OECD Guideline for Testing of
Chemicals #202 Daphnia sp. Acute Immobilization Test and Reproduction Test (1984). Test Type Static acute toxicity test GLP (Y/N) Y Year (Study Performed) 1993 Species/Strain Cladoceran, Daphnia magna Analytical Monitoring Total organic carbon (TOC) measurements of initial (0-h) test solutions and at test termination (48-h). Water samples were passed through 0.45-micron filter prior to TOC analysis using EPA Method
415.1. Exposure Period (unit) 48 hours Statistical methods Statistical analysis of data not warranted.

Remarks field for test conditions (fill as applicable) Test species: Juvenile daphnids less than 24-hours old were produced from laboratory in-house culture.

Test System: Individual WAFs were prepared for each test level and renewed daily. A measured weight of test material was added to a measured volume of dilution water (1-L) in a glass vessel and stirred for 24 hours. Stirring accomplished using a Telfon coated magnetic stir bar. Mixing speed adjusted su ch that a vortex formed between 30 to 50% of the distance to the bottom. Following the mixing period, the
test solutions were allowed to sta nd for 1 hour before the water phase was removed. To avoid removing test material from the surface or bottom, a siphon was placed in the mixing vessel prior to addition of
water and test substance with the lower end 1-2 inches off the bottom. The siphoned water phase (i.e., WAF) was used for the aquatic toxicity test.

Test conditions: Two 250-mL glass beakers that contained 200 mL of test solution were used per treatment. The 250-mL test vessels were50 loosely covered to reduce entry of dust.

Light: 16-hour light per day using cool-white fluorescent lights with an intensity of 20 uEin/m 2

.

Test temperature: 20.4 – 20.9 C

Dilution water: Filtered well water collected at Hampton, New Hampshire and adjusted to the appropriate hardness 176 mg/L as
CaCO 3 . The water was passed through activated carbon, a particle filter, and then an ultraviolet sterilizer and stored in a polyethylene
tank where it was aerated. TOC leve ls were 2 mg/L at the beginning and end of the test and <10 mg/L at the end of the test.

Water chemistry: Dissolved oxygen: 7.9 - 8.7 mg/L; pH: 7.2 - 8.1; conductivity: 860 – 880 umhos/cm.

Element: Immobilization/mortality

Test Levels: Control, 100, 300, & 1,000 mg/L WAF loading rates: 10 daphnids per replicate (20 per treatment). No undissolved test material was seen on the surface of the test vessels during the entire test.

Test Findings: At 24 hours, no immobilized or dead organisms were observed in the control or treatments. At 48-hours 5, 0, 20, and 5% immobilization were reported for c ontrol, 100, 300, and 1,000 mg/L, respectively.

Calculation of EL 50 s: Statistical analysis of survival data not
warranted.

Exposure period: 48 hours

Analytical Monitoring: TOC levels we re 2.8 – 3.5 mg/L in the control, 2.8 - 3.6 mg/l at 100, 3.0 - 3.7 mg/L at 300 mg/L, and 2.7 - 3.4 mg/L at 1,000 mg/L loading. TOC levels were not considered to be indicative
of actual test material concentrations and results are therefore based on nominal loading rates.
Results Nominal concentrations: 48-h EL 50 >1,000 mg/L. This is equivalent
to 48-h EL 0 = 1000 mg/L based on WAF loading rates.
Remarks Measured concentration: n/a

Unit: mg/L51 Statistical results: Not applicable.

Other:

• Test results reported in original study as “lethal concentrations” are reported in this summary as “lethal loading”, because test results are based on WAF loading rates.

• 20% immobilization/mortality seen at the middle test concentration of 300 mg/L (WAF) was not considered to be treatment related. This was based on findings of insignificant effects, or no effects, in both the highest and lowest test levels, respectively.

• Control response was satisfactory.
Conclusions The test material was not toxic to daphnids at loading rates tested. Percent survival/unaffected organisms was 95% in the control, 100% at 100, 80% at 300, and 95% at 1,000 mg/L. Data Quality (1) Reliable without restriction References Ward, T.J. (1993) Acute Toxicity of the Water Accommodated Fractions (WAFs) of CMA #604 to the Daphnid, Daphnia magna. T.R. Wilbury Study #9178-CMA/ESI-604.
Other Updated: 9-6-0052 Robust Summary 3-DAPH-2
Test Substance
CAS # 71486-79-8 Chemical Name Benzenesulfonic acid, mo no-C15-30-branched alkyl and di-C11-13- branched and linear alkyl deri vatives, calcium salts, overbased Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed Test protocol followed US EPA Toxic Substances Control Act Test Guideline #797.1300 (1985, 1987), OECD Guideline for Testing of
Chemicals #202 Daphnia sp. Acute Immobilization Test and Reproduction Test (1984). Test Type Static acute toxicity test GLP (Y/N) Y Year (Study Performed) 1993 Species/Strain Cladoceran, Daphnia magna Analytical Monitoring Total organic carbon (TOC) measurements of initial (0-h) test solutions and at test termination (48-h). Water samples were passed through 0.45-micron filter prior to TOC analysis using EPA Method
415.1. Exposure Period (unit) 48 hours Statistical methods Statistical analysis of data not warranted because immobilization seen at high dose not significant.
Remarks field for test conditions (fill as applicable) Test species: Juvenile daphnids less than 24-hours old were produced from laboratory in-house culture.

Test System: Individual WAFs were prepared for each test level and renewed daily. A measured weight of test material was added to a measured volume of dilution water (1-L) in a glass vessel and stirred for 24 hours. Stirring accomplished using a Telfon coated magnetic stir bar. Mixing speed adjusted su ch that a vortex formed between 30 to 50% of the distance to the bottom. Following the mixing period, the
test solutions were allowed to sta nd for 1 hour before the water phase was removed. To avoid removing test material from the surface or bottom, a siphon was placed in the mixing vessel prior to addition of
water and test substance with the lower end 1-2 inches off the bottom. The siphoned water phase (i.e., WAF) was used for the aquatic toxicity test.

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.

Light: 16-hour light per day using cool-white fluorescent lights with53 an intensity of 20 uEin/m.

Test temperature: 20.3 – 20.7 C

Dilution water: Filtered well water collected at Hampton, New Hampshire and adjusted to the appropriate hardness 176 mg/L as
CaCO 3 . The water was passed through activated carbon, a particle filter, and then an ultraviolet sterilizer and stored in a polyethylene
tank where it was aerated. TOC leve ls were 2 mg/L at the beginning and end of the test and <10 mg/L at the end of the test.

Water chemistry: Dissolved oxygen: 7.8 - 8.8 mg/L; pH: 7.4 - 8.5; conductivity: 850 – 900 umhos/cm.

Element: Immobilization/mortality

Test Levels: Control, 100, 300, & 1,000 mg/L WAF loading rates: 10 daphnids per replicate (20 per treatment). No undissolved test material was seen on the surface of the test vessels during the entire test.

Test Findings: At 24 hours, no immobilized or dead organisms were observed in the control or at 300 and 1,000 mg/L, but 5% immobilization /mortality was seen in the 100-mg/L treatment. At 48- hours 0, 10, 0, and 0% immobilizati on were reported for control, 100, 300, and 1,000 mg/L, respectively.

Calculation of EL 50 s: Statistical analysis of survival data not
warranted.

Exposure period: 48 hours

Analytical Monitoring: TOC levels we re 2.3 – 2.8 mg/L in the control, 2.8 - 2.9 mg/l at 100, 2.6 - 2.8 mg/L at 300 mg/L, and 2.6 - 3.0 mg/L at 1,000 mg/L loading. TOC levels were not considered to be indicative
of actual test material concentrations and results are therefore based on nominal loading rates.
Results Nominal concentrations: 48-h EL 50 >1,000 mg/L. This is equivalent
to 48-h EL 0 = 1000 mg/L based on WAF loading rates.
Remarks Measured concentration: n/a

Unit: mg/L

Statistical results: Not applicable because immobilization seen at high54 dose was not significant.

Other:

• Test results reported in original study as “lethal concentrations” are reported in this summary as “lethal loading”, because test results are based on WAF loading rates.

• Control response was satisfactory.
Conclusions The test material was not toxic to daphnids at loading rates tested. Percent survival/unaffected test organisms was 100% in the control, 90% at 100, 100% at 300, and 100% at 1,000 mg/L. Data Quality (1) Reliable without restriction References Ward, T.J. (1993) Acute Toxicity of the Water Accommodated Fractions (WAFs) of CMA #605 to the Daphnid, Daphnia magna. T.R. Wilbury Study #9178-CMA/ESI-605.
Other Updated: 9-6-0055 Robust Summary 3-DAPH-3
Test Substance
CAS # 71786-47-5 Chemical Name Benzenesulfonic acid, mono and dialkyl derivatives, magnesium salts Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed Test protocol followed US EPA Toxic Substances Control Act Test Guideline #797.1300 (1985, 1987), OECD Guideline for Testing of
Chemicals #202 Daphnia sp. Acute Immobilization Test and Reproduction Test (1984). Test Type Static acute toxicity test GLP (Y/N) Y Year (Study Performed) 1993 Species/Strain Cladoceran, Daphnia magna Analytical Monitoring Total organic carbon (TOC) measurements of initial (0-h) test solutions and at test termination (48-h). Water samples were passed through 0.45-micron filter prior to TOC analysis using EPA Method
415.1. Exposure Period (unit) 48 hours Statistical methods Statistical analysis of data not warranted because there was no immobilization occurred in this study.
Remarks field for test conditions (fill as applicable) Test species: Juvenile daphnids less than 24-hours old were produced from laboratory in-house culture.

Test System: Individual WAFs were prepared for each test level and renewed daily. A measured weight of test material was added to a measured volume of dilution water (1-L) in a glass vessel and stirred for 24 hours. Stirring accomplished using a Telfon coated magnetic stir bar. Mixing speed adjusted su ch that a vortex formed between 30 to 50% of the distance to the bottom. Following the mixing period, the
test solutions were allowed to sta nd for 1 hour before the water phase was removed. To avoid removing test material from the surface or bottom, a siphon was placed in the mixing vessel prior to addition of
water and test substance with the lower end 1-2 inches off the bottom. The siphoned water phase (i.e., WAF) was used for the aquatic toxicity test.

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.

Light: 16-hour light per day using cool-white fluorescent lights with56 an intensity of 20 uEin/m 2

.

Test temperature: 19.7 – 20.9 C

Dilution water: Filtered well water collected at Hampton, New Hampshire and adjusted to the appropriate hardness 176 mg/L as
CaCO 3 . The water was passed through activated carbon, a particle filter, and then an ultraviolet sterilizer and stored in a polyethylene
tank where it was aerated. TOC leve ls were 2 mg/L at the beginning and end of the test and <10 mg/L at the end of the test.

Water chemistry: Dissolved oxygen: 7.9 - 9.3 mg/L; pH: 7.0 - 8.4; conductivity: 870 – 910 umhos/cm.

Element: Immobilization/mortality

Test Levels: Control, 100, 300, and 1,000 mg/L WAF loading rates: 10 daphnids per replicate (20 per treatment). No undissolved test material was seen on the surface of the test vessels during the entire test.

Calculation of EL 50 s: Statistical analysis of survival data not
warranted.

Exposure period: 48 hours

Analytical Monitoring: TOC levels we re 1.8 – 2.8 mg/L in the control, 2.2 - 3.3 mg/l at 100, 2.1 - 3.2 mg/L at 300 mg/L, and 1.8 - 3.3 mg/L at 1,000 mg/L loading. TOC levels were not considered to be indicative
of actual test material concentrations and results are therefore based on nominal loading rates.
Results Nominal concentrations: 48-h EL 50 >1,000 mg/L. This is equivalent
to 48-h EL 0 = 1000 mg/L based on WAF loading rates (no immobilization noted).
Remarks Measured concentration: n/a

Unit: mg/L

Statistical results: Not applicable because there was no
immobilization.

Other:

• Test results reported in original study as “lethal concentrations” are reported in this summary as “lethal loading”, because test results are based on WAF loading rates.57 • Control response was satisfactory.
Conclusions The test material was not toxic to daphnids at loading rates tested. Percent survival/unaffected test organisms was 100% in the control, 100, 300, and 1,000 mg/L. Data Quality (1) Reliable without restriction References Ward, T.J. (1993) Acute Toxicity of the Water Accommodated Fractions (WAFs) of CMA #609 to the Daphnid, Daphnia magna. T.R. Wilbury Study #9178-CMA/ESI-609.
Other Updated: 9-6-0058 

### Robust Summary 3-DAPH- 4

Test Substance
CAS # 68608-26-4 Chemical Name Sulfonic acids, petroleum, sodium salts Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.
Method
Method/Guideline
followed OECD Guideline for Testing of Chemicals #202 Daphnia sp. Acute Immobilization Test and Reproduction Test (1984). Test Type Static acute toxicity test GLP (Y/N) Y Year (Study Performed) 2005 Species/Strain Daphnia magna Analytical Monitoring Test material concentra tions of exposure solutions were not determined. Exposure Period (unit) 48 hours
Positive Control Potassium dichromate at 0, 0. 32, 0.56, 1.0, 1.8, 3. 2 mg/L (Conducted during December 2004).
Statistical methods EL50 values calculated usi ng the trimmed Spearman-Karber method (ToxCalc software1999).
Remarks field for test conditions (fill as applicable) Twenty-four hours old Daphnia magna derived from in house cultures were used for the study. Individual wate r accommodated fractions (WAFs) were prepared for each test level. Appropriate amounts of test material was added
to a measured volume of reconstituted wate r in a glass vessel and stirred for 24 hours. Stirring was accomplished using a ma gnetic stirrer at a stirring rate that produced a slight dimple at the surface of the water. Following the mixing period, the test solutions were allowe d to stand for 1 hour before the water phase was gently siphoned (first 75-100 mL discarded) from the mixing vessel by mid-depth siphoning into the test vessels. The test chambers were covered, 250 ml vessels that contained 200 ml of test solution. Ten daphnids/time point were di stributed into each concentration for the range finding study. Five daphnids/r eplicate/time point (4 replicates) were used in the definitive study. Test v essels were covered to reduce evaporation
and were maintained at 20.6 to 21.1 o C with a photoperiod of 16 hours light and 8 hours dark. Daphnia were not fed nor were cultures aerated during exposure. Control groups were handled in the same manner as the test groups. Test preparations were not renewed during the exposure period. Water temperature was recorded daily throughout the test. Dissolved oxygen concentration and pH were recorded at the start and end of the study. Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. Da phnia were considered immobilized if they were unable to swim for approxima tely 15 seconds after gentle agitation. Test Concentrations Range Find Study A: 0, 10, 100 mg/L WAF Range Find Study B: 0, 1.0, 10 mg/L WAF Range Find Study C: 0, 1.0, 10, 100 mg/L WAF Definitive Study: 0, 100 mg/L WAF
Results The 48-hour EL 50 (Effective Loading rate) was determined to be >100 mg/L (WAF).59 Remarks Temperature was maintained at 20.3 to 21.0 o C throughout the test. No
treatment related differences were ob served in oxygen concentration or pH during the study.

After 24 hours stirring and a 1-hour standing period the 100 mg/L loading rate was a clear colorless water column with test material floating at the water surface. After siphoning and for the duration of the test, the loading rate was a clear colorless solution. No micro-disp ersions or undissolved test material were present upon microscopic examination.

Cumulative immobilization data was as follows:

Cumulative Immobilization (%)

Concentration mg/L (WAF) Number of Daphnia 24 Hours 48 Hours

Range Find Study A
0 10/intrerval 0 0
10 10/intrerval 4 10
100 10/intrerval 5 10

Range Find Studies
B and C
0 10/intrerval 0 0
1.0 10/intrerval 0 0
10 10/intrerval 0 0
100 10/intrerval 0 0

Definitive Study
100 20/intrerval 0 0

In the initial range find study immobiliz ation was observed at 10 and 100 mg/L WAF. However, in follow up range find studies B and C no immobilization was observed at concentrations ranging from 1.0 to 100 mg/L WAF. Based on these results the definitive study was conducted at 100 mg/L WAF. In the definitive study no immobilization was observed.

The results in the initial range find st udy could not be explained from an
examination of the data and were attribut ed to biological variation by the Study
Director.

The 24 and 48-hour EL 50 (Effective Loading rate) were determined to be >100 mg/L (WAF).

The no observed effect-loading rate at 24 and 48-hours was 100 mg/L (WAF).

The results of the positive control were within the normal range for potassium dichromate. The mean positive control 48-hour EC 50 was 0.78 mg/L.
Conclusions The 24 and 48-hour EL 50 (Effective Loading rate) was determined to be >10060 mg/L (WAF). The no observed effect-l oading rate at 24 and 48-hours was 100 mg/L (WAF). Data Quality Reliable with restriction (Klimisc h Code). Restriction due to findings noted during the initial range find study. References Acute Toxicity to Dapnia Magna SafePharm Laboratories Project No.: 1666/061 (21 Jul 2005) Other Updated: 8/15/200561 

### Robust Summary 3-DAPH- 5

Test Substance
CAS # 61789-86-4 Chemical Name Sulfonic acids, petroleum, calcium salts Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.
Method
Method/Guideline
followed OECD Guideline for Testing of Chemicals #202 Daphnia sp. Acute Immobilization Test and Reproduction Test (1984). Test Type Static acute toxicity test GLP (Y/N) Y Year (Study Performed) 2005 Species/Strain Daphnia magna Analytical Monitoring Test material concentra tions of exposure solutions were not determined. Exposure Period (unit) 48 hours
Positive Control Potassium dichromate at 0, 0. 32, 0.56, 1.0, 1.8, 3. 2 mg/L (Conducted during December 2004).
Statistical methods EL50 values calculated usi ng the trimmed Spearman-Karber method (ToxCalc software1999).
Remarks field for test conditions (fill as applicable) Twenty-four hours old Daphnia magna derived from in house cultures were used for the study. Individual wate r accommodated fractions (WAFs) were prepared for each test level. Appropriate amounts of test material was added
to a measured volume of reconstituted wate r in a glass vessel and stirred for 24 hours. Stirring was accomplished using a ma gnetic stirrer at a stirring rate that produced a slight dimple at the surface of the water. Following the mixing period, the test solutions were allowe d to stand for 1 hour before the water phase was gently siphoned (first 75-100 mL discarded) from the mixing vessel by mid-depth siphoning into the test vessels. The test chambers were covered, 250 ml vessels that contained 200 ml of test solution. Ten daphnids/time point were di stributed into each concentration for the range finding study. Five daphnids/r eplicate/time point (4 replicates) were used in the definitive study. Test v essels were covered to reduce evaporation
and were maintained at 20.3 to 21.0 o C with a photoperiod of 16 hours light and 8 hours dark. Daphnia were not fed nor were cultures aerated during exposure. Control groups were handled in the same manner as the test groups. Test preparations were not renewed during the exposure period. Water temperature was recorded daily throughout the test. Dissolved oxygen concentration and pH were recorded at the start and end of the study. Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. Da phnia were considered immobilized if they were unable to swim for approxima tely 15 seconds after gentle agitation. Test Concentrations Range Find Study: 0, 10, 100 mg/L WAF Definitive Study: 0, 100 mg/L WAF
Results The 48-hour EL 50 (Effective Loading rate) was determined to be >100 mg/L (WAF). Remarks Temperature was maintained at 20.3 to 21.0 o C throughout the test. No62 treatment related differences were ob served in oxygen concentration or pH during the study.

After 24 hours stirring and a 1-hour standing period the 100 mg/L loading rate was a clear colorless water column with brown oily test material floating at the water surface. After siphoning and for the duration of the test, the loading rate
was a clear colorless solution. No mi cro-dispersions or undissolved test material were present upon microscopic examination.

Cumulative immobilization data was as follows:

Cumulative Immobilization (%)

Concentration mg/L (WAF) Number of Daphnia 24 Hours 48 Hours

Range Find
0 10/intrerval 0 0
10 10/intrerval 0 0
100 10/intrerval 0 0

Definitive Study
100 20/intrerval 0 0

No immobilization was observed.

The 24 and 48-hour EL 50 (Effective Loading rate) were determined to be >100 mg/L (WAF).

The no observed effect-loading rate at 24 and 48-hours was 100 mg/L (WAF).

The results of the positive control were within the normal range for potassium dichromate. The mean positive control 48-hour EC 50 was 0.78 mg/L.
Conclusions The 24 and 48-hour EL 50 (Effective Loading rate) was determined to be >100 mg/L (WAF). The no observed effect-l oading rate at 24 and 48-hours was 100 mg/L (WAF). Data Quality Reliable without restriction (Klimisch Code). References Acute Toxicity to Daphnia Magna SafePharm Laboratories Project No.: 1666/064 (21 Jul 2005) Other Updated: 8/15/200563 

### 3.3 Toxicity to Aquatic Plants (e.g. Algae)

Robust Summary 3-ALG - 1
Test Substance
CAS # 115733-09-0 Chemical Name Benzenesulfonic acid, C14- C24 branched and linear alkyl derivatives,
calcium salts Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed Test protocol followed US EPA Toxic Substances Control Act Test Guideline #797.1050 (1985, 1987), OECD Guideline for Testing of Chemicals #201 Alga, Growth Inhibition Test (1984). Test Type Static acute toxicity test GLP (Y/N) Y Year (Study Performed) 1994 Species/Strain Freshwater algae, Pseudokirchneriella subcapitata formerly called Selenastrum capricornutum Element basis (# of cells/mL) approximately 10,000 cells/mL

Exposure period/duration 96 hours Analytical monitoring Total organic carbon (TOC) measurements of initial (0-h) high, low
and control test solutions and at t est termination (96-h). Water samples were passed through 0.45-micron f ilter prior to TOC analysis using
EPA Method 415.1 Statistical methods A parametric one-way an alysis of variance (ANOVA) and Dunnett’s
test were used to calculate the no-observed effect level.
Remarks field for test conditions (fill as applicable) Test Species: Cells taken from a log-growth phase in-house culture of Pseudokirchneriella subcapitata that was original ly purchased from University of Texas at Austin alga collection.

Test System: Individual WAFs were prepared for each test level and renewed daily. A measured weight of test material was added to a measured volume of dilution water (1-L) in a glass vessel and stirred for 24 hours. Stirring accomplished using a Telfon coated magnetic stir bar. Mixing speed adjusted su ch that a vortex formed between 30 to 50% of the distance to the bottom. Following the mixing period, the
test solutions were allowed to sta nd for 1 hour before the water phase was removed. To avoid removing test material from the surface or bottom, a siphon was placed in the mixing vessel prior to addition of
water and test substance with the lower end 1-2 inches off the bottom. The siphoned water phase (i.e., WAF) was used for the aquatic64 toxicity test.

Test Conditions: A static test was conducted; i.e., there was no daily renewal of test solution. Three 100-mL replicates per treatment, inoculum ~10,000 cells/mL. The 250-mL Erlenmeyer flasks were stoppered with foam plugs to reduce entry of dust, etc. During the test
all treatment and control flasks we re 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.

Light: Cool-white fluorescent lights pr ovided a light intensity of 47-50
uEin/m 2 sec 24-h per day.

Test temperature: 23.3 to 24.0 C.

Dilution Water: Sterile enriched alga growth media (US EPA, 1978, T.R. Wilbury SOP #6) adjusted to pH 7.5. Measured TOC and total suspended solids in fresh untreated alga media were <1.0 and <10 mg/L, respectively. Test media pH was 7.5 - 9.8 at 0-hour and 8.3 -
10.1 after 96 hours.

Test Levels: Control, 100, 300 & 1000 mg/L WAF loading rates. No
undissolved test material was seen on the surface of the test vessels during the entire aquatic toxicity test.

Calculation of EL 50 s and NOELs: Binomial nonlinear interpolation 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 level.

Method of calculating mean measure d concentrations: not applicable

Exposure period: 96 hours

Analytical monitoring: At the beginning and end of the test, TOC
measurements were betw een 2 - 8 mg/L in control, 2 – 4 mg/L at 100 mg/L, 2 – 3 mg/L at 300 mg/L and 1,000 mg/L. TOC levels were not
considered to be indicative of actua l test material concentrations and results are therefore based on nominal loading rates.65 Results Nominal concentrations: 72- & 96-h EL 50 >1,000 mg/L and 72- & 96-h
NOEL = 1,000 mg/L based on both growth rate and biomass

measurements.
Remarks Measured concentration: n/a

Unit: mg/L

Test Findings: At 72-hours biomass measurements in the treatments were 80%, 62%, and 70% of the control at 100, 300, and 1,000 mg/L, respectively. At 96-hours biomass measurements were 70, 66, and 88% of the control at 100, 300 and 1,000 mg/L, respectively.

Other:

• Test results reported in original study as “effect concentrations” and “no observed effect concentrations” are reported in this summary as “effect loading” and “no observed effect levels”, respectively, because test results are based on WAF loading rates.

• Control response was satisfactory.
Conclusions The test material was not toxic to freshwater alga at loading rates up to and including 1,000 mg/L. Data Quality (1) Reliable without restriction References Ward, T.J. (1994) Acute Toxicity of the Water Accommodated Fractions (WAFs) of CMA #604 to the Freshwater Alga, Selenastrum capricornutum. T.R. Wilbury Study #73-CM-604.

Stephan, C.E. (1983). Computer Program for the Calculation of LC50 Values. U.S. EPA. Duluth, MN. Personal Communication.
Other Updated: 9-6-0066 Robust Summary 3-ALG-2
Test Substance
CAS # 71486-79-8 Chemical Name Benzenesulfonic acid, mo no-C15-30-branched alkyl and di-C11-13- branched and linear alkyl deri vatives, calcium salts, overbased Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed Test protocol followed US EPA Toxic Substances Control Act Test Guideline #797.1050 (1985, 1987), OECD Guideline for Testing of Chemicals #201 Alga, Growth Inhibition Test (1984). Test Type Static acute toxicity test GLP (Y/N) Y Year (Study Performed) 1994 Species/Strain Freshwater algae, Pseudokirchneriella subcapitata formerly called Selenastrum capricornutum Element basis (# of cells/mL) approximately 10,000 cells/mL

Exposure period/duration 96 hours Analytical monitoring Total organic carbon (TOC) measurements of initial (0-h) high, low
and control test solutions and at t est termination (96-h). Water samples were passed through 0.45-micron f ilter prior to TOC analysis using
EPA Method 415.1 Statistical methods The computer program of Stephan (1983) was used to calculate EL 50 s. A parametric one-way analysis of variance (ANOVA) and Dunnett’s
test were used to calculate the no-observed effect concentration.
Remarks field for test conditions (fill as applicable) Test Species: Cells taken from a log-growth phase in-house culture of Pseudokirchneriella subcapitata that was original ly purchased from University of Texas at Austin alga collection.

Test System: Individual WAFs were prepared for each test level and renewed daily. A measured weight of test material was added to a measured volume of dilution water (1-L) in a glass vessel and stirred for 24 hours. Stirring accomplished using a Telfon coated magnetic stir bar. Mixing speed adjusted su ch that a vortex formed between 30 to 50% of the distance to the bottom. Following the mixing period, the
test solutions were allowed to sta nd for 1 hour before the water phase was removed. To avoid removing test material from the surface or bottom, a siphon was placed in the mixing vessel prior to addition of
water and test substance with the lower end 1-2 inches off the bottom. The siphoned water phase (i.e., WAF) was used for the aquatic toxicity test.

Test Conditions: A static test was conducted; i.e., there was no daily67 renewal of test solution. Three 100-mL replicates per treatment, inoculum ~10,000 cells/mL. The 250-mL Erlenmeyer flasks were stoppered with foam plugs to reduce entry of dust, etc. During the test
all treatment and control flasks we re 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.

Light: Cool-white fluorescent lights pr ovided a light intensity of 47-50
uEin/m 2 sec 24-h per day.

Test temperature: 24.0 to 24.2 C.

Dilution Water: Sterile enriched alga growth media (US EPA, 1978, T.R. Wilbury SOP #6) adjusted to pH 7.5. Measured TOC and total suspended solids in fresh untreated alga media were <1.0 and <10 mg/L, respectively. Test media pH was 7.3 - 10.8 at 0-hour and 9.7 -
10.8 after 96 hours.

Test Levels: Control, 100, 300, 1000 mg/L WAF loading rates. No
undissolved test material was seen on the surface of the test vessels during the entire aquatic toxicity test.

Calculation of EL 50 s and NOELs: Binomial nonlinear interpolation 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 level.

Method of calculating mean measure d concentrations: not applicable

Exposure period: 96 hours

Analytical monitoring: At the beginning and end of the test, TOC
measurements were betwee n non-detect (<1) - 3 mg/L in control, 1 – 2 mg/L at 100 mg/L, 3 mg/L at 300 mg/L, and 5 – 6 mg/L at 1,000 mg/L. TOC levels were not considered to be indicative of actual test
material concentrations and results are therefore based on nominal loading rates.
Results Nominal concentrations: 72- & 96-h EL 50 >1,000 mg/L, based on both growth rate and biomass measurements. 72- & 96-h NOEL = 1000 mg/L.
Remarks
Measured concentration: n/a68 Unit: mg/L

Test Findings: At 72-hours biomass measurements in the treatments were 133%, 75%, and 64% of the control at 100, 300, and 1,000 mg/L. At 96-hours biomass measurements were 93, 77, and 52% of the control at 100, 100% at 300, and 100% at 1,000 mg/L.

Statistical results: A parametric one-way analysis of variance (ANOVA) and Dunnett’s test were used to calculate the no-observed
effect level.

Other:

• Test results reported in original study as “effect concentrations” and “no observed effect concentrations” are reported in this summary as “effect loading” and “no observed effect levels”, respectively, because test results are based on WAF loading rates.

• Control response was satisfactory.
Conclusions The test material was not toxic to freshwater alga at loading rates up to and including 1,000 mg/L. Data Quality (1) Reliable without restriction References Ward, T.J. (1994) Acute Toxicity of the Water Accommodated Fractions (WAFs) of CMA #605 to the Freshwater Alga, Selenastrum capricornutum. T.R. Wilbury Study #73-CM-605.

Stephan, C.E. (1983). Computer Program for the Calculation of LC50 Values. U.S. EPA. Duluth, MN. Personal Communication.
Other Updated: 9-6-0069 Robust Summary 3-ALG-3
Test Substance
CAS # 71786-47-5 Chemical Name Benzenesulfonic acid, mono and dialkyl derivatives, magnesium salts Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed Test protocol followed US EPA Toxic Substances Control Act Test Guideline #797.1050 (1985, 1987), OECD Guideline for Testing of Chemicals #201 Alga, Growth Inhibition Test (1984). Test Type Static acute toxicity test GLP (Y/N) Y Year (Study Performed) 1994 Species/Strain Freshwater algae, Pseudokirchneriella subcapitata formerly called Selenastrum capricornutum Element basis (# of cells/mL) approximately 10,000 cells/mL

Exposure period/duration 96 hours Analytical monitoring Total organic carbon (TOC) measurements of initial (0-h) high, low
and control test solutions and at t est termination (96-h). Water samples were passed through 0.45-micron f ilter prior to TOC analysis using
EPA Method 415.1 Statistical methods The computer program of Stephan (1983) was used to calculate EL 50 s. A parametric one-way analysis of variance (ANOVA) and Dunnett’s
test were used to calculate the no-observed effect level.
Remarks field for test conditions (fill as applicable) Test Species: Cells taken from a log-growth phase in-house culture of Pseudokirchneriella subcapitata that was original ly purchased from University of Texas at Austin alga collection.

Test System: Individual WAFs were prepared for each test level and renewed daily. A measured weight of test material was added to a measured volume of dilution water (1-L) in a glass vessel and stirred for 24 hours. Stirring accomplished using a Telfon coated magnetic stir bar. Mixing speed adjusted su ch that a vortex formed between 30 to 50% of the distance to the bottom. Following the mixing period, the
test solutions were allowed to sta nd for 1 hour before the water phase was removed. To avoid removing test material from the surface or bottom, a siphon was placed in the mixing vessel prior to addition of
water and test substance with the lower end 1-2 inches off the bottom. The siphoned water phase (i.e., WAF) was used for the aquatic toxicity test.

Test Conditions: A static test was conducted; i.e., there was no daily70 renewal of test solution. Three 100-mL replicates per treatment, inoculum ~10,000 cells/mL. The 250-mL Erlenmeyer flasks were stoppered with foam plugs to reduce entry of dust, etc. During the test
all treatment and control flasks we re 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.

Light: Cool-white fluorescent lights pr ovided a light intensity of 47-50
uEin/m 2 sec 24-h per day.

Test temperature: 24.0 C.

Dilution Water: Sterile enriched alga growth media (US EPA, 1978, T.R. Wilbury SOP #6) adjusted to pH 7.5. Measured TOC and total suspended solids in fresh untreated alga media were <1.0 and <10 mg/L, respectively. Test media pH was 7.5 – 9.9 at 0-hour and 8.3 -
10.8 after 96 hours.

Test Levels: Control, 125, 250, 500, 1,000 and 1,500 mg/L WAF loading rates. No undissolved test material was seen on the surface of the test vessels during the entire aquatic toxicity test.

Calculation of EL 50 s and NOELs: Binomial nonlinear interpolation 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 level.

Method of calculating mean measure d concentrations: not applicable

Exposure period: 96 hours

Analytical monitoring: At the beginning and end of the test, TOC measurements were non-detect (<1) - 1 mg/L in control, 2 – 3 mg/L at 125 mg/L and between 5 – 6 mg/L at 1,000 mg/L. TOC levels were
not considered to be indicative of actual test material concentrations and results are therefore based on nominal loading rates.
Results Nominal concentrations: 72-h EL 50 s = 1,400 mg/L and >1,500 mg/L based on biomass and growth rate, respectively. 96-h EL 50 s = 1,100 mg/L and >1,500 mg/L, based on biomass and growth rate, respectively. The 72-h and 96-hr NOEL = 1,000 mg/L.

Remarks Measured concentration: n/a71 Unit: mg/L

Test Findings: At 72-hours biomass measurements in the treatments were 97, 100, 101, 95, and 35% of the control at 125, 250, 500, 1,000, and 1,500 mg/L, respectively. At 96-hours biomass measurements were 78, 61, 62, 58, and 31% of the control at 125, 250, 500, 1,000. and 1,500 mg/L, respectively. Logarithmic growth was observed at all treatments up to and including 1,000 mg/L; i.e., average biomass measurements 1,383 – 1,860 cells/mL x10 3 . Therefore, the 96-h NOELs were determined to be 1,000 mg/L although statistical analysis determined the 96-h NOELs to be 125 mg/L. But, the hypothesis test was biased towards the unusually high control growth (2,383 cells/mL
x10 3 ) between 72 – 96 hours upon which test concentration growth was compared. This produced an erroneous measurement of NOEL.

Other:

• Test results reported in original study as “effect concentrations” and “no observed effect concentrations” are reported in this summary as “effect loading” and “no observed effect levels”, respectively, because test results are based on WAF loading rates.

• Effects were determined to be algistatic based on the rapid regrowth of an aliquot of cells taken from 1,500 mg/L cultured in
fresh control media.

• Control response was satisfactory.
Conclusions The test material was not toxic to freshwater alga at loading rates up to and including 1,000 mg/L. Data Quality (1) Reliable without restriction References Ward, T.J. (1994) Acute Toxicity of the Water Accommodated Fractions (WAFs) of CMA #609 to the Freshwater Alga, Selenastrum capricornutum. T.R. Wilbury Study #73-CM-609.

Stephan, C.E. (1983). Computer Program for the Calculation of LC50 Values. U.S. EPA. Duluth, MN. Personal Communication.
Other Updated: 9-6-0072 Robust Summary 3-ALG-4
Test Substance
CAS # 68608-26-4 Chemical Name Sulfonic acids, petroleum, sodium salts Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed OECD Guideline for Testing of Chemicals #201 Alga, Growth Inhibition Test (1984). Test Type Static acute toxicity test (Water Accommodated Fraction-WAF) GLP (Y/N) Y Year (Study Performed) 2005

Species/Strain Freshwater algae, Scenedesmus subspicatus /CCAP 276/20 Element basis (# of cells/mL) Approximately 2.13 x 10 6 cells/mL, 5 mL used to inoculate 1 liter of medium for an initial cell density of 10 4 cells/mL. Exposure period/duration 72 hours Range find test Yes Analytical monitoring Not performed Statistical methods A Students t-test incorporati ng Bartlett’s test for homogeneity of variance was used to compare the area under the growth curve data of the treated and control groups at 72 hours.
Remarks field for test conditions (fill as applicable) Test Species: Cultures obtained from the Culture Collection of Algae and Protozoa (CCAP), Institute of Freshwa ter Ecology, The Ferry House, Far Sawrey, Ambleside, Cumbria, U.K.

Loading Concentration: Range Find Study: 0, 10 and 100 mg/L (WAF) Definitive Study: 0 and 100 mg/L (WAF)

Test System: A measured weight of test material was added to a measured volume of culture media in a glass vessel and stirred for 24 hours. Stirring was accomplished using a magnetic stirrer. Mixing speed was adjusted such that a slight vortex formed causing a slight di mple at the media surface. Following the mixing period, the test solution was allowed to stand for one hour. A small amount of the WAF was removed and examined microscopically for the presence of micro-dispersions or globules of test material. Microscopic examination of the 100 mg/L WAF confirmed that there was a significant amount of dispersed test material present in the media. The 100 mg/L WAF was therefore filtered through glass wool prio r to use. Microscopic examination post filtration showed there to be micro dispersions or undissolved test material present. Given that the test material exhibited no toxicity, this was considered to
have had no effect on the outcome of the study. The siphoned phase (i.e., WAF) was used for the aquatic toxicity test.

Test Conditions: A static test was conducte d; i.e., there was no daily renewal of test solution. Two (range find study) or six (definitive study) 100-mL replicates73 per treatment, inoculum ~10,000 cells/m L. The 250-mL conical flasks were plugged with polyurethane foam bungs. During the test all treatment and control flasks were randomly placed on an orbital shaker adjusted to approximately 150 cycles per minute under constant light (24 hours/day) for 72
hours. Cell densities were determined using a hemocytometer and light microscopic at 0, 24, 48 and 72 hours. pH was determined at 0 and 72 hours.

Light: Continuous illumination approximately 7000 lux.

Test temperature: 24.0 o C. Culture Media: As specified in the guideline.

Method of calculating mean measure d concentrations: Not applicable. Exposure period: 72 hours
Results Range Find Study: No effect on growth at 10 or 100 mg/L WAF.

Definitive Study: Neither growth nor biomass were affected by the presence of the test material over a 72 hour period at 100 mg/L WAF.

The E b L 50 , the loading rate that reduced bioma ss by 50%, was >100 mg/L WAF.
The E
r L 50, the loading rate that reduced specific growth by 50%, was >100 mg/L WAF.

The No Observed Effect Loading Rate (NOEL) was 100 mg/L WAF.

The cell concentrations of the control cultures increased by a factor of 43 during the study meeting the guideline requirement of at least a factor of 16 after 72
hours.

All test and control cultures were inspected microscopically at 72 hours. No abnormalities were observed in any of the control or treated cultures. Control culture pH increased from 7.3 at 0 hour to 7.8 at 72 hours.
Conclusions Both the growth and the biomass of Scenedesmus subspicatus (CCAP 276/20) were unaffected by the presence of the test material over the 72-hour exposure period. The E b L 50 , the loading rate that reduced biomass by 50% was >100 mg/L WAF. The E
r L 50, the loading rate that redu ced specific growth by 50% was >100 mg/L WAF. The No Observed Effect Loading Rate (NOEL) was 100 mg/L WAF. Data Quality (1) Reliable without restriction References Algal Inhibition Test SafePharm Laboratories Project No.: 1666/060 (20 Jul 2005)
Other Updated: 8/16/200574 Robust Summary 3-ALG-5
Test Substance
CAS # 61789-86-4 Chemical Name Sulfonic acids, petroleum, calcium salts Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed OECD Guideline for Testing of Chemicals #201 Alga, Growth Inhibition Test (1984). Test Type Static acute toxicity test (Water Accommodated Fraction-WAF) GLP (Y/N) Y Year (Study Performed) 2005

Species/Strain Freshwater algae, Scenedesmus subspicatus /CCAP 276/20 Element basis (# of cells/mL) Approximately 2.13 x 10 6 cells/mL, 5 mL used to inoculate 1 liter of medium for an initial cell density of 10 4 cells/mL. Exposure period/duration 72 hours Range find test Yes Analytical monitoring Not performed Statistical methods A Students t-test incorporati ng Bartlett’s test for homogeneity of variance was used to compare the area under the growth curve data of the treated and control groups at 72 hours.
Remarks field for test conditions (fill as applicable) Test Species: Cultures obtained from the Culture Collection of Algae and Protozoa (CCAP), Institute of Freshwa ter Ecology, The Ferry House, Far Sawrey, Ambleside, Cumbria, U.K.

Loading Concentration: Range Find Study: 0, 10 and 100 mg/L (WAF) Definitive Study: 0 and 100 mg/L (WAF)

Test System: A measured weight of test material was added to a measured volume of culture media in a glass vessel and stirred for 24 hours. Stirring was accomplished using a magnetic stirrer. Mixing speed was adjusted such that a slight vortex formed causing a slight di mple at the media surface. Following the mixing period, the test solution was allo wed to stand for one hour. The test
media was clear and colorless with test material floating on the surface. A
small amount of the WAF was removed a nd examined microscopically for the presence of micro-dispersions or globules of test material. Microscopic examination of the WAF confirmed that there were no micro-dispersions of test material present. The siphoned phase (i.e., WAF) was used for the aquatic toxicity test.

Test Conditions: A static test was conducte d; i.e., there was no daily renewal of test solution. Two (range find study) or six (definitive study) 100-mL replicates per treatment, inoculum ~10,000 cells/m L. The 250-mL conical flasks were75 plugged with polyurethane foam bungs. During the test all treatment and control flasks were randomly placed on an orbital shaker adjusted to approximately 150 cycles per minute under constant light (24 hours/day) for 72
hours. Cell densities were determined using a Coulter Multisizer Particle Counter at 0, 24, 48 and 72 hours. pH was determined at 0 and 72 hours.

Light: Continuous illumination approximately 7000 lux.

Test temperature: 24.0 o C. Culture Media: As specified in the guideline.

Method of calculating mean measure d concentrations: Not applicable. Exposure period: 72 hours
Results Range Find Study: No effect on growth at 10 or 100 mg/L WAF.

Definitive Study: Neither growth nor biomass were affected by the presence of the test material over a 72 hour period at 100 mg/L WAF.

The E b L 50 , the loading rate that reduced biomass by 50%, was >100 mg/L WAF.
The E
r L 50, the loading rate that reduced specific growth by 50%, was >100 mg/L WAF.

The No Observed Effect Loading Rate (NOEL) was 100 mg/L WAF.

The cell concentrations of the control cultures increased by a factor of 40 during the study meeting the guideline requirement of at least a factor of 16 after 72
hours.

All test and control cultures were inspected microscopically at 72 hours. No abnormalities were observed in any of the control or treated cultures. Control culture pH increased from 7.2 at 0 hour to 7.6 at 72 hours.
Conclusions Both the growth and the biomass of Scenedesmus subspicatus (CCAP 276/20) were unaffected by the presence of the test material over the 72-hour exposure period. The E b L 50 , the loading rate that reduced biomass by 50% was >100 mg/L WAF. The E
r L 50, the loading rate that redu ced specific growth by 50% was >100 mg/L WAF. The No Observed Effect Loading Rate (NOEL) was 100 mg/L WAF. Data Quality (1) Reliable without restriction References Algal Inhibition SafePharm Laboratories Project No.: 1666/063 (20 Jul 2005)
Other Updated: 8/16/200576 

### 4. Mammalian Toxicity Category: Alkaryl Sulfonates

4.1 Acute Toxicity

4.1.1 Acute Oral Toxicity

Robust Summary 3-Acute Oral -1
Test Substance CAS # Analog of 70024-69-0 Chemical Name C20-24 alkaryl calcium salt derivative Remarks This substance is an analog fo r the group of substances in HERTG’s Final Submission for the Alkaryl Sulfonates Category. It is chemically similar to members of this CAS number, but does not belong to this CAS number. For more information on this chemical, see Section 2.0
“General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed OECD Guideline 401 Test Type Acute oral toxicity GLP (Y/N) Y Year (Study Performed) 1989 Species/Strain Rats/Sprague-Dawley strain
Sex Male and female

No. of animals/sex/dose 5

Vehicle None Route of administration Oral (intragastric) Dose level 5 g/kg Dose volume Not provided Control group included Yes
Remarks field for test
conditions A single dose of 5.0 g/kg of the undiluted test material was administered intragastrically to five fasted (over night) male and female rats. An untreated control group of 5/sex was included. The animals were observed for signs of physiological or behavioral changes frequently on the day of treatment. Thereafter all animals were examined for signs of toxicity once per day. Individual body weights were recorded on the day of dosing and at 2, 7 and 14 days after dosing. The surviving animals were euthanized at the conclusion of the observation period. Gross autopsies were performed on all animals after 14 days.77 Results LD50 > 5.0 g/kg (males and females) Remarks No mortality was observed. Di arrhea and reduced food intake were observed in one treated female on Day 1. No other signs of toxicity were observed. Body weights were unremarkable.

Conclusions The test article, when administered as received to 5 male and 5 female Sprague-Dawley rats, had an acute or al LD50 of greater than 5.0 g/kg. Data Quality Reliable without restriction (Klimisch Code) References Unpublished confidential business information
Other Updated: 2/10/00 (RTA-004)78 Robust Summary 3-Acute Oral-2
Test Substance
CAS # CAS# 61789-86-4
Chemical Name Petroleum derived calcium salt Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed OECD Guideline 401 Test Type Acute oral toxicity (Limit Test) GLP (Y/N) Y Year (Study Performed) 1985 Species/Strain Rats/Sprague-Dawley Crl:CD® (SD)BR
Sex Male and female

No. of animals/sex/dose 5

Vehicle Peanut oil Route of administration Oral (intragastric) Dose level 5 g/kg Dose volume 7 ml/kg Vehicle control group Yes Chemical analysis of dosing solution Yes

Remarks field for test
conditions A single dose of 5.0 g/kg of the test material diluted in peanut oil at a concentration of 714 mg/ml was administered intragastrically to five fasted (over night) male and female rats. The concentration of the test material in the vehicle was analyzed for homogeneity and for stability. The test material was administered at a dose volume of approximately 7 ml/kg body weight. A vehicle control group consisting of 5-fasted animals/sex was dosed with 7 ml/kg of peanut oil. The animals were observed for signs of toxicity or behavioral changes frequently on the day of treatment. Thereafter, all animals were examined for signs of toxicity twice per day (once/day on weekends), for the 13-day observation period and once on day 14 prior to sacrifice. Individual weights were recorded immediately prior to dosing and at 2, 7 and 14 days after dosing. The surviving animals were euthanized at the conclusion of the observation period. Gross autopsies were performed on all animals after 14 days. Histopathological evaluations were performed on grossly abnormal tissues only.
Results LD50 > 5.0 g/kg (males and females) Remarks Analysis confirmed that the dosing solution was homogeneous and stable for the period of use and that it was prepared at the appropriate concentration. No deaths were observed during the 14-day79 observation period. Diarrhea was observed in one treated male and in one control male 5 hours post dosing, only. Alopecia with or without thinned fur was seen in one vehicle control male (Days 12-14) and in one vehicle control female (Days 10-13) Other than the previous observations, all animals appeared normal throughout the 14-day observation period. No body weight effects occurred. No test material related macroscopic or microscopic findings were evident.

Conclusions The test article, when administered in peanut oil to 5 male and 5 female Sprague-Dawley rats, had an acute oral LD50 of greater than 5.0 g/kg. Data Quality Reliable without restriction (Klimisch Code) References Unpublished confidential business information
Other Updated: 2/9/00 (RTA-001)80 Robust Summary 3-Acute Oral-3
Test Substance
CAS # CAS# 61790-48-5
Chemical Name Petroleum derived barium salt Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed FHSA 16CFR1500.3 Test Type Acute oral toxicity (Limit Test) GLP (Y/N) Y Year (Study Performed) 1982 Species/Strain Rats/Sprague-Dawley strain
Sex Male and female

No. of animals/sex/dose 5

Vehicle None Route of administration Oral (intragastric) Dose level 2 g/kg Dose volume 2.4 ml/kg Control group included No
Remarks field for test
conditions A single dose of 2.0 g/kg of the undiluted test material was administered intragastrically to five fasted (over night) male and female rats. A control group was not included. The animals were observed for signs of toxicity or behavioral changes frequently on the day of treatment. Thereafter, all animals were examined for signs of toxicity twice per day. Individual weights were recorded on the day of dosing and weekly thereafter. The surviving animals were euthanized at the conclusion of the observation period. Gross autopsies were performed on all animals after 14 days.
Results LD50 > 2.0 g/kg (males and females)
Remarks One treated female died on Test Day 5 without exhibiting any clinical symptoms. All remaining animals su rvived to study termination. The animals exhibited ruffled fur 3 hours post dosing. Urine staining was observed within 24-48 hours of dosing. After 72 hours all animals essentially recovered. No body weight effects were observed. Gross necropsy findings were unremarkable for all animals.

Conclusions The test article, when administered as received to 5 male and 5 female Sprague-Dawley rats, had an acute or al LD50 of greater than 2.0 g/kg. Data Quality Reliable without restriction (Klimisch Code) References Unpublished confidential business information
Other Updated: 2/10/00 (RTA-003)81 Robust Summary 3-Acute Oral-4

Test Substance

CAS # CAS# 68608-26-4
Chemical Name Petroleum derived sodium salt Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed FHSA 16CFR1500.3

Test Type Acute oral toxicity GLP (Y/N) Y Year (Study Performed) 1983 Species/Strain Rats/Sprague-Dawley strain
Sex Male and female

No. of animals/sex/dose 5

Vehicle None Route of administration Oral (intragastric) Dose level 5 g/kg Dose volume 5 ml/kg Control group included No

Remarks field for test
conditions A single dose of 5.0 g/kg of the undiluted test material was administered intragastrically to five fasted (over night) male and female rats. A control group was not included. The animals were observed for signs of toxicity or behavioral changes frequently on the day of treatment. Thereafter, all animals were examined for signs of toxicity twice per day. Body weights were recorded on the day of dosing and weekly thereafter. The surviving animals were euthanized at the conclusion of the observation period. Gross autopsies were performed on all animals after 14 days.
Results LD50 > 5.0 g/kg (males and females) Remarks No deaths or clinical signs of toxicity were observed during the 14-day observation period. The treated males exhibited a slight body weight decrease during the first week post dosing. These body weights recovered during the second week. Body weight gain in the females was normal at week 1 but was less than expected during the second week of the study. Gross necropsy findings were unremarkable.82 Conclusions The test article, when administered as received to 5 male and 5 female Sprague-Dawley rats, had an acute or al LD50 of greater than 5.0 g/kg. Data Quality Reliable without restriction (Klimisch Code) References Unpublished confidential business information
Other Updated: 6/16/00 (RTA-036)83 Robust Summary 3-Acute Oral-5
Test Substance
CAS # CAS# 68783-96-0 Chemical Name Petroleum derived calcium salt, overbased Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed OECD Guideline 401 Test Type Acute oral toxicity (Limit Test) GLP (Y/N) Y Year (Study Performed) 1984 Species/Strain Rats/Sprague-Dawley
Sex Male and female No. of animals/sex/group 5

Vehicle None Route of administration Oral (intragastric) Dose level 5 g/kg Dose volume 4.3 ml/kg Control group included Yes
Remarks field for test
conditions A single dose of 5.0 g/kg of the undiluted test material was administered intragastrically to five fasted (over night) male and
female rats. Five fasted undosed animals of each sex served as the controls. The animals were observed frequently for any physiological or behavioral abnormalities on the day of dosing and at least twice each weekday for 13 days after treatment. On weekends, observations were made once daily. On day 14 the animals were observed once prior to sacrifice. Individual body we ights were recorded on the day of dosing and on days 2, 7 and 14 after dosing. The surviving animals were euthanized at the conclusion of the observation period. Gross autopsies were performed on all animals on Day 14.
Results LD50 > 5.0 g/kg (males and females) Remarks No mortality was observed. Body weights were unremarkable. Slightly reduced food consumption was observed in one treated male (Day 2) and female (Day 1). There were no macroscopic findings
associated with treatment.84 Conclusions The test article, when administered as received to 5 male and 5 female Sprague-Dawley rats, had an acute or al LD50 of greater than 5.0 g/kg. Data Quality Reliable without restriction (Klimisch Code) References Unpublished confidential business information
Other Updated: 2/17/00 (RTA-018)85 Robust Summary 3-Acute Oral-6
Test Substance
CAS # CAS# 71549-79-6 Chemical Name Mixed C15-C30 and C11-13 alkaryl derivative Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed FHSA 16CFR1500.3 Test Type Acute oral toxicity GLP (Y/N) N Year (Study Performed) 1978 Species/Strain Rats/Sherman-Wistar strain
Sex Male

No. of animals/dose 5

Vehicle None Route of administration Oral (intragastric) Dose level 1, 2, 4, 8, and 16 ml/kg Dose volume 1, 2, 4, 8, and 16 ml/kg Control group included No
Remarks field for test
conditions A single dose of the undiluted test material was administered intragastrically to five fasted (over night) male rats at each treatment level. A control group was not included. The animals were observed for signs of toxicity or behavioral changes daily. Individual weights were recorded on the day of dosing and at termination. All animals were euthanized at the conclusion of the observation period. Gross autopsies were performed on all animals after 14 days.
Results LD50 14.9 g/kg (males) Remarks Three 16.0 g/kg animals died on test day 3. Animals at this dose level were depressed at 1-hour post dosi ng and remained in poor health for approximately 7 days before recove ring. A reduced mean body weight compared to the other treated groups was observed in this group at termination. No clinical findings or body weight effects were evident in the other dose groups. Gross necropsy findings were unremarkable
for all animals.
Conclusions The test article, when administered as received to male ShermanWistar rats, had an acute oral LD50 of 14.9 g/kg. Data Quality Reliable without restriction (Klimisch Code) References Unpublished confidential business information
Other Updated: 6/15/00 (RTA-035)86 Robust Summary 3-Acute Oral-7
Test Substance

CAS # CAS# 71786-47-5 Chemical Name Alkaryl magnesium salt derivative Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed FHSA 16CFR1500.3

Test Type Acute oral toxicity GLP (Y/N) N Year (Study Performed) 1980 Species/Strain Rats/Sherman-Wistar strain
Sex Male

No. of animals /dose 5

Vehicle None Route of administration Oral (intragastric) Dose level 1.0, 2.0, 4.0, 8.0 and 16 g/kg Dose volume 1.0, 2.0, 4.0, 8.0 and 16 ml/kg Control group included No

Remarks field for test
conditions A single dose of the undiluted test material was administered intragastrically to five fasted (over night) male rats at each dose level. A control group was not included. All animals were examined for signs of toxicity daily for 14 days. Individual weights were recorded on the day of dosing and at termination. The surviving animals were
euthanized at the conclusion of th e observation period. Gross autopsies were performed on all animals after 14 days.
Results LD50 > 16.0 g/kg (males) Remarks No deaths were observed during the 14-day observation period. The animals at 8 and 16 g/kg exhibited ruffled fur for 18-24 hours post dosing. Within 48 hours all animals appeared normal. No body weight effects were observed. Gross necropsy findings were unremarkable.

Conclusions The test article, when administered as received to male ShermanWistar rats, had an acute oral LD50 of greater than 16.0 g/kg. Data Quality Reliable without restriction (Klimisch Code) References Unpublished confidential business information
Other Updated: 6/16/00 (RTA-034)87 Robust Summary 3-Acute Oral-8
Test Substance CAS # Analog of 78330-12-8 Chemical Name C15-C21 alkaryl sodium salt derivative Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed OECD Guideline 401

Test Type Acute oral toxicity GLP (Y/N) Y Year (Study Performed) 1982 Species/Strain Rats/Sprague-Dawley strain
Sex Male and female

No. of animals/sex/dose 5

Vehicle None Route of administration Oral (intragastric) Dose level 5 g/kg Dose volume 4.5 ml/kg Control group included No

Remarks field for test
conditions A single dose of 5.0 g/kg of the undiluted test material was administered intragastrically to five fasted (over night) male and female rats. A control group was not included. The animals were observed for signs of toxicity or behavioral changes frequently on the day of treatment. Thereafter, all animals were examined for signs of toxicity twice per day. Individual weights were recorded on the day of dosing and weekly thereafter. The surviving animals were euthanized at the conclusion of the observation period. Gross autopsies were performed on all animals after 14 days.
Results LD50 > 5.0 g/kg (males and females) Remarks No deaths were observed during the 14-day observation period. The animals exhibited ruffled fur 3 hours post dosing. Within 24 hours all animals appeared normal. No body weight effects were observed. Gross necropsy findings were unremarkable.

Conclusions The test article, when administered as received to 5 male and 5 female Sprague-Dawley rats, had an acute or al LD50 of greater than 5.0 g/kg. Data Quality Reliable without restriction (Klimisch Code) References Unpublished confidential business information
Other Updated: 2/10/00 (RTA-002)88 

### Robust Summary 3-Acute Oral-9
Test Substance
CAS # CAS# 115733-09-0 Chemical Name C14-24 alkaryl calcium salt derivative Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed OECD Guideline 401 Test Type Acute oral toxicity GLP (Y/N) Y Year (Study Performed) 1981 Species/Strain Rats/Sprague-Dawley strain
Sex Male and female

No. of animals/sex/dose 5

Vehicle None Route of administration Oral (intragastric) Dose level 5 g/kg Dose volume Adjusted for specific gravity of 0.94 g/ml Control group included No
Remarks field for test
conditions A single dose of 5.0 g/kg of the undiluted test material was administered intragastrically to five fasted (approximately 16 hours) male and female rats. The animals were observed for signs of physiological or behavioral changes at 2 and 4 hours post dose. Thereafter all animals were examined for signs of toxicity twice per day. Individual body weights were recorded on the day of dosing and at termination. The surviving animals were euthanized at the conclusion of the observation period. Gross autopsies were performed on all animals after 14 days.
Results LD50 > 5.0 g/kg (males and females) Remarks No mortality was observed. Two rats exhibited slight diarrhea two hours post dosing. On Day 1., most ra ts had slight diarrhea and anal stains. Two rats exhibited slight bloody nasal discharge on Days 2 and 3. All rats were normal on days 4 through 14. No other signs of toxicity were observed. Body weights and necropsy findings were
unremarkable.89 Conclusions The test article, when administered as received to 5 male and 5 female Sprague-Dawley rats, had an acute or al LD50 of greater than 5.0 g/kg. Data Quality Reliable without restriction (Klimisch Code) References Unpublished confidential business information
Other Updated: 6/18/0190 

### 4.1.2 Acute Dermal Toxicity

Robust Summary 3-Acute Dermal-1
Test Substance
CAS # CAS# 115733-09-0

Chemical Name C14-24 alkaryl calcium salt derivative Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed OECD Guideline 402 Test Type Acute dermal toxicity (Limit Test) GLP (Y/N) Y Year (Study Performed) 1981 Species/Strain Rabbits/New Zealand White
Sex Male and female No. of animals/sex/group 5

Vehicle None

Route of administration Dermal Dose level 5 g/kg Dose volume 5 ml/kg Control group included No
Remarks field for test
conditions This study was conducted prior to the development of Test Guideline 402. This study deviated from Guideline 402 in that animals were abraded prior to dosing.
This deviation was not considered suffi cient to change the outcome of the study.

Approximately 24 hours prior to topical application of the test material, the hair of each animal was closely clipped. On the day of dosing the skin was abraded prior to test material administration. A single dose of 5 g/kg of the undiluted test material was administered dermally to five male and female animals. The test material was kept in contact with the skin for a period of 24 consecutive hours under a gauze patch and elastic bandage. The application site was wiped clean of residual test material at the end of the 24-hour exposure period. The animals were observed for abnormal clinical signs frequently on the day of dosing and once daily for 14 days after treatment. Individual body weights were recorded on the day of dosing and on days 7 and 14. The surviving animals we re euthanized at the conclusion of the observation period. Gross necropsies were performed on all animals on Day 14.
Results LD50 > 5.0 g/kg (males and females Remarks Rabbits were normal at 0, 2 and 4 hours post dosing. Upon bandage removal at
24 hours rabbits were distressed. Skin at the dose site was red, swollen and stained with test material. Irritation subsided by day 9, however the skin91 remained dry, flaky and stained throughout the observation period. All animals gained weight during the study. No systemic toxicity was observed. At necropsy 9 rabbits exhibited alopecia, matted fur and flaky skin at or around the test site. One animal had a friable, white, mottled left front liver lobe. One rabbit had a small right testis.
Conclusions The test article, when administered dermally as received to 5 male and 5 female
New Zealand white rabbits had an acute de rmal LD50 of greater than 5.0 g/kg. No evidence of systemic toxicity was observed. Data Quality Reliable without restriction (Klimisch Code) References Unpublished confidential business information
Other Updated: 6/16/00 (RTA-033)92 Robust Summary 3-Acute Dermal-2
Test Substance
CAS # CAS# 61789-86-4

Chemical Name Petroleum derived calcium salt Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed OECD Guideline 402 Test Type Acute dermal toxicity (Limit Test) GLP (Y/N) Y Year (Study Performed) 1985 Species/Strain Rabbits/New Zealand White
Sex Male and female No. of animals/sex/group 5

Vehicle None

Route of administration Dermal Dose level 5 g/kg Dose volume 5 ml/kg Control group included Yes
Remarks field for test
conditions Approximately 24 hours prior to topical application of the test material, the hair of each animal was closely clipped. Elizabethan type collars were placed on the
neck of each rabbit. The skin was left intact. Collars remained on for 24 hours post dosing. Animals were reclipped as needed. A single dose of 5 g/kg of the
undiluted test material was administered dermally to five male and female animals. The test material was kept in contact with the skin for a period of 24 consecutive hours under a plastic wrap, which was over wrapped with paper toweling. The application site was wiped cleaned of residual test material at the end of the 24-hour exposure period. Five clipped, untreated animals/sex were wrapped as described above and served as sham controls. The animals were observed for abnormal clinical signs fre quently on the day of dosing and twice daily for 13 days after treatment. On Day 14 the animals were observed once prior to sacrifice. Dermal examinations (Draize) were performed on day 1, 7 and 14. Individual body weights were recorded on the day of dosing and on days 2, 7 and 14. The surviving animal s were euthanized at the conclusion of the observation period. Gross necropsies were performed on all animals on Day 14. Selected tissues, including the skin were examined microscopically.
Results LD50 > 5.0 g/kg (males and females) Remarks No mortality was observed. Redu ced food intake and nasal discharge were
observed in treated and control animals. Slight to moderate erythema and edema were observed in both sexes 24 hours after treatment. Slight erythema was observed in three animals on Day 7. Dry and flaky skin was observed in all treated animals on Days 6 and 14. Body weights were unremarkable. Gross93 pathological findings included dry, fla ky skin at the dose site of all treated animals. Microscopic examination re vealed the presence of trace to mild hyperkeratosis. There were no other tr eatment related gross or microscopic findings.

Conclusions The test article, when administered dermally as received to 5 male and 5 female
New Zealand white rabbits had an acute de rmal LD50 of greater than 5.0 g/kg. No evidence of systemic toxicity was observed. Data Quality Reliable without restriction (Klimisch Code) References Unpublished confidential business information
Other Updated: 2/17/00 (RTA-020)94 Robust Summary 3-Acute Dermal-3
Test Substance
CAS # CAS# 68783-96-0

Chemical Name Petroleum derived calcium salt, overbased Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed OECD Guideline 402 Test Type Acute dermal toxicity (Limit Test) GLP (Y/N) Y Year (Study Performed) 1993 Species/Strain Rabbits/New Zealand White
Sex Male and female No. of animals/sex/group 5

Vehicle None

Route of administration Dermal Dose level 2 g/kg Dose volume 1.8 ml/kg Control group included No
Remarks field for test
conditions Approximately 24 hours prior to topical application of the test material, the hair of each animal on the dorsal surface from the shoulder region to the lumbar region was closely clipped. Elizabethan t ype collars were placed on the neck of
each rabbit. The skin was left intact. Collars remained on for the duration of the study. Animals were reclipped as needed. A single dose of 2 g/kg of the
undiluted test material was administered dermally to five male and female animals. The test material was kept in contact with the skin for a period of 24 consecutive hours, on approximately 10% of the total body surface under a semi-occlusive bandage that was cove red with an elastic bandage. The application site was wiped clean of residual test material with water at the end of the 24-hour exposure period. The animals were observed for abnormal clinical signs 2 and 4 hours after dosing and once daily for the 14-day study period. Cutaneous examinations (Drai ze) were performed on day 1 (45 minutes after patch removal) and on Days 3, 7, 10 and 14. Individual body weights were recorded on the day of dosing and on day 7 and 14. The surviving animals
were euthanized at the conclusion of the observation period. Gross necropsies were performed on all animals on Day 14.
Results LD50 > 2.0 g/kg (males and females) Remarks No mortality was observed. Clinical observations were unremarkable in 7 of 10
treated animals. Three treated animals exhibited findings consistent with stress and collaring. Findings included sores/scabs in the mouth and in the dorsal cervical area and stool abnormalities. Erythemia was observed in all animals on day 1. Very slight to well defined erythemia was observed in 9 of 10 treated95 animals on Day 3. On Day 7 one animal exhibited slight erythemia and one animal exhibited well-defined erythemia. Slight erythemia was observed in two animals on Days 10 and 14. Edema was not observed in any of the animals. Desquamation was observed in all animals on Day 7 By Day 14 desquamation
was observed in six treated animals. Al l animals exhibited body weight gains during the treatment period. At necropsy 6 of 10 treated animals exhibited desquamation. One animal was noted with tan striations on the liver.

Conclusions The test article, when administered dermally as received to 5 male and 5 female
New Zealand white rabbits had an acute de rmal LD50 of greater than 2.0 g/kg. No evidence of systemic toxicity was observed. Data Quality Reliable without restriction (Klimisch Code) References Unpublished confidential business information
Other Updated: 2/17/00 (RTA-019)96 Robust Summary 3-Acute Dermal-4
Test Substance CAS # Analog of 70024-69-0

Chemical Name C20-C24 alkaryl calcium salt derivative Remarks This substance is an analog for the group of substances in HERTG’s Final Submission for the Alkaryl Sulfonates Category. It is chemically similar to members of this CAS number, but does not belong to this CAS number. For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category..

Method

Method/Guideline
followed OECD Guideline 402 Test Type Acute dermal toxicity (Limit Test) GLP (Y/N) Y Year (Study Performed) 1989 Species/Strain Rats/Sprague Dawley
Sex Male and female No. of animals/sex/group 5

Vehicle None

Route of administration Dermal Dose level 2 g/kg Dose volume Not specified Control group included Yes
Remarks field for test
conditions Approximately 24 hours prior to topical application of the test material, the hair of each control and treated animal was closely clipped. A single dose of 2 g/kg of the undiluted test material was administered dermally to five male and
female animals. The test material was ke pt in contact with the skin for a period of 24 consecutive hours under a plastic film and elastic bandage. Plastic collars were used during the dosing phase. The application site was wiped clean of residual test material at the end of the 24-hour exposure period. The animals
were observed for abnormal clinical si gns frequently on the day of dosing and once daily for 14 days after treatment. Individual body weights were recorded on the day of dosing and on days 2, 7 and 14. The surviving animals were
euthanized at the conclusion of the observation period. Gross necropsies were performed on all animals on Day 14.
Results LD50 > 2.0 g/kg (males and females) Remarks No signs of systemic toxicity were observed. All treated animals exhibited skin irritation. Significant differences in mean body weight were observed between treated and control males on Days 2, 7 a nd 14. At necropsy, multiple pinpoint
scabs were observed in three treated males and one treated female.97 Conclusions The test article, when administered dermally as received to 5 male and 5 female Sprague Dawley rats had an acute dermal LD50 of greater than 2.0 g/kg. No evidence of systemic toxicity was observed. Data Quality Reliable without restriction (Klimisch Code) References Unpublished confidential business information
Other Updated: 10/4/00 (RTA-068)98 

### 4.1.3 Acute Inhalation Toxicity

Robust Summary 3-Acute Inhalation-1
Test Substance
CAS # CAS# 68783-96-0 Chemical Name Petroleum derived calcium salt, overbased Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed OECD Guideline 403 Test Type Acute Inhalation toxicity (Limit Test) GLP (Y/N) Y Year (Study Performed) 1986 Species/Strain Rats/Sprague-Dawley
Sex Male and female

No. of animals/sex 5

Vehicle Oil based material dosed undiluted

Route of administration Aerosol inhalati on (single 4 hour whole body exposure) Dose level 1.9 mg/L (actual maximum attainable concentration) Vehicle control group No Chamber analysis Yes
Remarks field for test
conditions One group of five rats/sex was exposed for 4 hours to the test material as a liquid droplet aerosol genera ted by a pressure spray apparatus delivered into a 100-liter plexi-glass exposure chamber. The actual exposure concentration as measured by gravimetric analysis was 1.9mg/L. Particle size analyses were performed once/hour using a multistage cascade impactor. Animal observations for toxicological signs and mortality were recorded peri odically during exposure and twice daily during the 14 day observation period. Individual body weights were recorded on Day1 (immediately prior to exposure) and on Days 2, 3, 5, 8 and 15. Animals were euthanized by exsanguination under ether anesthesia. All animals were subjected to a complete gross

necropsy. Results LC50 > 1.9 mg/L (males and fe males)(maximum attainable concentration) Remarks The mass median aerodynamic diameter was 4.2 microns with a geometric standard deviation of 1.9 (estimated percent of particles <10 microns=93%). All animals survived the exposure and observation periods. Observations recorded during exposure included reduced activity, matted coat and closed eyes. Observations noted post exposure on Day 1 included lacrimation, nasal discharge, salivation, , rales, matted coat, hunched appearance, soft stool and closed eyes.99 These findings decreased in inciden ce over the next week. Animals were free of symptoms of exposure during the second week of
observation. Several animals exhib ited very slight body weight losses on Day 2. Body weights recovered and were unremarkable by Day 5. There were no abnormal postmortem findings evident in any of the animals at study termination.

Conclusions Following 4-hour whole body exposure to a liquid droplet aerosol of the test material the LC50 in male and female Sprague Dawley rats was >1.9 mg/L. This was the maximum concentration attainable. Data Quality Reliable without restriction (Klimisch Code) References Unpublished confidential business information
Other Updated: 2/18/00 (RTA-023)100 

### 4.2 Repeated Dose Toxicity

Robust Summary 3-Repeated Tox - 1
Test Substance CAS # Analog of 70024-69-0 Chemical Name C20-24 alkaryl calcium salt derivative Remarks This substance is an analog for the group of substances in HERTG’s Final Submission for the Alkaryl Sulfonates Category. It is chemically similar to members of this CAS number, but does not belong to this CAS number. For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed OECD 407

Test Type 28-day oral toxicity study in rats GLP (Y/N) Y Year (Study Performed) 1989 Species Rat Strain Sprague-Dawley CD, 41 days old at initiation of treatment Route of administration Oral ga vage (syringe and dosing tube) Duration of test 29 days of treatment followed by 14 day recovery period in the control and high dose satellite recovery groups Doses/concentration levels 0, 100, 500 and 1000 mg/kg/day
Sex Males and females Exposure period 29-day treatment dur ation with a 14 day recovery Frequency of treatment 7 days/week Control group and

treatment 6 rats/sex/group for each dose, and satellite recovery groups of 6 animals/sex for the control and 1000 mg/kg/day dose. Control group received daily doses of peanut oil at 2.0 ml/kg, and treatment groups received the indicated dose of test material diluted in peanut oil at a dose volume of 2.0 ml/kg Post exposure observation period 14-days

Statistical methods Body weight, food consumption, feed efficiency, 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 parame tric ANOVA with a Dunnett’s post-hoc test, non-parametric Kruskal-Wallis and a Mann- Whitney U-test, Bartlett’s test for equal variances, a Student’s t -test and Dixon’s test for rejection of outlying
values. Dose rangefinding study Yes (Pilot two-week repeated dose oral toxicity study)
Remarks field for test
conditions Single oral doses were administered for 29 consecutive days using a gavage
needle. Clinical observations were made daily. Viability checks were performed twice daily. Body weight were recorded twice weekly during treatment and weekly during recovery. Terminal body weights were recorded.101 Food consumption were recorded during treatment and recovery. Hematology, clinical chemistry and urinalysis parame ters were evaluated at termination of treatment and recovery. Macroscopic examinations were performed on all animals. Select organs were weighed. A range of tissues was examined microscopically.

Significant deviations from the OECD 407 test guidelines include:

• A function observational battery for neurotoxicity was not performed since this test was not part of the OECD 407 guideline at the time the study was performed.

• Microscopic pathology was perfo rmed as required by OECD 407 guideline in place at the time the study was conducted.

Results Remarks An NOEL of 500 mg/kg/day was established for this study. No test material related mortality w as observed. One low dose male was found dead on Day 9. This was attributed to a probable misdosing. A second low dose male was replaced, due to a possible misdosing, on the first day of treatment. Mean serum cholesterol levels were significantly reduced in the 1000 mg/kg males and females at termination of dosing and inthe 1000 mg/kg females at the end of the 14-day recovery period. No treatmentrelated effects were observed on mortalit y, clinical observations, body weight and body weight gain, food consumption, feed efficiency, hematology, urinalysis, absolute and relative organ weights and macroscopic or microscopic pathology. Statistically significant differences from control were observed for some hematology and clinical chemistry para meters. These values were within clinically normal limits and were not associated with corresponding histopathological changes. They were not considered biologically significant. Chemical analysis of dosing solutions co nfirmed that they were homogeneously prepared at the desired concentrations.

Conclusions Little subchronic toxicity was observed over the range of doses administered in this study. Based on a reduction in m ean cholesterol values in the males and females treated at the 1000 mg/kg dose level, the NOEL was 500 mg/kg. Data Quality Reliable without restriction (Klimisch Code) References Unpublished confidential business information
Other Updated: 2/11/00 (RTA-006)102 Robust Summary 3-Repeated Tox -2
Test Substance
CAS # CAS# 68783-96-0 Chemical Name Petroleum derived calcium salt, overbased Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed OECD 410

Test Type 28-day dermal toxicity study in rats GLP (Y/N) Y Year (Study Performed) 1995 Species Rat Strain Sprague-Dawley CD, 8-9 weeks of age at initiation of treatment Route of administration Dermal, 6 hour/day, to the clipped, unabraided, dorsal surface. Duration of test 28 days of treatment followed by 14 day recovery period in the high dose satellite recovery group only. Doses/concentration levels 0, 100, 300 and 1000 mg/kg/day
Sex Males and females Frequency of treatment Once/day, 7 days/week
Control and treatment

groups 5 rats/sex in the control group, in each dose level and in the satellite recovery group at the1000 mg/kg/day dose. The control group received no treatment (sham control). The test material was administered undiluted to the treated animal based on individual animal body weight. Post exposure observation period 14-days (High dose group only)

Dose rangefinding study Dose levels were selected b ased on results of a rangefinding study conducted at dose levels up to 1000mg/kg/day. No signs of toxicity were observed. Statistical methods Body weight, food consumption, hematology and clinical chemistry parameters, organ weights and organ/body weight ratios were analyzed. Mean values of all dose groups were compared to control at each time interval. Tests included parametric ANOVA with a Dunnett’s test and regression analysis for linear response, non-parametric Kruskal-Wallis and Dunn’s Summed Rank Test,
Jonckheere’s test for monotonic trend. A Student’s t -test was used to compare the satellite group’s main study termin ation and recovery blood values and organ weights.
Remarks field for test
conditions The test material was applied to the clipped, unabraided dorsal surface of the rats for 6 hours/day, 7 days/week for 28 days. The gauze patch was secured to the trunk with non-irritating tape and wra pped with an elastic sleeve. After at least 6 hours the test material residue was removed from the skin with peanut oil and a paper towel. Clinical obser vations were made daily. Dermal responses were evaluated (Draize) prior to dosing on days 0, 1, 4, 7, 11, 14, 18, 21, and 25; prior to blood collection on day 28 and after sleeve removal on day
0. Satellite animals were also evalua ted on Days 32, 35, 40 and 42. Body103 weight and food consumption were reco rded during treatment and recovery. Hematology and clinical chemistry parame ters were evaluated at termination of treatment and recovery. Macroscopic examinations were performed on all animals. Select organs were weighed. A range of tissues was examined microscopically.
Results Remarks A NOAEL of 1000 mg/kg was establishe d for this study. No mortality occurred during this study. Low incidences of very slight erythemia, desquamation and/or pinpoint scabbing were observed sporadically in the treated animals. All animals were free of edema during the study. Body weights and food consumption data were unremarkable during the treatment and recovery periods. There were no treatment-rela ted differences from control observed in the hematology data of the treated animals following the dosing or recovery periods. Differences from control we re noted for several hematology parameters including a statistically significant increase in the mean percentage of neutrophils of the 300 and 1000 mg /kg females and a decrease in mean percentage of lymphocytes in the 1000 mg/kg females compared to control on Day 28. There was a statistically signifi cant decrease in mean percentage of basophils in the satellite females from Da y 28 to 42. However these values were within the normal range. In the absence of differences from control in absolute white blood cell counts, these findings were considered unrelated to treatment. There was a statistically significan t decrease in the mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration of the male satellite animals from Day 28 to 42. In the absence of other significant findings in mean hemoglobin or red blood cell pa rameters, these small differences were not considered clinically significant. Serum chemistry values were unremarkable in the treated animals at termination of the treatment and recovery periods. There was a slight increase in the mean aspartate aminotransferase and alanine aminotransferase of the high dose females at Day 28. These increases
were attributed to two females with high values. Similar changes were not
observed in the satellite females or in the males at Day 28. These increases
were not considered related to treatment. There were several differences from control noted at the end of recovery. These values were within the range of
normal and similar differences were not evident at the end of the treatment period indicating that these findings were not clinically significant or treatment related. Gross postmortem findings were limited to one 300 mg/kg male with small testes, one control female with discolored lungs and liver and black material in the stomach; and single o ccurrences of scabs in the 100 and 1000 mg/kg and recovery males. These findings were considered incidental and unrelated to treatment. Tape irritation was observed in a number of animals. There were no alterations in organ weights that were attributed to treatment with the test material. Slight alterations were noted in several organ weights at termination of dosing or recovery. There was a statistically significant decrease in mean absolute brain weight of the 3 00 mg/kg females compared to control. This finding lacked a dose response a nd was not considered biologically significant. There was a statistically significant decrease in mean relative adrenal and testes weights of the male satellite animals at termination of recovery compared to control at end of treatment. Compared to the high dose at104 study termination there was a statistical ly significant decrease in mean relative adrenal, brain and testes weight of the male satellite animals and mean relative adrenal and brain weight of the female sa tellite animals at recovery termination. These alterations in organ weights were a ttributed to the cessation of the stress associated with wrapping (adrenal) and the animals continued increase in body weight while organ weights remained cons tant in adult animals. In the absences of significant organ weight findings following treatment or correlating effects with histopathology these findings were not considered clinically significant.
There were no test material related mi croscopic findings noted in any group. Livers from female rats of all groups (i ncluding control) sacrificed after 28 days of treatment exhibited focal necrosis. This finding did not exhibit a dose response. This finding has been seen in other dermal studies and has been attributed to trauma and/or ischemia to the liver resulting from the wrapping and manipulation of the animals. Liver necrosis was not evident in any of the satellite recovery animals. This finding was not considered treatment related.
The treated skin of most animals reveal ed variable amounts of thickening of the epidermis due to acanthosis and hyperkeratosis, sebaceous gland hyperplasia
and focal dermal inflammation. These ch anges occurred in all groups including control. However the severity of these changes tended to be increased in the male treated group rats and in the fe males of the 300 and 1000 mg/kg groups, suggesting a mild irritating effect of the test material. Following recovery these findings were less severe.
Conclusions A NOAEL of 1000 mg/kg was established fo r this study. Under the conditions of this study dermal application of this test material resulted in no signs of overt systemic toxicity. Data Quality Reliable without restriction (Klimisch Code) References Unpublished confidential business information
Other Updated: 2/18/00 (RTA-024)105 Robust Summary 3-Repeated Tox-3
Test Substance
CAS # CAS# 68783-96-0 Chemical Name Petroleum derived calcium salt, overbased Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed OECD 412

Test Type 28-day inhalation toxicity study in rats GLP (Y/N) Y Year (Study Performed) 1986 Species Rat Strain Sprague-Dawley CD, 6-7 weeks of age at initiation of treatment Route of administration Aerosol inhalation, whole body exposure Duration of exposure 6 hours/day Doses/concentration levels 49.5, 156, 260 mg/m 3 (measured concentration)
Sex Males and females Frequency of treatment 5 days/week for 4 weeks
Control and treatment

groups 5 rats/sex

Post exposure recovery period None

Statistical methods Body weight, food consumption, hematology and clinical chemistry parameters, organ weights and organ/body weight ratios were analyzed. Mean values of all dose groups were compared to control at each time interval. Tests included parametric ANOVA with a Dunnett’s test and regression analysis for linear response, non-parametric Kruskal-Wallis and Dunn’s Summed Rank Test,
Jonckheere’s test for monotonic trend Dose rangefinding study No
Remarks field for test
conditions Treated animals were exposed to the test material as a liquid droplet aerosol generated by a pressure spray apparatu s delivered into a 1000-liter glass and stainless steel exposure chamber. Chamber airflow rates was approximately
200 liters/minute with a chamber 99% e quilibration time of 22 minutes. Control animals were exposed to room air only. Chamber exposure concentrations were measured by gravimetric analysis at one and one half-hour intervals. Particle size analyses were performed once/week using a multi-stage cascade impactor. Animal observations for toxicological signs and mortality were recorded periodically during exposure and twice daily during the 14 day observation period. Individual body weights were recorded weekly. Hematology and clinical chemistry evaluations were perf ormed on all animals prior to terminal sacrifice. Animals were euthanized by exsanguination under ether anesthesia. Macroscopic examinations were performed on all animals. Select organs were weighed. A range of tissues was examined microscopically.106 Results Remarks The actual exposure concentrations measured by gravimetric analysis were 49.5, 156, 260 mg/m 3 . The mass median aerodynamic diameter of the aerosol ranged from 3.3 to 3.7 microns, with an average geometric standard deviation range of 2.0 to 2.1. These data confirme d that the aerosol was respirable in the rat (estimated percent of particles <10 microns=93%). There was no test material exposure related mortality du ring the study. One low dose animal escaped from its cage and was euthanized. One control male died during blood collection immediately prior to its scheduled sacrifice. Red nasal discharge, matted coat and decreased activity were noted at the two higher concentrations. The mean body weight gain of the high dose males was slightly reduced over the four weeks of study. Body weights and gains in the other groups were unremarkable. Clinical chemistry and hematology data exhibited no patterns indicative of a treatment-related effect. Several incidental statistically significant differences from control we re observed these included: increased hematocrit (low dose females), creatinine phosphokinase (low and high dose females) and sodium (high dose females). These differences were not attributed
to treatment. Dose related increases in absolute and relative (to body weight) lung weights were observed in the mid and high dose males and females. Increases were statistically significant, with the exception of mid dose female absolute lung weight. Microscopically the accumulation of intraalveolar macrophages (males: 3,5,5,5; females: 5,5,5,5) and hyperplasia/hypertrophy of bronchiole epithelium (males: 3,4,5,5; female s: 4,5,5,5) were seen in the control and treated groups. While these findings were observed in control and treated animals the severity of the lesions exhibited a dose response in the mid and high dose groups and was considered treatment related. Differences in severity between the control and low dose group were equivocal. Based on these findings the lowest dose level (49.5 mg/m 3 ) is considered the NOAEL by this reviewer.

Conclusions Under the conditions of this study inhalation exposure of this test material resulted in minimal toxicity over the range of doses administered. A NOAEL of 49.5 mg/m 3 was established for this study based on the slight, dose related increase observed in the severity of microscopic pulmonary findings and increased lung weights. Data Quality Reliable without restriction (Klimisch Code) References Unpublished confidential business information
Other Updated: 2/24/00 (RTA-025)107 Robust Summary 3-Repeated Tox - 4
Test Substance
CAS # CAS# 71786-47-5 Chemical Name Alkaryl magnesium salt derivative Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed OECD 410

Test Type 28-day dermal toxicity study in rabbits GLP (Y/N) Y Year (Study Performed) 1981 Species Rabbit Strain New Zealand White (SPF) (approximate ly 2 kg in body weight at initiation) Route of administration Dermal, 6 hour/day, 5 days /week, to the clipped, unabraided, dorsal surface. Duration of test 20 days of treatment followed by 4 week recovery period Doses/concentration levels 0, 25 and 100% (w/v) (OECD Guideline 410 suggests three treated groups and a control be included in this study design. The lowest dose level should be free of toxic effects. These suggestions were not met in this study.)
Vehicle control Primol 205 Dose volume 2 mL/kg/day
Sex Males and females Frequency of treatment Once/day, 5 days/week for a total of 20 doses.
Vehicle control and treatment groups 15 rabbits/sex in the vehicle control group and in both treated groups. Five of the initial 15 animals/sex/group served as recovery animals. The control group
received the vehicle. An untreated cont rol group was not included in the study.
The test material was administered undilu ted to the treated animal in the high dose group. The animals in the low dose group received the test material
diluted in the vehicle. Doses were administered based on individual animal body weights. Post exposure observation period 4 weeks

Statistical methods Body weight, food consumption, hematology and clinical chemistry parameters, organ weights and organ/body weight ratios were analyzed. Mean values of all dose groups were compared to control at each time interval. Tests included parametric ANOVA with a Dunnett’s test and regression analysis for linear response, non-parametric Kruskal-Wa llis and Dunn’s Summed Rank Test,
Jonckheere’s test for monotonic trend.
Remarks field for test
conditions The test material was applied to the clipped, unabraided dorsal surface of the rabbits for 6 hours/day, 5 days/week for 20 days. Elizabethan collars were used to prevent ingestion. (OECD Guidelin e 410 suggests the use of a gauze patch
over the treatment site secured to the trunk with non-irritating tape and wrapped with an elastic sleeve. This procedure w as not used during this study. This is considered a minor deviation from the Guideline.) After approximately 6 hours108 the test material residue was removed from the skin with a paper towel, if necessary. Clinical observations were made weekly. Dermal responses were evaluated daily during treatment (7 days/week; prior to dosing on dosing days) and recovery. Body weight was recorded weekly during treatment and recovery. (OECD Guideline 410 suggests the recording of food consumption. This parameter was not recorded during this study. This is considered a minor deviation from the guideline.) Hematology and clinical chemistry parameters were evaluated pretest and at termin ation of treatment and recovery. Macroscopic examinations were performed on all animals. Select organs were weighed. A range of tissues was examined microscopically in the control and high dose animals sacrificed at the end of the treatment period and for all found dead and moribund sacrifice animals. In addition the liver, testes and epididymides were evaluated in all low dose animals.

Results Remarks One control and four high dose animal s died or were sacrificed early during this study. One control female was sacrificed moribund during recovery (test day 35). Two high dose males were sacrificed moribund during the treatment period (test days 23 and 32). One high dose male was found dead (test day 49) during recovery. One high dose female was sacrificed moribund during recovery (test day 39). The cause of death of these animals was not established. Alopecia was observed in many of the low and high dose males and females during the last two to three weeks of tr eatment and during the first two to three weeks of recovery. Several high dose males and females exhibited this finding throughout recovery. Erythema, edema, atonia, desquamation, fissuring and exfoliation were observed in all of the low and high dose animals throughout the treatment period. Most of these findi ngs were evident during recovery with a decreasing severity and incidence. These data did not exhibit a strong dose response. Erythema and desquamation we re observed in the control males and females during the treatment and recovery periods. These findings were less
severe then those observed in the treated animals. As in the treated groups severity and incidences decreased with time during recovery. The mean body weights of the low dose males and fema les were slightly lower (~5%) than control during the last two weeks of treatment and during the first week of recovery. The mean body weights of the high dose males and females were lower than control (5-15%) during the last two weeks of treatment and throughout recovery. Some of the difference from control observed during recovery may be due to the small number of animals (2-5) available in each group and the normal variability expected in rabbit weight.

The mean total leukocyte count of the low and high dose males and females were statistically significantly lower than control at termination of the treatment period. Low and high dose males and high dose females were also slightly reduced at the end of recovery. In addition the mean hemoglobin and hematocrit values and the mean erythrocyte count of the high dose females were significantly reduced following treatment but not following recovery. The low and high dose males and females exhibited slight or statistically significant, dose-related decreases in total protein and globulin and increased109 albumin/globulin ratios at termination of treatment. In addition albumin was slightly reduced in the high dose females. At termination of the recovery period the mean globulin level of the low do se females was significantly reduced and the albumin/globulin ratios of the low and high dose females were slightly (statistically significantly) increased compared to control. At termination of treatment the low and high dose males exhibited increases in mean SGOT and alkaline phosphatase. The low and high dose females exhibited increases in SGOT and SGPT. These enzymes were unremarkable following recovery. (The changes observed in SGOT and SG PT were not discussed in the original final report of this study.)

Treatment related decreases were observed in the absolute and relative (to body weight) testes and epididymides weights of the low and high dose males at the end of the treatment and recovery periods. Absolute testes weights were decreased -21 and -35%, compared to control, in the low and high dose groups following treatment and -22 (low dose) and -58% (high dose) following recovery. Treatment related increases were observed in the absolute and relative (to body weight) liver weights of the low and high dose males (+5/+30%- absolute weight) and females (+12/+23% -absolute weight) following treatment and in the high dose males (+14%-absolute weight) following recovery.

Macroscopic examinations revealed de rmal findings consistent with those observed during the in life examinations. The testes of many low and high dose animals were noted to be small in size at the end of the treatment period. This observation was recorded in one high dose recovery animal. These data are consistent with the reduced testes weights observed in the low and high dose

groups.

Microscopic evaluations revealed treatme nt related morphologic changes in the skin, testes, epididymides and possibly the liver. Compound related microscopic lesions were seen in the tr eated skin of the high dose animals at termination of the treatment period (hig h dose recovery and low dose treatment and recovery animals were not examined). Treated skin findings included slight to moderately severe hyperkeratosis a nd epithelial hyperplasia. Findings in males and females were comparable. Possible treatment related liver findings were observed in the high dose group on ly. Findings present at termination of dosing but not following recovery includ ed the presence of multifocal areas of minimal to moderate hepatocellular degeneration usually accompanied by
multifocal areas of necrosis and/or multifocal areas of coarse cytoplasmic vacuolation of hepatocytes. Testicular changes were observed in the high dose males only following treatment and recovery. No changes were evident in the low dose males. Alterations observed in the high dose included aspermatogenesis, reduced numbers of spermatids, and multifocal to diffuse tubular hypoplasia. Epithelial hypopl asia of the epididymis accompanied the testicular changes in many animals at te rmination of treatment but not following recovery. There were no other findings observed in this study that were
considered treatment related.110 Conclusions Based on the findings observed during this study this reviewer has concluded that an NOAEL was not established for this study. Data Quality Reliable without restriction (Klimisch Code) References Unpublished confidential business information
Other Updated: 4/13/00 (RTA-029)111 Robust Summary 3-Repeated Tox - 5
Test Substance
CAS # CAS# 71786-47-5 Chemical Name Alkaryl magnesium salt derivative Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed OECD 410

Test Type 28-day dermal toxicity study in rats GLP (Y/N) Y Year (Study Performed) 1995 Species Rat Strain Sprague-Dawley CD, 8-9 weeks of age at initiation of treatment Route of administration Dermal, 6 hour/day, to the clipped, unabraided, dorsal surface. Duration of test 28 days of treatment followed by 14 day recovery period in the high dose satellite recovery group only. Doses/concentration levels 0, 100, 300 and 1000 mg/kg/day
Sex Males and females Frequency of treatment Once/day, 7 days/week
Control and treatment

groups 5 rats/sex in the control group, in each dose level and in the satellite recovery group at the1000 mg/kg/day dose. The control group received no treatment (sham control). The test material was administered undiluted to the treated animal based on individual animal body weight. Post exposure observation period 14-days (High dose group only)

Statistical methods Body weight, food consumption, hematology and clinical chemistry parameters, organ weights and organ/body weight ratios were analyzed. Mean values of all dose groups were compared to control at each time interval. Tests included parametric ANOVA with a Dunnett’s test and regression analysis for linear response, non-parametric Kruskal-Wallis and Dunn’s Summed Rank Test,
Jonckheere’s test for monotonic trend. A Student’s t -test was used to compare the satellite group’s main study termin ation and recovery blood values and organ weights. Dose rangefinding study Dose levels were selected b ased on results of a rangefinding study conducted at dose levels up to 1000mg/kg/day. No signs of toxicity were observed.
Remarks field for test
conditions The test material was applied to the clipped, unabraided dorsal surface of the rats for 6 hours/day, 7 days/week for 28 days. The gauze patch was secured to the trunk with non-irritating tape and wra pped with an elastic sleeve. After at least 6 hours the test material residue was removed from the skin with peanut oil and a paper towel. Clinical obser vations were made daily. Dermal responses were evaluated (Draize) prior to dosing on days 0, 1, 4, 7, 11, 14, 18, 21, and 25; prior to blood collection on day 28 and after sleeve removal on day
0. Satellite animals were also evalua ted on Days 32, 35, 40 and 42. Body weight and food consumption were reco rded during treatment and recovery.112 Hematology and clinical chemistry parame ters were evaluated at termination of treatment and recovery. Macroscopic examinations were performed on all animals. Select organs were weighed. A range of tissues was examined microscopically.

Results Remarks A NOAEL of 1000 mg/kg was establishe d for this study. No treatment related mortality was observed. One 300 mg/kg female was found dead on Day 19. This death was attributed to the wrapping procedure. One 1000 mg/kg male died following blood collection at study termination. Desquamation was observed in one 300 mg/kg female on days 4 and 7. No other significant clinical in life or dermal observations were observed. Body weights and food consumption data were unremarkable during the treatment and recovery periods. There were no treatment-rela ted differences from control observed in the hematology data of the treated animals following the dosing or recovery periods. Differences from control we re noted for several hematology parameters including a decrease in mean percentage of eosinophils in low and
mid dose males at termination of treatment. These values were within the normal range and did not exhibit a dose response. Following the recovery period there were several statistically significant differences from control noted in the hematology parameters of the satellite animals. These included decreases in mean white blood cell count, absolute lymphocytes and basophils in the females; an increase in mean percentage of large unclassified cells in males and females; and an increase in mean corpuscular hemoglobin in the females. All of these differences were within the expected range of normal and were not considered clinically significant. Increases observed in mean prothrombin time and activated partial thromboplastin time (APTT) in the male recovery animals and in mean APTT of the female recovery animals were attributed to variations in bleeding technique. Serum chemis try values were unremarkable in the treated animals at termination of the treatment period. Following recovery there were a number of small, but statisti cally significant differences observed in serum chemistry parameters of the sate llite animals. These included a decrease in mean blood urea nitrogen (males), sodium (males) and chloride (females) and increases in phosphorus and billirubin (mal es) and calcium, total billirubin and triglycerides (females). All of these findings were within the range of normal values and were comparable to control following the termination of dosing. These findings were not considered clinically significant or related to treatment. One female (300 mg/kg) which died on Day 19 exhibited an enlarged liver, ascites in the abdominal cavity and a reddened jejunum. This death was attributed to the wrapping procedure. There were no gross postmortem observations or alterations in organ weig hts that were attributed to treatment with the test material. Slight alterations were noted in several organ weights at termination of dosing or recovery. These included a statistically significant increase in absolute and relative liver weight in the 100 mg/kg females and statistically significant decreases in relative brain and ovary weights in the 1000mg/kg females at termination of recovery. These findings did not correlate with any histopathological findings and were not attributed to treatment. There were no test material related microscopic findings noted in any group. One male113 and one female (1000 mg/kg) has epider mal acanthosis/hyperkeratosis. The male also exhibited slight focal epith elial spongiosis. Similar lesions were observed in the skin of one untreated control female. These findings were attributed to the repeated clipping and tape irritation in both treated and sham control animals. No skin changes were noted following recovery. Livers from rats of all groups (including control) sacrificed after 28 days of treatment
exhibited focal or multifocal necrosis. This was an acute change which was characterized by coagulative necrosis of hepatocytes and occurred in a random fashion. This finding has been seen in other dermal studies and has been attributed to trauma and/or ischemia to the liver resulting from the wrapping and manipulation of the animals. Liver necrosis was not evident in any of the satellite recovery animals.
Conclusions A NOAEL of 1000 mg/kg was established for this study. Dermal application of this test material resulted in no signs of overt toxicity. Data Quality Reliable without restriction (Klimisch Code) References Unpublished confidential business information
Other Updated: 2/15/00 (RTA-009)114 . 

### Robust Summary 3-Repeated Tox - 6
Test Substance
CAS # CAS# 115733-09-0 Chemical Name Benzenesulfonic acid, C1 4-C24 branched & linear alkyl derivs.
Calcium salts Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed OECD 407; OPPTS 870.3050

Test Type 28-day oral toxicity study in rats GLP (Y/N) Y Year (Study Performed) 2002-2003 Species Rat Strain Sprague-Dawley CD, 6-7 weeks of age at initiation of treatment Route of administration Oral gavage Duration of test 28 days of treatment followed by 14 day recovery period in the control and high dose groups Doses/concentration levels 0, 50 , 150, 500 and 1000 mg/kg/day
Dose Formulation Analysis Analysis performed for dosing solution stability, homogeneity and
concentration.
Sex Males and females Exposure period 28-day treatment dur ation with a 14 day recovery Frequency of treatment Once daily, 7 days/week Control group and

treatment 5 rats/sex/group for each dose, and recovery groups of 5 animals/sex for the control and 1000 mg/kg/day dose. Control group received daily doses of corn oil at 5.0 ml/kg, and treatment groups received the
indicated dose of test material diluted in corn oil at a dose volume of 5.0 ml/kg Post exposure observation period 14-days

Statistical methods Body weight, body wei ght change, food cons umption, functional observational battery observations, he matology and clinical chemistry parameters, organ weights, organ/ body and organ to brain weight ratios were analyzed. Mean values of all dose groups were compared to control at each time interval. Tests included parametric ANOVA with a Tukey-Kramer test, non-pa rametric Kruskal-Wallis and a Dunn’s test, Fisher’s Exact test, Chi-Square test, and Levene’s test as appropriate.
Remarks field for test
conditions Single oral gavage doses were administered for 28 consecutive days.
Clinical observations were made daily, between one-half and two hours following dosing and once daily during recovery. Viability checks were performed twice daily. Ophthalmology examinations were performed during study w eeks 3 and 5. An abbreviated functional observation battery (FOB) was performed once prior to115 dosing initiation and once during weeks 0, 1 and 2. A full FOB assessment (home cage, removal from home cage, open field, manipulative tests and motor activity) was performed during weeks 3
and 5. All FOB assessments were performed blind. Body weights were recorded on test days –2, 0, 7, 14, 21, 27, 35 and 41.Fasted body weights were recorded at necropsy. Food consumption was recorded during treatment and recovery. Hematology, clinical chemistry and urinalysis parameters were evaluate d at termination of treatment and recovery. Macroscopic examinations were performed on all animals. Select organs were weighed. All tissues were examined microscopically in the control and high dose groups. Stomachs were
evaluated in all animals.
Results Remarks An NOAEL (no observed adverse effect levels) of 1000 mg/kg/day was established for males and of 150 mg/kg/day was established for females for this study.

Body weight effects were limited to the males at 500 and 1000 mg/kg/day and included decreased weight gain during the third study week. Overall weight gain was redu ced, compared to control, in the 500 and 1000 mg/kg males by approximately 9 and 6% at the end of treatment. The weight gain of the 1000 mg/kg/day males was approximately 6% lower than control at the end of recovery. Statistically significant reductions in mean food consumption were observed in the 500 mg/kg/day males during week 3 of the treatment period and in the 1000 mg/kg/day females during week 2 of the treatment period.

Notable microscopic changes were limited to irritation of the nonglandular stomach in the 500 and 1000 mg/kg/day males and in the 150, 500 and 1000 mg/kg/day females. This finding was transient and was not evident in the recovery animals. In the 500 mg/kg/day males,
minimal edema in the submucosa was observed in 2 of 5 animals. In the 1000 mg/kg/day males minimal to mild edema in the submucosa and minimal epithelial hyperplasia were observed in 3 of 5 animals at
the end of treatment. No stomach abnormalities were evident after

recovery.

In the 150 mg/kg/day females, mi nimal edema in the submucosa was observed in 2 of 5 animals. In the 500 mg/kg/day females, mild edema in the submucosa, minimal hemorrhage, minimal epithelial hyperplasia, mild inflammation and a mild ulcer were observed in 1 of 5 animals. In the 1000 mg/kg/day females minimal edema in the
submucosa was observed in 1 of 5 animals and minimal to mild epithelial hyperplasia was observed in 2 of 5 animals at the end of
treatment. No stomach abnormalities we re evident after recovery. No stomach abnormalities were evident after recovery.116 No mortality, significant clinical abnormalities, meaningful neurological changes, ophthalmoscopic changes, hematology, clinical chemistry, urinalysis, absolute and relative organ weights and macroscopic pathology were observed during treatment or recovery.

Chemical analysis of dosing solu tions confirmed that they were homogeneously prepared and stable at the desired concentrations. Weekly concentration analysis confirmed that the dosing solutions were prepared appropriately.
Conclusions Little subchronic toxicity was observed over the range of doses administered in this study. Based on the microscopic data the Study Director concluded that the NOAEL was 1000 mg/kg/day in males and 150 mg/kg/day in females. Data Quality Reliable without restriction (Klimisch Code) References Unpublished confidential business information
Other Updated: 6/7/2005; SLI Study No.: 3580.1117 

### Robust Summary 3-Repeated Tox - 7
Test Substance
CAS # CAS# 68783-96-0 Chemical Name Petroleum derived calcium salt, overbased Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed OECD 410

Test Type 28-day dermal toxicity study in rats GLP (Y/N) Y Year (Study Performed) 1995 Species Rat Strain Sprague-Dawley CD, 8-9 weeks of age at initiation of treatment Route of administration Dermal, 6 hour/day, to the clipped, unabraided, dorsal surface. Duration of test 28 days of treatment followed by 14 day recovery period in the high dose satellite recovery group only. Doses/concentration levels 0, 100, 300 and 1000 mg/kg/day
Sex Males and females Frequency of treatment Once/day, 7 days/week
Control and treatment

groups 5 rats/sex in the control group, in each dose level and in the satellite recovery group at the1000 mg/kg/day dose. The control group received no treatment (sham control). The test material was
administered undiluted to the treated animal based on individual animal body weight. Post exposure observation period 14-days (High dose group only)

Dose rangefinding study Dose levels were select ed based on results of a rangefinding study conducted at dose levels up to 1000mg/ kg/day. No signs of toxicity
were observed. Statistical methods Body weight, food consum ption, hematology and clinical chemistry parameters, organ weights and organ/body weight ratios were analyzed. Mean values of all dose gr oups were compared to control at each time interval. Tests included parametric ANOVA with aDunnett’s test and regression analysis for linear response, nonparametric Kruskal-Wallis and Dunn’s Summed Rank Test,
Jonckheere’s test for monotonic trend. A Student’s t -test was used to compare the satellite group’s main study termination and recovery blood values and organ weights.
Remarks field for test
conditions The test material was applied to the clipped, unabraided dorsal surface of the rats for 6 hours/day, 7 days/week for 28 days. The gauze patch
was secured to the trunk with non-i rritating tape and wrapped with an
elastic sleeve. After at least 6 hours the test material residue was118 removed from the skin with peanut oil and a paper towel. Clinical observations were made daily. Dermal responses were evaluated (Draize) prior to dosing on days 0, 1, 4, 7, 11, 14, 18, 21, and 25; prior to blood collection on day 28 and after sleeve removal on day 0. Satellite animals were also evaluated on Days 32, 35, 40 and 42. Body weight and food consumption were recorded during treatment and recovery. Hematology and clinical chemistry parameters were evaluated at termination of treatment and recovery. Macroscopic examinations were performed on all animals. Select organs were weighed. A range of tissues was examined microscopically.
Results Remarks A NOAEL of 1000 mg/kg was established for this study. No mortality occurred during this study. Low inci dences of very slight erythemia, desquamation and/or pinpoint scabbing were observed sporadically in the treated animals. All animals were free of edema during the study. Body weights and food consumption data were unremarkable during the treatment and recovery periods. There were no treatment-related differences from control observed in the hematology data of the treated animals following the dosing or recovery periods. Differences from control were noted for several hematology parameters including a statistically significant increase in the mean percentage of neutrophils of the 300 and 1000 mg/kg females and a decrease in mean percentage of lymphocytes in the 1000 mg/kg females compared to control on Day 28. There was a statistically significant decrease in mean percentage of basophils in the satellite females from Day 28 to 42. However these values were within the normal range. In the
absence of differences from control in absolute white blood cell counts, these findings were considered unrelated to treatment. There was a statistically significant decrease in the mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration of the male satellite animals from Day 28 to 42. In the absence of other significant findings in mean hemogl obin or red blood cell parameters, these small differences were not considered clinically significant. Serum chemistry values were unremarkable in the treated animals at termination of the treatment and recovery periods. There was a slight increase in the mean aspartate aminotransferase and alanine aminotransferase of the high dose females at Day 28. These increases were attributed to two females with high values. Similar changes were not observed in the satellite females or in the males at Day 28. These
increases were not considered related to treatment. There were several differences from control noted at the end of recovery. These values were within the range of normal and similar differences were not evident at the end of the treatment period indicating that these findings were not clinically significant or treatment related. Gross postmortem findings were limited to one 300 mg/kg male with small testes, one control female with discolored lung s and liver and black material in the stomach; and single occurrences of scabs in the 100 and 1000 mg/kg and recovery males. These findings were considered incidental119 and unrelated to treatment. Tape irritation was observed in a number
of animals. There were no altera tions in organ weights that were attributed to treatment with the test material. Slight alterations were noted in several organ weights at termination of dosing or recovery. There was a statistically significan t decrease in mean absolute brain weight of the 300 mg/kg females compared to control. This finding lacked a dose response and was not considered biologically significant. There was a statistica lly significant decrease in mean relative adrenal and testes weights of the male satellite animals at termination of recovery compared to control at end of treatment. Compared to the high dose at study termination there was a statistically significant decrease in mean relative adrenal, brain and testes weight of the male satellite animals and mean relative adrenal and brain weight of the female satellite animals at recovery
termination. These alterations in or gan weights were attributed to the cessation of the stress associated with wrapping (adrenal) and the animals continued increase in body weight while organ weights
remained constant in adult animal s. In the absences of significant organ weight findings following treatment or correlating effects with histopathology these findings were not considered clinically significant. There were no test material related microscopic findings noted in any group. Livers from fe male rats of all groups (including control) sacrificed after 28 days of treatment exhibited focal necrosis. This finding did not exhibit a dose response. This finding has been
seen in other dermal studies and has been attributed to trauma and/or ischemia to the liver resulting from the wrapping and manipulation of
the animals. Liver necrosis was not evident in any of the satellite recovery animals. This finding was not considered treatment related.
The treated skin of most animal s revealed variable amounts of thickening of the epidermis due to acanthosis and hyperkeratosis, sebaceous gland hyperplasia and focal dermal inflammation. These changes occurred in all groups including control. However the severity of these changes tended to be increased in the male treated group rats and in the females of the 300 and 1000 mg/kg groups, suggesting a mild irritating effect of the test material. Following recovery these findings were less severe.
Conclusions A NOAEL of 1000 mg/kg was established for this study. Under the conditions of this study dermal appli cation of this test material resulted in no signs of overt systemic toxicity. Data Quality Reliable without restriction (Klimisch Code) References Unpublished confidential business information
Other Updated: 2/18/00 (RTA-024)120 

### 4.3 Toxicity to Reproduction

Robust Summary 5-ReproTox-1

Test Substance
CAS # CAS# 115733-09-0 Chemical Name Benzenesulfonic acid, C1 4-C24 branched & linear alkyl derivs.
Calcium salts Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed OECD 415

Test Type Oral (Gavage) One-Generation Reproductive Toxicity Study GLP (Y/N) Y Year (Study Performed) 2003-2004 Species Rat Strain Sprague-Dawley Crl: CD®(SD) IG S BR rats, Males approximately 7 weeks of age at initiation of treatment. Females approximately 8 weeks of age at initiation of treatment. Route of administration Orally by gastric intubation

Duration of test F 0 males- 70 days premating; mating period through completion of parturition.
F 0 females- 14 days premating; mating; 25 days of gestation and 20 days of lactation.
F 1 pups- gestation through day 20 of lactation. Doses/concentration levels 0, 50, 167 and 500 mg/kg/day
Vehicle control Corn Oil
Sex Males and Females Frequency of treatment Once/day, 7 days/week Analytical confirmation of
concentration. Homogeneity, stability and weekly dose concentration confirmation.

Control and treatment

groups 28 F 0 rats/sex/group in the control, low, mid and high dose groups.

Mating 1 male mated to 1 female from the same group until evidence of mating (presence of copulatory plug or sperm) was observed. If evidence of mating was not observed mating was discontinued after
three weeks. Post exposure observation period None

Statistical methods Body weights, body we ight changes, food consumption, semen parameters, organ weights, number of days to mating, gestation length, pup viability data, total pups deliv ered, pup body weights and mean live litter size were analyzed by ANOVA followed, as needed, by121 Dunnett’s test. Count data were analyzed by Chi-Square test followed by Fisher’s Exact Test for copulation and fertility indices, pup sex ratios, number of live and dead pups/group and pup survival. All analysis were two-tailed with a minimum significance level of 5%. Dose rangefinding study Yes (28 Day oral gava ge study conducted according to OECD Test Guideline 407.)
Remarks field for test
conditions F 0 Generation:
All F 0 males were dosed for 70 days prior to mating and through the completion of parturition. All F 0 females were dosed for 14 days prior to mating and through day 20 of lactation. All F 0 animals were examined twice daily for appearance and behavior. Detailed clinical observations were performed weekly and cage side observations were performed daily approximately 30 to 120 minutes post dosing. Body weights were recorded weekly for both sexes prior to mating; maternal body weights were recorded on gestation days 0, 7, 14 and 21 as well as on lactation days 1, 4, 7, 14 and 21. Food consumption was recorded on the same days as body weights except during the mating period and during lactation. Animals were paired 1:1 for mating. Positive evidence of mating was confirmed by the presence of sperm or a vaginal copulatory plug (day 0 of gestation). If evidence of mating was not present after three weeks, mating was discontinued. All of the surviving F 0 females were allowed to deliver and rear their pups to lactation day 21. The offspring were potentially exposed to
the test substance in utero and thro ugh nursing during lactation days 1- 21 until euthanization on post-natal day 21. The surviving F 0 dams were necropsied on lactation day 21, following a minimum of 60 days of dosing. The surviving F 0 males were necropsied at the conclusion of parturition following a minimum of 96 days of dosing. F 0 females that failed to deliver were necropsie d on post-mating day 25 (with evidence of mating) or 25 days following the termination of the mating period (with no evidence of mating).

Organ weights were determined and microscopic examinations
were conducted for all survivin g control and high dose F 0 animals. Tissues examined microscopically included the liver, kidney, brain, right epididymides, cervix, coagulation gland, ovaries, pituitary, prostrate, seminal v esicles, testes, uterus, vagina and gross lesions.

F 0 animals from all groups found dead or sacrificed early were subjected to a gross necropsy and the microscopic evaluation of all
tissues.

Sperm was collected from all surviving F 0 males and evaluated for sperm count, concentration, mo tility and morphology assessment.

F 1 Generation:122 On lactation day 4 each litter was randomly culled to a maximum of eight pups, 4/sex/litter, when possible. Detailed pup examinations were performed on lactation days 0, 4, 7, 14 and 21. Pup sex was determined on lactation day 0 and verified on lactation days 4, 7, 14 and 21. Individual pup weights were determined on lactation days 1, 4, 7 14 and 21. Pups that were stillborn, cannibalized or found dead were subjected to a gross necropsy with emphasis on developmental morphology. Pups culled on day 4 were subjected to an abbreviated gross necropsy with emphasis on the reproductive system. All surviving pups were euthanized on lactation day 21 and examined macroscopically. All internal gr oss lesions were preserved for possible future microscopic examination.

Results Results of the homogeneity analysis indicate that the test article was homogeneous in the vehicle and stable for ten days when stored under
ambient conditions. Concentration an alysis confirmed that the test article was at the appropriate con centration in the dosing solutions.

F 0 Generation:
F 0 males exhibited a dose related incr ease in post dosing salivation and dark material around the nose in the mid and high dose groups The remaining F 0 male parameters were unr emarkable including: mean body weight and food consumption, mating and fertility indicies, absolute and relative organ weights, sperm evaluation parameters and macro and microscopic pathology.

The clinical signs of the Fo females were generally unremarkable. There were no toxicologically meaningful differences between the control low, mid and high dose groups with respect to F 0 female mean body weights, body weight change , food consumption, mating and fertility indicies, precoital intervals or gestation length. A macroscopic finding observed in two high dose and one mid female sacrificed on post mating day 25 was a finding of negative ammonium sulfide staining in animals that failed to deliver and were euthanized on gestation day 25.

No other remarkable findings were noted in the F 0 females at necropsy and no meaningful microscopic lesions were observed in any of the
treated F 0 females.

F 1 Generation: No treatment related findings were noted in the F 1 pups during lactation. No treatment related gross necropsy findings were evident in any of the F 1 pups examined (stillborn, dead during lactation, culled or examined at scheduled sacrifice on lactation day 21.)123 Conclusions Based on the results of this study the Study Director concluded that the 500 mg/kg/day dose level was the no observed adverse effect level (NOAEL) for parental F 0 and F 1 pup toxicity. Data Quality Reliable without restriction (Klimisch Code)
References An Oral (Gavage) Study One – Generation Reproduction Toxicity Study in Sprague Dawley Rats with CASRN 115733-09-0. SLI Study No.: 3626.1 (March 11, 2004)
Other Updated: 4/26/2005124 

### 4.4 Genetic Toxicity:

Robust Summary 3-Gentox-1
Test Substance CAS # Analog of 70024-69-0 Chemical Name C20-24 alkaryl calcium salt derivative Remarks This substance is an analog for the group of substances in HERTG’s Final Submission for the Alkaryl Sulfonates Category. It is chemically similar to members of this CAS number, but does not belong to this CAS number. For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed OECD Guideline 474

Test Type Mammalian Erythrocyte Micronucleus Test GLP (Y/N) Y Year (Study Performed) 1989 Species Mouse Strain Swiss Albino Crl: CD-1 (ICR) BR 50 days of age at initiation of treatment Route of administration Intraperitoneal Duration of test Single dose followed by 72-hour evaluation period. Doses/concentration levels 0, 100, 200, 400 and 500 mg/kg Dose volume 5 ml/kg
Sex Males and females Frequency of treatment Once
Control and treatment

groups Peanut oil vehicle control: 18/sex; triethylenemelamine positive control: 0.25 mg/kg, 5/sex; 100 and 500 mg/kg: 15/sex; 200 and 400 mg/kg: 18/sex
Statistical methods Animal to animal variab ility in spontaneous frequency of micronucleated polychromatic erythrocytes were evaluate d in vehicle controls. Statistically significant differences were evaluate d in the frequency of micronucleated polychromatic erythrocytes between tr eated groups and vehicle controls. NCE/PCE (normochromatic erythrocytes/polychromatic erythrocytes) ratios in treated and control groups were compar ed. Tests included dispersion test of Amphlett and Delow, and Margolin, Fish ers exact test, binomial approximation, Cochran-Armitage test for trend, a one -way analysis of variance and Dunnett’s procedure. Dose rangefinding study A rangefinding study was conduc ted at 200, 400 and 600 mg/kg. Mortality and physical observations were evaluated.
Remarks field for test
conditions All animals were observed frequently for physiological or behavioral abnormalities on the day of dosing and at least twice daily thereafter. Body weights taken on first day of the study prior to treatment and at sacrifice. Macroscopic pathology performed on all animals at sacrifice. Five/sex from each treatment group and vehicle cont rol group were sacrificed for bone marrow sampling 24, 48 and 72 hours post treatment. Positive controls sampled at 24 hours only. NCE/PCE ratio and %PCE of total erythrocytes were125 calculated by counting a total of > 1000 erythrocytes/animal. A total of 1000 PCE /animal were evaluated for the presence of micronuclei. (Guideline calls for 2000/animal to be evaluated.) The number of micronuclei in NCEs was also
determined.
Results Remarks During the dose rangefinding study mortality (9 of 10 animals) was observed at 600 mg/kg but not at lower dose levels. Signs of toxicity observed at all dose levels included reduced feces, reduced food consumption, hyperactivity and phonation. Decreased motor activity was observed at 400 and 600 mg/kg.
Based on these results dose levels of 100, 200, 400 and 500 mg/kg were selected for the main study.

During the main study toxicity was observed at 400 and 500 mg/kg. At 500 mg/kg 5 males and 4 females of 15/sex died prior to the scheduled sampling time. At 400 mg/kg 1 of 18 treated females died on Day 3. Other clinical signs of toxicity included palpebral closure, decreased motor activity and weakness. Cytotoxicity was observed in both sexes. A statistically significant increase in NCE/PCE ratio was observed in males at 500 mg/kg at 24 hours. Elevated ratios were also observed in individual animals of both sexes in other groups. Altered proportions of erythrocytes to nucleated cells were noted for both sexes in the treated groups. No biological or statistical significant increase in the number of micronucleated-PCE was observed in any treated group compared to
the vehicle control. All values for indi vidual animals were within the expected range of micronucleated-PCE/1000 PCE expected for control animals. The variability in response observed in the treated animals was similar to that observed in the vehicle controls. The po sitive control exhibited a statistically significant increase in micronuclei as expected. Chemical analysis confirmed that the dosing solution preparation proce dure utilized for this study resulted in homogeneous solutions of a ppropriate concentration.
Conclusions Under the conditions of this study the test material did not induce micronuclei in bone marrow erythrocytes of mice. The genotoxicity NOEL was 500 mg/kg. Data Quality Reliable without restriction (Klimisch Code) References Unpublished confidential business information
Other Updated: 2/10/00 (RTA-005)126 Robust Summary 3-Gentox-2
Test Substance
CAS # CAS# 68783-96-0 Chemical Name Petroleum derived calcium salt, overbased Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed OECD Guideline 474

Test Type Mammalian Erythrocyte Micronucleus Test GLP (Y/N) Y Year (Study Performed) 1995 Species Mouse Strain Swiss Albino CD-1; 10-12 weeks of age at initiation of dosing Route of administration Oral gavage Duration of test Three treatment days followed by a 24-hour holding period.
Doses/concentration levels 0, 500, 1000, 2000 mg/kg Dose volume 10 ml/kg
Sex Males and females Frequency of treatment Three treatments administered approximately 24 hours apart.
Control and treatment

groups Peanut oil vehicle control: 5/sex; Cy clophosphamide positive control: 20 mg/kg (in water), 5/sex; 500, 1000 and 2000 mg/kg: 5/sex Statistical methods Data summarized by sex and dose group/time point. Analysis performed using an analysis of variance, Dunnett’s test, Cochran-Armitage test for linear trend Wilk’s Criterion or Kolomogorov-Smir nov statistic, Kruska l-Wallis, Dunn’s Summed Rank Test and Jonkheere’s test of ordered response. Dose Rangefinding
Studies Doses: 0.5, 1.0 and 2.0 g/kg; 2/sex/ dose sacrificed 24 hours after dosing. Percent polychromatic erythrocytes (PCE) was determined by counting 1000
cells. Number of micronuclei/1000 PCE determined.
Remarks field for test
conditions All animals were observed after dosing for signs of toxicity. Animals were examined twice daily for viability. Body weights were recorded prior to initiation of dosing. The animals from each group were sacrificed for bone marrow sampling 24 hours after the third dose. Necropsies were not performed.
2000 PCEs from each animal were exam ined for the presence of micronuclei. The percent of PCE in the total population of erythrocytes was determined for each animal by counting a total of 1000 polychromatic and normochromatic erythrocytes. If the test article induced neither a statistically significant dose response nor a statistically significant increase at any dose level above concurrent vehicle, at any sampling point, it was considered negative.
Results Remarks All dose rangefinding animals survived and were free of clinical signs. Bone127 marrow toxicity was not observed at any dose levels tested. Therefore 2000 mg/kg was selected as the high dose for the micronucleus assay. The mid and low doses were selected to be 1/2 and 1/4 of the high dose. In the main study, all vehicle, positive control and treated animals were normal after dosing and remained healthy until sacrifice. There were no dose related
increases or statistical differences in micronuclei formation observed at any dose level. Cytotoxicity was not obser ved since there were no statistically significant decreases in the percentage of polychromatic erythrocytes compared to the vehicle control. The positive co ntrol induced a statistically significant
increase in mean micronucleated PCEs in both sexes compared to the vehicle controls which indicated the positive control was clastogenic and responded appropriately. The positive control also induced cytotoxicity. Chemical analysis confirmed the uniformity and stability of the test material in peanut oil for at least 9 days at all three concentrations. Concentration verification analysis confirmed that each dose level was within 3% of nominal
concentration.

Conclusions The test material was not genotoxic under the conditions of this study. The genotoxicity NOEL was 2000 mg/kg. Data Quality Reliable without restriction (Klimisch Code) References Unpublished confidential business information

Other Updated: 2/18/00 (RTA-022)128 Robust Summary 3-Gentox -3
Test Substance
CAS # CAS# 68783-96-0 Chemical Name Petroleum derived calcium salt, overbased Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed OECD Guideline 476

Test Type Mouse Lymphoma Mutagenicity Screen GLP (Y/N) Y Year (Study Performed) 1984

Test System L5178Y-3.7.2C mouse lymphoma cells Culture Preparation and
Maintenance Cells were stored frozen in liquid nitr ogen. Cultures were incubated at 37 o C with shaking. Cultures were diluted dail y to a cell density of approximately 3 x
10 5 cells/mL. Cultures were checked for bacterial and fungal contamination.
Prior to use cultures were treated with methotrexate to reduce the frequency of spontaneously occurring TK
- / - cells. Exposure Method Dilution

Test Substance
Doses/concentration levels Concentrations of 500, 1000, 1500, 2 000, 4000 and 5000 ug/mL were evaluated
with and without metabolic activation.
Metabolic Activation Aroclor induced rat liver Vehicle Dimethyl sulfoxide (DMSO) 10 ul/mL
Positive Control concentration levels by
activation status With activation: 7,12-dimethylbenzanthracene (DMBA) 5 ug/mL Without activation: ethylmethanesulfonate (EMS) 744 ug/mL

Statistical Analysis Means and sta ndard deviations were determined. Test Substance Solubility Test substance solubility in the vehicle was determined during a solubility test. Dose range finding study Test substance (dose levels from 1 to 10,000 ug/mL) and vehicle control tested
with and without activation. Cultures we re exposed to the test substance and incubated for approximately four hours, th en washed and cultured for two days. Cell culture density was determined 24 a nd 48 hours post exposure. Treated cell suspension growth at each dose leve l was compared to the negative solvent
control.
Remarks field for test
conditions Prior to study initiation the solubility of the test substance and of the positive control materials in the vehicle (DMSO) was confirmed. A pretest dose range finding study was conducted at concentr ations up to 10,000 ug/mL with and
without metabolic activation.

In the main study there were two treatment sets for each concentration of test substance, with (+S9) and without (-S9) metabolic activation. DMBA (positive control) was tested with activation and EMS (positive control) was tested without activation. The test material was prepared so that the highest and lowest concentrations would yield perc ent total growth of approximately 10%129 and 90% respectfully. The test material was added to cells with and without
activation and incubated for four hours. Cells were then washed and placed in suspension cultures for two days with a cell population adjustment at 24 hours. The cells were then plated in a restri ctive media containing trifluorothymidine (TFT) which allows TK
- / - cells to grow. Cells were also plated in a nonrestrictive media that indicated cell viability. Plates were incubated at 37 O C in
a humidified 5% CO 2 atmosphere for 10-12 days. Following incubation all plates were scored for total number of colonies/plate. The frequency of mutation by dose was determined by comparing the average number of colonies in the mutagenicity plates to the average number of colonies in the corresponding viability plates. For the study to be acceptable the following criteria must be met: mutation frequency of positive controls with or without
activation should be twice that of th e solvent control; the negative control spontaneous mutation frequency should be in the range of 0.2 to 2.0/10 4 cells; negative control plating efficiency shou ld be at or above 50% and the test
material should be tested to the level of approximately 10% total growth or to the limits of solubility or to a high dose of 100 mg/mL.
Results The test substance was not mutagenic in this assay with or without metabolic
activation. Remarks The dose rangefinding study indicated significant toxicity (<90% total growth) at 500 ug/mL with and without metabolic activation. Based on these results the test material was evaluated for mutageni city at concentrations ranging from 500 to 5000 ug/mL. Six cultures with and without activation were selected for cloning at 500, 1000, 1500, 2000, 4000 and 5000 ug/mL. None of the cultures
treated with test material with or without activation exhibited mutant frequencies significantly different from the average mutant frequency of the negative (solvent) controls at a percent to tal growth of 10% or greater. Positive and vehicle control group responses we re appropriate and met the criteria
outlined above.
Conclusions The test material was not genotoxic under the conditions of this study. Data Quality Reliable without restriction (Klimisch Code) References Unpublished confidential business information
Other Updated: 4/11/00 (RTA-028)130 Robust Summary 3-Gentox-4
Test Substance
CAS # CAS# 68783-96-0 Chemical Name Petroleum derived calcium salt, overbased Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed OECD Guideline 471

Test Type Bacterial Reverse Mutation Assay GLP (Y/N) Y Year (Study Performed) 1995 Test System Salmonella typhimurium Strains Tested TA98, TA100, TA1535, TA1537, TA1538 Exposure Method Plate incorporation

Test Substance
Doses/concentration levels 250, 500, 1000, 2500 and 5000 ug/plate (initial assay) 1000, 2000, 3000, 4000 and 5000 ug/plate (repeat assay) Metabolic Activation With and without (0.5 mL S9 fraction mix of livers of Aroclor 1254 pretreated Sprague Dawley rats. –S9 gr oups received 0.5mL saline).

Vehicles Tetrahydrofuran (THF, for test material), Dimethylsulfoxide (DMSO, for positive control substances)
Positive Controls and concentration levels by
tester strain and activation

status 9-Aminoacridine (9AA), 100 ug/plate- TA1537 without S9 2-Aminoanthracine (2AA), 2.5 ug/plate-all strains with S9 N-Methyl-N-Nitro-N-Nitrosoguanidine (MNNG), 10 ug/plate-TA100, TA1535
without S9 2-Nitrofluorene (2NF), 5 ug/plate-TA98, TA1538 without S9 Vehicle Controls Tetrahydrofuran 25 uL/plate Dimethylsulfoxide 100 uL/plate Statistical Analysis Mean revertant colony count and standard deviation were determined for each dose point. Dose Range finding Study Conducted using tester strain TA100 at concentrations up to 5000 ug/plate with and without metabolic activation. Cytotoxicity was evaluated.
Remarks field for test
conditions This study was conducted according to OECD Guideline 471 (1983). Revision to this Guideline in 1997 suggests the addition of the E. coli WP2 uvrA or S. typhimurium TA 102 tester strains. Since this study was conducted prior to this revision, these strains were not included.

Prior to study initiation the solubility of the test substance in the vehicle (tetrahydrofuran) was confirmed. A pretest dose range finding study was conducted using tester strain TA100 at c oncentrations up to 5000 ug/plate with
and without metabolic activation. In th e main study there were two treatment sets for each tester strain, with and wit hout metabolic activation. Each of the
five tester strains was dosed with five concentrations of test substance (250, 500, 1000, 2500 and 5000 ug/plate), two vehicle controls (THF and DMSO), a131 nontreated control and a positive control. Three plates/dose group/strain/treatment set were evaluated. The results of the initial assay were verified by repeating the assay at do se levels of 1000, 2000, 3000, 4000 and 5000 ug/plate. After 2 days of incuba tion all plates in the initial and repeat assays were evaluated for gross toxic effects and total revertant colony
numbers.
Results The test substance was not mutagenic in this assay with or without metabolic
activation. Remarks Toxicity (notable reduction in b ackground lawn and/or 50% reduction in the number of revertant colonies compared to vehicle control) was not observed at any concentration tested with or with out metabolic activation in the range finding study. However at the 5000 and 2000 ug/plate levels a haze attributed to the test substance was present. These findings resulted in the selection of concentrations of 250, 500, 1000, 2500 and 5000 ug/plate for the initial study.

The test substance did not induce signi ficant increases in revertant colonies (equal to or greater than three times the TH F control) in any of the tester strains, at any dose level, with or without metabolic activation in the initial or repeat assays. Beading of the test substance was observed at 5000 ug/plate in all tester strains (with/without activation) and at 4000 ug/plate in tester strain TA1537 (with/without activation) in the repeat as say. The positive controls produced at
least a three-fold increase in revertant colonies when compared with the DMSO control in each respective strain. The no ntreated and vehicle controls responded appropriately. The 5000 ug/plate concentr ation of test substance in THF was evaluated analytically for concentration in both the initial and repeat assays. Analysis conformed that the test substa nce concentration was within 7% of the nominal concentration for both assays.
Conclusions The test substance was not mutagenic in any strain of Salmonella typhimurium tested, including at least one dose above th e solubility of the test substance. The genotoxicity NOEL was 5000 ug/plate. Data Quality Reliable without restriction (Klimisch Code) References Unpublished confidential business information
Other Updated: 2/18/00 (RTA-021)132 Robust Summary 3-Gentox-5
Test Substance
CAS # CAS# 71786-47-5 Chemical Name Alkaryl magnesium salt derivative Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed OECD Guideline 473

Test Type In Vitro Chromosomal Aberration Assay in CHO Cells GLP (Y/N) Y Year (Study Performed) 1995

Test System Chinese hamster ovary cells
Clone Tested WBL Culture Preparation and
Maintenance Cells were thawed and cultured in Mc Coy's 5A Medium containing 10% fetal bovine serum and 2 mM L-glutamine at 37 o C, in 4-6% CO 2 in air. Cultures
were seeded at 1.2 x 10 6 cells (16-hour harvest) and 0.8 x 10 6 (40-hour harvest) approximately 1 day prior to dosing. Fetal bovine serum was excluded from
activated cultures. Exposure Method Dilution

Test Substance
Doses/concentration levels A 50 uL sample of concentrations of 10, 20, 40, 80, 120, 160 ug/mL was
evaluated with and without metabolic activation. Metabolic Activation With and without (0.015 mL/ mL serum free medium) S9 fraction mix of livers of Aroclor 1254 pretreated Sprague Dawley rats and 0.06 mL/ mL serum free medium cofactor mix (13.4 mg/mL NADP and 25 mg/mL DL-Isocitric Acid in distilled water). Vehicles Tetrahydrofuran (THF, for test material), acetone (for positive control substances)
Vehicle and Positive
Control concentration levels by activation status Acetone, 5 ug/mL with and without activation Tetrahydrofuran, 5 ug/mL w ith and without activation N-Methyl-N-Nitro-N-Nitrosoguanidine (MNNG), 0.6 ug/mL without activation 7,12-Dimethylbenz[a]anthracene (DMBA), 10 ug/mL with activation Statistical Analysis The number of cells with at least one aberrant chromosome and the number of cells examined in each replicate were us ed for statistical analysis. The number of aberrant individual chromosomes/cell was not analyzed. Positive control groups were compared to vehicle control by Fisher Exact Test. Each pair of replicates was compared by Fisher Exact Test. Differences between control and treated groups were compared using Fisher Exact Test and if necessary a 2x2 Fisher Tests. A permutation test was performed to test for dose related trends. Significance levels of less than 0.05 were reported. Test Substance Solubility Test substance solubility in the vehicle was determined. Culture Medium Solubility
Test The solubility of the test substance in the culture medium was established at concentrations of 10, 20, 39, 78, 156, 313, 625, 1250, and 2500 ug/mL. Visual and microscopic examinations were made for precipitation at 0, 30 and 180133 minutes post preparation. Concentrations showing signs of insolubility at any of these time points were considered unsuitable for dosing.

Dose range finding study Test substance and vehicle c ontrols tested in duplicate cultures each with and
without activation. Test substance tested at concentrations of 2.5, 5, 10, 20, 40, 60, 80, 120, and 160 ug/ml. Cytotoxici ty and mitotic indices were evaluated.
Remarks field for test
conditions Prior to study initiation the solubility of the test substance and of the positive control materials in the vehicles (tetrahydrofuran/acetone) was confirmed. A pretest dose range finding study was conducted at concentrations up to 160 ug/mL with and without metabolic activati on. In the main study there were two treatment sets for each concentration of test substance, with (+S9) and without (-S9) metabolic activation. DMBA (positiv e control) was tested with activation and MNNG (positive control) was tested without activation. Prepared cultures
were treated with test substance or c ontrol material and were incubated for 16 hours. A repeat assay was performed using 16 and 40 hour harvest time points. Vehicle, MNNG and DMBA cultures were incubated for 16 hours only. Two to three hours prior to the 16 and 40 hour harvest the spindle inhibitor, Colcemid,
was added to each culture to obtain a final concentration of 0.2 ug/mL. Harvested cells were evaluated microscopically for percent confluency, morphology and estimated number of mi totic cells prior to harvest.

The test substance treated groups, select ed for chromosome analysis based on cell count data and the presence of participate, were as follows:

Initial assay +S9 (16 hour harvest) 20, 40 80 ug/mL Initial assay -S9 (16 hour harvest) 20, 40 80 ug/mL Repeat assay +S9 (16 hour harvest) 20, 40 80 ug/mL Repeat assay -S9 (16 hour harvest) 20, 40 80 ug/mL Repeat assay +S9 (40 hour harvest) 80, 120, 160 ug/mL Repeat assay -S9 (40 hour harvest) 80, 120, 160 ug/mL

Slides were prepared for these groups using Giemsa stain. Two slides/treatment group were evaluated. 200 metaphase cells (100 per culture) each containing 19-23 chromosomes per treatment group were scored. Chromosomes were
counted for each cell. Chromosome aberrations, either chromosome or chromatid type were recorded. The fo llowing observations were recorded and excluded from the total aberration frequency: gaps, polyploid and endoreduplicated cells, pulverized chromo somes, Robertsonian translocations,
translocations and abnormal monocentr ic chromosomes. The percent of aberrant cells and the frequency of ab erration (%) per treatment group were
determined. In order for a test substa nce to be considered to have induced a positive response compared to vehicle control a statistically significant dose related increase in the percentage of aberrant cells along with a mean percentage of aberrant cells in excess of 5% in at least one treatment group were required. Or, a reproducible and statistica lly significant response in at least one treatment group with a mean % of aberrant cells exceeding 5% was observed.
Test substance concentration verifica tion was performed on the highest stock concentration in both the initial and repeated assays. Results were within 8% of134 nominal.
Results The test substance was not mutagenic in this assay with or without metabolic
activation.
Remarks In the culture medium solubility test precipitate and/or cloudiness were present
with and without metabolic activation at concentrations of 39 ug/mL and 78 ug/mL and greater. In the pretest toxicity assay, a greater than 50% reduction in cell counts or mitotic activity was not observed at concentrations up to 160 ug/mL. The doses selected for the initial assay were 10, 20, 40, 80, 120 and 160 ug/mL.

Cell survival was not significantly redu ced when compared to the vehicle control in the initial assay. Cell survival was reduced by at least 50% compared to vehicle control in the repeat assay (40-hr harvest) without metabolic activation at the 160 ug/mL concentration. A greater than 50% reduction in mitotic index was not observed in either the initial or repeat assays at any concentration tested. Precipitation was observed at concentrations greater than 80 ug/mL in the chromosomal aberra tion assay. Therefore, the highest
concentration evaluated at 16 hours was 80 ug/mL. There were no statistically significant differences in the number of chromosomal aberrations at 16 hours
with activation and at 40 hours with an d without metabolic activation. In the
initial 16-hour harvest without activation a statistically significant increase was
observed with one dose level different fro m the vehicle control. However this finding was not evident in the repeat 16-hour harvest without activation. The observed initial increase was not reproducible and was not considered biologically significant. Positive and vehicle control group responses were as expected. The positive control group had a statistically significant higher percentage of aberrant cells than th e vehicle control group with and without
activation at each harvest interval.
Conclusions The test material was not genotoxic under the conditions of this study. Data Quality Reliable without restriction (Klimisch Code) References Unpublished confidential business information
Other Updated: 4/10/00 (RTA-026)135 Robust Summary 3-Gentox-6
Test Substance
CAS # CAS# 71786-47-5 Chemical Name Alkaryl magnesium salt derivative Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed OECD Guideline 474

Test Type Mammalian Erythrocyte Micronucleus Test GLP (Y/N) Y Year (Study Performed) 1995 Species Mouse Strain CD-1, 10-12 weeks of age at initiation of treatment Route of administration Oral gavage

Duration of test Three treatments administered approximately 24 hours apart followed by a 24- hour hold period prior to bone marrow sample collection. Doses/concentration levels 0, 500, 1000 and 2000 mg/kg Dose volume 10 ml/kg
Sex Males and females Frequency of treatment Three treatments administered approximately 24 hours apart.
Control and treatment

groups Peanut oil vehicle control: 5/sex; cycl ophosphamide (in water) positive control: 20 mg/kg, 5/sex; 500, 1000, 2000 mg/kg, 5/sex/kg. Statistical methods The following parameters were recorded and evaluated; the ratio of polychromatic to normochromatic erythrocytes, number of polychromatic erythrocytes with micronuclei and num ber of polychromatic erythrocytes scored. Statistical analysis included means and standard deviations of the micronuclei data and a test of equality of group means. Tests included a oneway analysis of variance, Duncan’s Multip le Range test and regression analysis. Residuals from the ANOVA were analyzed by Wilk’s Criterion or the Kolomogorov-Smirnov statistic. Nonparame tric analyses included the KruskalWallis one way ANOVA followed by Dunn’s Summed Rank Test. Dose response was evaluated by Jonkheere’s test of ordered response. Dose range finding study A dose range finding stud y was conducted at 500, 1000 and 2000 mg/kg. Percent polychromatic erythrocytes (PCE ) were determined by counting 1000
cells. Number of micronuclei/1000 PCE determined.
Remarks field for test
conditions All animals observed for viability twice daily during the dosing period. Detailed clinical observations recorded after each test substance administration. Body weights recorded prior to initiation of dosing. Twenty-four hours after the third dose the animals were sacrificed for bone marrow sampling. Necropsies were not performed. A total of 2000 polyc hromatic erythrocytes/animal were evaluated for the presence of micronuclei. The percent of PCE in the total population of erythrocytes was determined for each animal by counting the total polychromatic and normochromatic erythrocytes.136 Results Remarks All dose rangefinding animals survived and were free of clinical signs. Bone marrow toxicity was not observed at any dose levels tested. Therefore 2000 mg/kg was selected as the high dose for the micronucleus assay. The mid and low doses were selected to be 1/2 and 1/4 of the high dose.

All animals survived to scheduled sacrifi ce and were free of clinical signs. The responses of the vehicle control and pos itive control groups were appropriate and support the validity of the assay results. The positive control induced a significant increase in mean number of micronucleated polychromatic erythrocytes. In addition it induced cy totoxicity. There were no dose-related
increases or statistical differences in micronuclei formation observed at any dose level of the test material. Cytotoxicity was not observed. There were no statistical decreases in the percentage of polychromatic erythrocytes compared to the vehicle control. Chemical analysis of dosing solutions confirmed that they were homogeneously prepared at the desi red concentrations and that they were stable for the intended period of use.

Conclusions Under the conditions of this study the test material did not induce micronuclei in bone marrow erythrocytes and did not induce cytotoxicity in the bone marrow of CD-1 mice. The genotoxicity NOEL was 2000 mg/kg. Data Quality Reliable without restriction (Klimisch Code) References Unpublished confidential business information
Other Updated: 2/11/00 (RTA-007)137 Robust Summary 3-Gentox-7
Test Substance
CAS # CAS# 71786-47-5 Chemical Name Alkaryl magnesium salt derivative Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed OECD Guideline 471

Test Type Bacterial Reverse Mutation Assay GLP (Y/N) Y Year (Study Performed) 1995 Test System Salmonella typhimurium Strains Tested TA98, TA100, TA1535, TA1537, TA1538 Exposure Method Plate incorporation

Test Substance
Doses/concentration levels 62.5, 125, 250, 500 and 1000 ug/plate

Metabolic Activation With and without (0.5 mL S9 fraction mix of livers of Aroclor 1254 pretreated Sprague Dawley rats. –S9 groups received 0.5mL saline.

Vehicles Tetrahydrofuran (THF, for test material), Dimethylsulfoxide (DMSO, for positive control substances)
Positive Controls and concentration levels by
tester strain and activation

status 9-Aminoacridine (9AA), 100 ug/plate- TA1537 without S9 2-Aminoanthracine (2AA), 2.5 ug/plate-all strains with S9 N-Methyl-N-Nitro-N-Nitrosoguanidine (MNNG), 10 ug/plate-TA100, TA1535
without S9 2-Nitrofluorene (2NF), 5 ug/plate-TA98, TA1538 without S9 Vehicle Controls Tetrahydrofuran 25 uL/plate Dimethylsulfoxide 100 uL/plate Statistical Analysis Mean revertant colony count and standard deviation were determined for each dose point. Dose rangefinding study Conducted using tester strain TA100 at concentrations up to 5000 ug/plate with
and without metabolic activation.
Remarks field for test
conditions This study was conducted according to OECD Guideline 471 (1983). Revision to this Guideline in 1997 suggests the addition of the E. coli WP2 uvrA or S. typhimurium TA 102 tester strains. Since this study was conducted prior to this revision, these strains were not included.

Prior to study initiation the solubility of the test substance in the vehicle (tetrahydrofuran) was confirmed. A pretest dose range finding study was conducted using tester strain TA100 at c oncentrations up to 5000 ug/plate with
and without metabolic activation. In th e main study there were two treatment sets for each tester strain, with (+S9) and without (-S9) metabolic activation.
Each of the five tester strains was do sed with five concentrations of test substance, two vehicle controls (THF and DMSO), a nontreated control and a positive control. Three plates/dose group/strain/treatment set were evaluated.138 The results of the initial assay were verified by repeating the assay. After 2 days of incubation all plates in the initial assay and the TA1537 and TA1538 plates in the repeat assay were refrige rated. These plates were evaluated for gross toxic effects and total revertant colony numbers on the following day. In the repeat assay TA98, TA100 and TA1 535 were evaluated after 2 days of
incubation.
Results The test substance was not mutagenic in this assay with or without metabolic
activation. Remarks In the range finding study toxicity (n otable reduction in background lawn and/or 50% reduction in the number of revertant colonies compared to vehicle control) was not observed at any concentration tested with or without metabolic
activation. However the 5000 and 2000 ug/plate levels were difficult to
evaluate due to test substance inte rference. At 1000 and 500 ug/plate precipitate was observed on the plates. These findings resulted in the selection of concentrations ranging from 62.5 to 1000 ug/plate for the main study.

The test substance did not induce signi ficant increases in revertant colonies (equal to or greater than three times the TH F control) in any of the tester strains, at any dose level, with or without metabolic activation in the initial or repeat assays. A greater than 50% reduction in mean number of revertant colonies compared to THF were observed in the initial assay in TA1537 without activation at 250 ug/plate. In TA1535 with activation, no background /no revertants was noted in the initial assay for all three plates at 250 and 500 and in two plates at 1000 ug/plate. The significance of these reductions is difficult to interpret since the findings were incons istent between assays and dose levels. Precipitate was seen on all plates at 1000 ug/plate (+/- S9) in the initial and repeat assays. The positive controls produ ced at least a three-fold increase in revertant colonies compared with the DMSO control in their respective strains.
Nontreated and vehicle controls were a cceptable and were consistent with data from previous assays. The 1000 ug/plate concentration of test substance in THF was evaluated analytically for stability, concentration and homogeneity. Analysis conformed that the test subs tance was stable and homogeneous in THF for the intended period of use. The 1000 ug/plate solution was prepared and assayed twice during the study. The result for the first preparation was 121%
above nominal. The result of the second preparation was 15% above nominal.

Conclusions The test substance was not mutagenic in any strain of Salmonella typhimurium tested, including at least one dose above th e solubility of the test substance. The genotoxicity NOEL was 1000 ug/plate. Data Quality Reliable without restriction (Klimisch Code) References Unpublished confidential business information
Other Updated: 2/14/00 (RTA-008)139 Robust Summary 3-Gentox-8
Test Substance CAS # CAS# Analog of 78330-12-8 Chemical Name C15-C21 alkaryl sodium salt derivative Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed OECD Guideline 471

Test Type Bacterial Reverse Mutation Assay GLP (Y/N) Y Year (Study Performed) 1983 Test System Salmonella typhimurium
Strains Tested Salmonella typhimurium tester strains TA98, TA100, TA1535, TA1537 and TA
1538 Exposure Method Plate incorporation

Test Substance
Doses/concentration levels 0.1, 0.3, 1.0, 3.0 and 10 mg/plate

Metabolic Activation With and without 25 ul/pla te S9 fraction mix of livers of Aroclor 1254 pretreated Sprague Dawley rats)

Vehicle Sterile distilled water Tester strain, activation status, Positive Controls
and concentration level TA98 +S9 2-aminoanthracene 2.0 ug/plate TA98 -S9 2-nitroflourene 10.0 ug/plate TA100 +S9 2-aminoanthracene 2.0 ug/plate TA100 -S9 sodium azide 1.0 ug/plate TA1535 +S9 2-aminoanthracene 2.0 ug/plate TA1535 -S9 sodium azide 1.0 ug/plate TA1537 +S9 2-aminoanthracene 2.0 ug/plate TA1537 -S9 9-Aminoacridine 50.0 ug/plate TA1538 +S9 2-aminoanthracene 2.0 ug/plate TA1538 -S9 2-nitroflourene 10.0 ug/plate
Vehicle Control Sterile distilled water Statistical Analysis Mean revertant colony count and standard deviation were determined for each dose point. Dose Range finding Study Conducted using tester strain TA100 at dose levels of test material ranging from 0.005 to 10 mg/plate without S9. S9 Optimization Study Conducted using tester st rains TA98 and TA100, and a dose level of test material of 10 mg/plate and concentrati ons of S9 mix ranging from 25 to 250 ul S-9/plate.
Remarks field for test
conditions This study was conducted according to OE CD Guideline 471 (1983). Revisions to this Guideline in 1997 suggest the addition of the E. coli WP2 uvrA or S. typhimurium TA 102 tester strains. Since this study was conducted prior to this revision, these strains were not included.140 In the main study there were two treatment sets for each tester strain, with (+S9) and without (-S9) metabolic activation. Each of the tester strains was dosed
with five concentrations of test subs tance, vehicle controls, and a positive control. Three plates/dose group/strain/treatment set were evaluated. 0.1 ml of test material, positive control or vehicle control were added to each plate along with 0.1 ml of tester strain, S9 mix (if needed) and 2.0 ml of top agar. This was overlaid onto the surface of 25 ml minimal bottom agar in a petri dish. Plates
were incubated for 48 hours at 37 o C. The condition of the bacterial background lawn was evaluated for cytotoxicity and test article precipitate.
Results The test substance was not mutagenic in this assay with or without metabolic
activation. Remarks Slight cytotoxicity was observed in the dose range finding study with tester strain TA100 without metabolic activation. The S9 optimization study was performed using TA98 and TA100 at 10 mg/p late and concentrations of S9 mix of 25-250 ul. In the absence of any effect 25 ul S9 mix/plate was used in the mutagenicity study.

In the main study the test material was not mutagenic to any strain. It was slightly cytotoxic to TA100 in the ab sence of metabolic activation. Positive control responses were acceptable.
Conclusions Under the conditions of this study, the t est material was not mutagenic with or
without metabolic activation. Data Quality Reliable without restriction (Klimisch Code) References Unpublished confidential business information
Other Updated: 10/4/00 (RTA-070)141 Robust Summary 3 – Gentox-9
Test Substance CAS # Analog of 70024-69-0 Chemical Name C20-C24 alkaryl calcium salt derivative
Remarks The tested substance is an anal og for the group of substances referred to as C16-C24 alkyl calcium salt overbased derivative in the HERTG’s Final Submission for Alkaryl Sulfonates Category. It is chemically similar to members of this CAS number, but does not belong to this CAS number. For more information on this chemical, see Section 2.0
“General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed OECD Guideline 471

Test Type Bacterial Reverse Mutation Assay GLP (Y/N) Y Year (Study Performed) 1989 Test System Salmonella typhimurium and Escherichia Coli
Strains Tested Salmonella typhimurium tester strains TA98, TA100, TA1535, TA1537; Escherichia Coli tester strain WP2 uvr A Exposure Method Plate incorporation

Test Substance
Doses/concentration levels 0.1, 0.33, 1.0, 3.33 and 10 mg/plate

Metabolic Activation With and without (500 ul of 10% S9 fraction mix of livers of Aroclor 1254 pretreated Sprague Dawley rats)

Vehicle Pluronic F127 25% w/w in ethanol Tester strain, activation status, Positive Controls
and concentration level TA98 +S9 2-aminoanthracene 2.0 ug/plate TA98 -S9 2-nitroflourene 10.0 ug/plate TA100 +S9 2-aminoanthracene 2.0 ug/plate TA100 -S9 sodium azide 1.0 ug/plate TA1535 +S9 2-aminoanthracene 2.0 ug/plate TA1535 -S9 sodium azide 1.0 ug/plate TA1537 +S9 2-aminoanthracene 2.0 ug/plate TA1537 -S9 ICR-191 2.0 ug/plate
WP2 uvr A +S9 2-aminoanthracene 80.0 ug/plate
WP2 uvr A –S9 ICR-191 50.0 ug/plate
Vehicle Control Pluronic F127 25% w/w in ethanol Statistical Analysis Mean revertant colony co unt and standard devia tion were determined for each dose point. Dose Rangefinding Study Conducted using tester strains TA98 and TA100, and dose levels of test material ranging from 0.003 to 10 mg/plate. S9 Optimization Study Conducted using tester strains TA98 and TA100, and a dose level of test material of 10 mg/plate and c oncentrations of S9 mix ranging from 25 to 400 ul S-9/plate. Cytotoxicity was evaluated. Remarks field for test In the main study there were two treatment sets for each tester strain,142 conditions with (+S9) and without (-S9) metabolic activation. Each of the tester
strains was dosed with five concentr ations of test substance, vehicle controls, and a positive control. Three plates/dose group/strain/treatment set were eval uated. The results of the initial assay were confirmed in a second independent experiment. 100 ul of test material, positive control or vehicle control were added to each plate along with 100 ul of tester strain, S9 mix (if needed) and 2.0 ml of top agar. This was overlaid onto the surface of 25 ml minimal bottom agar in a petri dish. Plates were incubated for 48 hours at
37 o C. The condition of the bacteria l background lawn was evaluated for cytotoxicity and test article precipitate.
Results The test substance was not genotoxic in this assay with or without
metabolic activation. Remarks No cytotoxicity was observed in the dose rangefinding study with
tester strains TA100 and WP2 uvr A with or without metabolic activation as evidenced by normal background lawn and no reduction in the number of revertants/plate. The S9 optimization study was performed using TA98 and TA100 with the highest non-cytotoxic dose of test article, (10,000 ug/plate) a nd concentrations of S9 mix of 25- 400 ul. In the absence of any effect 25 ul S9 mix/plate was used in the mutagenicity study.

The test material formed a stable emulsion with the vehicle and the dilutions were well dispersed in the top agar. However after incubation test material was visible at all dose levels in the top layer. The test material was not cytotoxic to any tester strain. In the repeat study statistically significant increases in revertant colonies were
observed in TA1535 without meta bolic activation and in WP2 uvr A
with metabolic activation. Howe ver since these findings were not found during the first experiment they were not considered biologically significant. The positiv e control for each respective test strain exhibited at least a 3-fold increase (with or without S9) over the mean value of the vehicle control for a given strain, confirming the expected positive control response. Dosing solution analysis confirmed that high dose concentration was acceptable.

Conclusions Under the conditions of this study, the test material was not mutagenic
with or without metabolic activation. Data Quality Reliable without restriction (Klimisch Code) References Unpublished confidential business information
Other Updated: 10/4/00 (RTA-069)143 

### Robust Summary 3-Gentox - 10
Test Substance
CAS # CAS# 68783-96-0 Chemical Name Petroleum derived calcium salt, overbased Remarks For more information on this chemical, see Section 2.0 “General Substance Information” in HERTG’s Final Submission for Alkaryl Sulfonate Category.

Method

Method/Guideline
followed OECD Guideline 473

Test Type In Vitro Chromosomal Aberration Assay in CHO Cells GLP (Y/N) Y Year (Study Performed) 1995

Test System Chinese hamster ovary cells
Clone Tested WBL Culture Preparation and
Maintenance Cells were thawed and cultured in Mc Coy's 5A Medium containing 10% fetal bovine serum and 2 mM L-glutamine at 37 o C, in 4-6% CO 2 in air. Cultures
were seeded at 1.2 x 10 6 cells (16-hour harvest) and 0.8 x 10 6 (40-hour harvest) approximately 1 day prior to dosing. Fetal bovine serum was excluded from
activated cultures. Exposure Method Dilution

Test Substance
Doses/concentration levels A 50 uL sample of concentrations of 10, 20, 40, 80, 120, 160 ug/mL was
evaluated with and without metabolic activation. Metabolic Activation With and without (0.015 mL/ mL serum free medium) S9 fraction mix of livers of Aroclor 1254 pretreated Sprague Dawley rats and 0.06 mL/ mL serum free medium cofactor mix (13.4 mg/mL NADP and 25 mg/mL DL-Isocitric Acid in distilled water). Vehicles Tetrahydrofuran (THF, for t est material), acetone (for positive control substances)
Vehicle and Positive
Control concentration levels by activation status Acetone, 5 ug/mL with and without activation Tetrahydrofuran, 5 ug/mL w ith and without activation N-Methyl-N-Nitro-N-Nitrosoguanidine (MNNG), 0.6 ug/mL without activation 7,12-Dimethylbenz[a]anthracene (DMBA), 10 ug/mL with activation Statistical Analysis The number of cells with at least one aberrant chromosome and the number of cells examined in each replicate were us ed for statistical analysis. The number of aberrant individual chromosomes/cell was not analyzed. Positive control groups were compared to vehicle control by Fisher Exact Test. Each pair of replicates was compared by Fisher Exact Test. Differences between control and treated groups were compared using Fisher Exact Test and if necessary a 2x2 Fisher Tests. A permutation test was performed to test for dose related trends. Significance levels of less than 0.05 were reported. Test Substance Solubility Test substance solubility in the vehicle was determined. Culture Medium Solubility
Test The solubility of the test substance in the culture medium was established at concentrations of 10, 20, 39, 78, 156, 313, 625, 1250, and 2500 ug/mL. Visual and microscopic examinations were made for precipitation at 0, 30 and 180144 minutes post preparation. Concentrations showing signs of insolubility at any of these time points were considered unsuitable for dosing.

Dose rangefinding study Test substance and vehicle c ontrols tested in duplicate cultures each with and
without activation. Test substance tested at concentrations of 0.625, 1.25, 2.5, 5, 10, 20, 40, 80 and 160 ug/ml. Cy totoxicity and mitotic indices were
evaluated.
Remarks field for test
conditions Prior to study initiation the solubility of the test substance and of the positive control materials in the vehicles (tetrahydrofuran/acetone) was confirmed. A pretest dose range finding study was conducted at concentrations up to 160 ug/mL with and without metabolic activati on. In the main study there were two treatment sets for each concentration of test substance, with (+S9) and without (-S9) metabolic activation. DMBA (positiv e control) was tested with activation and MNNG (positive control) was tested without activation. Prepared cultures
were treated with test substance or c ontrol material and were incubated for 16 hours. A repeat assay was performed using 16 and 40 hour harvest time points. Vehicle, MNNG and DMBA cultures were incubated for 16 hours only. Two to three hours prior to the 16 and 40-hour harvest the spindle inhibitor, Colcemid,
was added to each culture to obtain a final concentration of 0.2 ug/mL. Harvested cells were evaluated microscopically for percent confluency, morphology and estimated number of mi totic cells prior to harvest.

The test substance treated groups, select ed for chromosome analysis based on cell count data and the presence of participate, were as follows:

Initial assay +S9 (16 hour harvest) 10, 20, 40 ug/mL Initial assay -S9 (16 hour harvest) 10, 20, 40 ug/mL Repeat assay +S9 (16 hour harvest) 10, 20, 40 ug/mL Repeat assay -S9 (16 hour harvest) 10, 20, 40 ug/mL Repeat assay +S9 (40 hour harvest) 10, 20, 40 ug/mL Repeat assay -S9 (40 hour harvest) 10, 20, 40 ug/mL

Slides were prepared for these groups using Giemsa stain. Two slides/treatment group were evaluated. 200 metaphase cells (100 per culture) each containing 19-23 chromosomes per treatment group were scored. Chromosomes were
counted for each cell. Chromosome aberrations, either chromosome or chromatid type were recorded. The fo llowing observations were recorded and excluded from the total aberration frequency: gaps, polyploid and endoreduplicated cells, pulverized chromo somes, Robertsonian translocations,
translocations and abnormal monocentr ic chromosomes. The percent of aberrant cells and the frequency of ab erration (%) per treatment group were
determined. In order for a test substa nce to be considered to have induced a positive response compared to vehicle control a statistically significant dose related increase in the percentage of aberrant cells along with a mean percentage of aberrant cells in excess of 5% in at least one treatment group were required. Or, a reproducible and statistica lly significant response in at least one treatment group with a mean % of aberrant cells exceeding 5% was observed. Test substance concentration verification, uniformity and stability were145 performed on the highest stock concentra tion in both the initial and/or repeated assays. Results were within 6% of nom inal. Samples were homogeneous and stable for the intended period of use.
Results The test substance was not mutagenic in this assay with or without metabolic
activation.
Remarks In the culture medium solubility test precipitate and/or cloudiness were present
with and without metabolic activation at concentrations of 39 ug/mL and greater. In the pretest toxicity assay there was an 81% reduction (compared to vehicle control) in cell survival at 160 ug/mL without metabolic activation. The doses selected for the initial assay were 10, 20, 40, 80, 120 and 160 ug/mL.

A greater than 50% reduction in cell su rvival and/or mitotic index was not observed in either the initial or repeat assays. Precipitation was observed at concentrations greater than 40 ug/m L in the chromosomal aberration assay. Therefore, 40 ug/mL was considered to be the limit of solubility for the test substance and was selected as the highest t est concentration to be evaluated. There were no statistically significant differences in the number of
chromosomal aberrations between the tr eated and vehicle control groups in either the initial or repeat assay at any dose level evaluated (10, 20 and 40 ug/mL with and without metabolic activ ation). In the initial 16-hour harvest, there were statistically significant increa ses with dose in the percent of aberrant
cells for both the activated and nonactivat ed evaluations. These trends were not reproducible in the repeat 16-hour harvest and therefore were not considered biologically significant. Positive and vehicle control group responses were as expected. The positive control groups ha ve frequencies of aberrations outside the normal range of the vehicle control and at least twice the vehicle control
value.
Conclusions The test material was not genotoxic under the conditions of this study. Data Quality Reliable without restriction (Klimisch Code) References Unpublished confidential business information
Other Updated: 4/11/00 (RTA-027)