1

Substance Group: Group 3: Alkaryl Sulfonates 

Summary prepared by: Petroleum Additives Panel 

 Health & Environmental Research Task Group 

 October 2005 
2

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 summaries 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 modeled using a structure based modeling program (see 

reference) and that the values selected are based on a structure that is representative 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 30o

C for 17.5 hours 

and standing at 20o

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°C 
4

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 30o

C for 3 hours and 

standing at 20o

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. Based 
6

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 on 

chemical 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 30o

C for 3 hours and 

standing at 20o

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 detector 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 water 
8

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 acid,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 30o

C for 3 hours and 

standing at 20o

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 30o

C for 3 hours and 

standing at 20o

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 percentage 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 considered 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 domestic 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 substance, sodium benzoate, 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: 9-6-00 
15

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 domestic 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/A 
16

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 (sodium 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: 9-6-00 
17

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 controls 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 biodegradation 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 t1/2: <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: 9-6-00 
19

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 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 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 domestic 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 controls 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 substances 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 controls 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 concentrations: 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 similar, 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 

NA 
21

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: 9-6-00 
22

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 controls 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 biodegradation 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 t1/2: <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: 9-6-00 
24

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 (TOC) measurements of each freshly prepared 

test solution and control and after 24-h on test just before daily 

renewal with fresh test solution. 

Exposure Period (unit) 96 hours 

Statistical methods Statistical analysis of survival 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 hours 
25

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 

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 LL50 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 LC50 was 

1.2 mg/L. No information provided on method of calculation. 

Results Nominal concentrations: 96-h LL50 >10,000 mg/L. This is equivalent 

to 96-h LL0 = 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: 9-6-00 
27

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 analog 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 (TOC) measurements of each freshly prepared 

test solution and control and after 24-h on test just before daily 

renewal with fresh test solution. 

Exposure Period (unit) 96 hours 

Statistical methods Statistical analysis of survival 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 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 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 LL50 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 LC50 was 

1.2 mg/L. No information provided on method of calculation. 

Results Nominal concentrations: 96-h LL50 >10,000 mg/L. This is equivalent 

to 96-h LL0 = 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: 9-6-00 
30

Robust Summary 3-Fish - 3 

Test Substance

CAS # 71486-79-8 

Chemical Name Benzenesulfonic acid, mono-C15-30-branched alkyl and di-C11-13-

branched and linear alkyl derivatives, 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 survival 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: juvenile, 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. Mixing 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 

CaCO3. The water was passed through activated carbon, a particle 

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

polyethylene tank where it was aerated. The water was characterized 

as moderately hard water. 

 

Light: 16-h light per day using cool-white fluorescent lights with an 

intensity of 20 uEin/m2

. 

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 surface 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 therefore based on nominal loading 

rates. 

 

Reference Substance: No 

Results Nominal concentrations: 96-h LL50 >1,000 mg/L. This is equivalent 

to 96-h LL0 = 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: LL50 and LL0 reported 
32

as LC50 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: 9-6-00 
33

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 survival 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. Mixing 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 

CaCO3. The water was passed through activated carbon, a particle 

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

polyethylene tank where it was aerated. The water was characterized 

as moderately hard water. 

 

Light: 16-h light per day using cool-white fluorescent lights with an 

intensity of 20 uEin/m2

. 

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 LL50 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 therefore based on nominal loading 

rates. 

 

Reference Substance: No 

Results Nominal concentrations: 96-h LL50 >1,000 mg/L. This is equivalent 

to 96-h LL0 = 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: 9-6-00 
36

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 (TOC) measurements of each freshly prepared 

test solution and control and after 24-h on test just before daily 

renewal with fresh test solution. 

Exposure Period (unit) 96 hours 

Statistical methods Statistical analysis of survival 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 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 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 LC50 was 

1.2 mg/L. No information provided on method of calculation. 

Results Nominal concentrations: 96-h LL50 >10,000 mg/L. This is equivalent 

to 96-h LL0 = 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: 9-6-00 
39

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 Chemicals #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 (LL50) 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. Following 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 covered 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 and 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 LL50 (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 LL50s (loading levels likely to cause 50% mortality) 

were as follows: 

Time 

 (hours) 

LL50 

(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 LL50 (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: 3/22/2005 
41

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 continuously 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 preparations were renewed daily. 

Vehicle None 

Statistical Analysis LL50 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 CaCO3

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 14OC during holding period 

14.5-14.9OC during exposure period 
42

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 

Rate50 (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 (LL50) 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 the 96-hour Lethal Loading rate (LL50) 

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: 8/15/2005 

 
45

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 continuously 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 preparations were renewed daily. 

Vehicle None 

Statistical Analysis LL50 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 CaCO3

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 14OC during holding period 

13.6-14.4OC 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 

Rate50 (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 (LL50) 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 the 96-hour Lethal Loading rate (LL50) 

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: 8/15/2005 

 
47

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 continuously 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 preparations were renewed daily. 

Vehicle None 

Statistical Analysis LL50 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 CaCO3

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 OC during holding period 

13.9-14.5 OC 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 

Rate50 (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 (LL50) 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 the 96-hour Lethal Loading rate (LL50) 

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: 8/15/2005 

 
49

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 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 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 
50

loosely covered to reduce entry of dust. 

Light: 16-hour light per day using cool-white fluorescent lights with 

an intensity of 20 uEin/m2

. 

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 

CaCO3. The water was passed through activated carbon, a particle 

filter, and then an ultraviolet sterilizer and stored in a polyethylene 

tank where it was aerated. TOC levels were 2 mg/L at the beginning 

and end of the test and <10 mg/L 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 control, 100, 300, and 1,000 mg/L, 

respectively. 

Calculation of EL50 s: Statistical analysis of survival data not 

warranted. 

Exposure period: 48 hours 

Analytical Monitoring: TOC levels were 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 EL50 >1,000 mg/L. This is equivalent 

to 48-h EL0 = 1000 mg/L based on WAF loading rates. 

Remarks Measured concentration: n/a 

Unit: mg/L 
51

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: 9-6-00 
52

Robust Summary 3-DAPH-2 

Test Substance

CAS # 71486-79-8 

Chemical Name Benzenesulfonic acid, mono-C15-30-branched alkyl and di-C11-13-

branched and linear alkyl derivatives, 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 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 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 with 
53

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 

CaCO3. The water was passed through activated carbon, a particle 

filter, and then an ultraviolet sterilizer and stored in a polyethylene 

tank where it was aerated. TOC levels were 2 mg/L at the beginning 

and end of the test and <10 mg/L 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% immobilization were reported for control, 100, 

300, and 1,000 mg/L, respectively. 

Calculation of EL50 s: Statistical analysis of survival data not 

warranted. 

Exposure period: 48 hours 

Analytical Monitoring: TOC levels were 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 EL50 >1,000 mg/L. This is equivalent 

to 48-h EL0 = 1000 mg/L based on WAF loading rates. 

Remarks Measured concentration: n/a 

Unit: mg/L 

Statistical results: Not applicable because immobilization seen at high 
54

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: 9-6-00 
55

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 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 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 with 
56

an intensity of 20 uEin/m2. 

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 

CaCO3. The water was passed through activated carbon, a particle 

filter, and then an ultraviolet sterilizer and stored in a polyethylene 

tank where it was aerated. TOC levels were 2 mg/L at the beginning 

and end of the test and <10 mg/L 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 EL50 s: Statistical analysis of survival data not 

warranted. 

Exposure period: 48 hours 

Analytical Monitoring: TOC levels were 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 EL50 >1,000 mg/L. This is equivalent 

to 48-h EL0 = 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: 9-6-00 
58

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 concentrations 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 using 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 water accommodated fractions (WAFs) were 

prepared for each test level. Appropriate amounts of test material was added 

to a measured volume of reconstituted water in a glass vessel and stirred for 24 

hours. Stirring was accomplished using a magnetic stirrer at a stirring rate that 

produced a slight dimple at the surface of the water. Following the mixing 

period, the test solutions were allowed 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 distributed into each concentration for 

the range finding study. Five daphnids/replicate/time point (4 replicates) were 

used in the definitive study. Test vessels were covered to reduce evaporation 

and were maintained at 20.6 to 21.1o

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. Daphnia were considered immobilized if 

they were unable to swim for approximately 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 EL50 (Effective Loading rate) was determined to be >100 mg/L 

(WAF). 
59

Remarks Temperature was maintained at 20.3 to 21.0oC throughout the test. No 

treatment related differences were observed 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-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 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 immobilization 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 study could not be explained from an 

examination of the data and were attributed to biological variation by the Study 

Director. 

The 24 and 48-hour EL50 (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 EC50 was 0.78 mg/L. 

Conclusions The 24 and 48-hour EL50 (Effective Loading rate) was determined to be >100 
60

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

mg/L (WAF). 

Data Quality Reliable with restriction (Klimisch 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: 8/15/2005 
61

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 concentrations 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 using 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 water accommodated fractions (WAFs) were 

prepared for each test level. Appropriate amounts of test material was added 

to a measured volume of reconstituted water in a glass vessel and stirred for 24 

hours. Stirring was accomplished using a magnetic stirrer at a stirring rate that 

produced a slight dimple at the surface of the water. Following the mixing 

period, the test solutions were allowed 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 distributed into each concentration for 

the range finding study. Five daphnids/replicate/time point (4 replicates) were 

used in the definitive study. Test vessels were covered to reduce evaporation 

and were maintained at 20.3 to 21.0o

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. Daphnia were considered immobilized if 

they were unable to swim for approximately 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 EL50 (Effective Loading rate) was determined to be >100 mg/L 

(WAF). 

Remarks Temperature was maintained at 20.3 to 21.0oC throughout the test. No 
62

treatment related differences were observed 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 micro-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 EL50 (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 EC50 was 0.78 mg/L. 

Conclusions The 24 and 48-hour EL50 (Effective Loading rate) was determined to be >100 

mg/L (WAF). The no observed effect-loading 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: 8/15/2005 
63

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 test termination (96-h). Water samples 

were passed through 0.45-micron filter prior to TOC analysis using 

EPA Method 415.1 

Statistical methods 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 originally 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 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 for the aquatic 
64

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 were randomly placed on an orbital 

shaker adjusted to approximately 100 cycles per minute under constant 

light (24 hours/day). Daily cell counts were made visually by means of 

direct microscopic examination with a hemocytometer. 

Light: Cool-white fluorescent lights provided a light intensity of 47-50 

uEin/m2

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 EL50 s and NOELs: Binomial nonlinear interpolation 

methods (Stephan, 1983) were used to calculate EC50s (i.e., EL50s). A 

parametric one-way analysis of variance (ANOVA) and Dunnett’s test 

were used to calculate the no-observed effect level. 

 

Method of calculating mean measured concentrations: not applicable 

Exposure period: 96 hours 

Analytical monitoring: At the beginning and end of the test, TOC 

measurements were between 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 actual test material concentrations and 

results are therefore based on nominal loading rates. 
65

Results Nominal concentrations: 72- & 96-h EL50 >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: 9-6-00 
66

Robust Summary 3-ALG-2 

Test Substance

CAS # 71486-79-8

Chemical Name Benzenesulfonic acid, mono-C15-30-branched alkyl and di-C11-13-

branched and linear alkyl derivatives, 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 test termination (96-h). Water samples 

were passed through 0.45-micron filter prior to TOC analysis using 

EPA Method 415.1 

Statistical methods The computer program of Stephan (1983) was used to calculate EL50s. 

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 originally 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 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 for the aquatic 

toxicity test. 

Test Conditions: A static test was conducted; i.e., there was no daily 
67

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 were randomly placed on an orbital 

shaker adjusted to approximately 100 cycles per minute under constant 

light (24 hours/day). Daily cell counts were made visually by means of 

direct microscopic examination with a hemocytometer. 

Light: Cool-white fluorescent lights provided a light intensity of 47-50 

uEin/m2

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 EL50 s and NOELs: Binomial nonlinear interpolation 

methods (Stephan, 1983) were used to calculate EC50s (i.e., EL50s). A 

parametric one-way analysis of variance (ANOVA) and Dunnett’s test 

were used to calculate the no-observed effect level. 

 

Method of calculating mean measured concentrations: not applicable 

Exposure period: 96 hours 

Analytical monitoring: At the beginning and end of the test, TOC 

measurements were between 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 EL50 >1,000 mg/L, based on both 

growth rate and biomass measurements. 72- & 96-h NOEL = 1000 

mg/L. 

Remarks 

Measured concentration: n/a 
68

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: 9-6-00 
69

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 test termination (96-h). Water samples 

were passed through 0.45-micron filter prior to TOC analysis using 

EPA Method 415.1 

Statistical methods The computer program of Stephan (1983) was used to calculate EL50s. 

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 originally 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 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 for the aquatic 

toxicity test. 

Test Conditions: A static test was conducted; i.e., there was no daily 
70

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 were randomly placed on an orbital 

shaker adjusted to approximately 100 cycles per minute under constant 

light (24 hours/day). Daily cell counts were made visually by means of 

direct microscopic examination with a hemocytometer. 

Light: Cool-white fluorescent lights provided a light intensity of 47-50 

uEin/m2

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 EL50 s and NOELs: Binomial nonlinear interpolation 

methods (Stephan, 1983) were used to calculate EC50s (i.e., EL50s). A 

parametric one-way analysis of variance (ANOVA) and Dunnett’s test 

were used to calculate the no-observed effect level. 

 

Method of calculating mean measured 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 EL50s = 1,400 mg/L and >1,500 mg/L 

based on biomass and growth rate, respectively. 96-h EL50s = 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/a 
71

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 x103

. 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 

x103

) 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: 9-6-00 
72

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 106 cells/mL, 5 mL used to inoculate 1 liter of medium 

for an initial cell density of 104

 cells/mL. 

Exposure period/duration 72 hours 

Range find test Yes 

Analytical monitoring Not performed 

Statistical methods A Students t-test incorporating 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 Freshwater 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 dimple 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 prior 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 conducted; i.e., there was no daily renewal of 

test solution. Two (range find study) or six (definitive study) 100-mL replicates 
73

per treatment, inoculum ~10,000 cells/mL. 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: 24.0o

 C. 

Culture Media: As specified in the guideline. 

Method of calculating mean measured 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 EbL50, the loading rate that reduced biomass by 50%, was >100 mg/L WAF. 

The ErL50, 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 EbL50, the loading rate that reduced biomass by 50% was >100 

mg/L WAF. The ErL50, 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. 

Data Quality (1) Reliable without restriction 

References Algal Inhibition Test 

SafePharm Laboratories Project No.: 1666/060 (20 Jul 2005) 

Other Updated: 8/16/2005 
74

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 106 cells/mL, 5 mL used to inoculate 1 liter of medium 

for an initial cell density of 104

 cells/mL. 

Exposure period/duration 72 hours 

Range find test Yes 

Analytical monitoring Not performed 

Statistical methods A Students t-test incorporating 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 Freshwater 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 dimple at the media surface. Following the 

mixing period, the test solution was allowed 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 and 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 conducted; 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/mL. The 250-mL conical flasks were 
75

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: 24.0o

 C. 

Culture Media: As specified in the guideline. 

Method of calculating mean measured 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 EbL50, the loading rate that reduced biomass by 50%, was >100 mg/L WAF. 

The ErL50, 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 EbL50, the loading rate that reduced biomass by 50% was >100 

mg/L WAF. The ErL50, 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. 

Data Quality (1) Reliable without restriction 

References Algal Inhibition 

SafePharm Laboratories Project No.: 1666/063 (20 Jul 2005) 

Other Updated: 8/16/2005 
76

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 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 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. Diarrhea 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 oral 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-day 
79

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 survived 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 oral 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 oral 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 weights 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 oral 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 dosing and remained in poor health for 

approximately 7 days before recovering. 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 the 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 oral 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 rats 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 oral LD50 of greater than 5.0 g/kg. 

Data Quality Reliable without restriction (Klimisch Code) 

References Unpublished confidential business information 

Other Updated: 6/18/01 

 
90

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 sufficient 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 were 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 skin 
91

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 dermal 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 frequently 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 animals 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. Reduced 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. Gross 
93

pathological findings included dry, flaky skin at the dose site of all treated 

animals. Microscopic examination revealed the presence of trace to mild 

hyperkeratosis. There were no other treatment 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 dermal 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 type 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 covered 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 (Draize) 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 treated 
95

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. All 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 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: 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 kept 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 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 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 and 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 inhalation (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 generated by a pressure spray apparatus 

delivered into a 100-liter plexi-glass exposure chamber. The actual 

exposure concentration as measured by gravimetric analysis was 1.9 

mg/L. Particle size analyses were performed once/hour using a multistage 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 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 females)(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 incidence over the next week. Animals 

were free of symptoms of exposure during the second week of 

observation. Several animals exhibited 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 gavage (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 duration 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 parametric 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 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. 

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 performed 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 was 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 in 

the 1000 mg/kg females at the end of the 14-day recovery period. No treatmentrelated effects were observed on mortality, 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 parameters. 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 confirmed 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 mean 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 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 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 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-irritating tape and wrapped 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 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 
103

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 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-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 hemoglobin 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 lungs 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 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 300 mg/kg females compared to control. 

This finding lacked a dose response and 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 at 
104

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 organ 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 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 microscopic findings noted in any group. 

Livers from female 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 animals 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 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/m3 (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 apparatus delivered into a 1000-liter glass and 

stainless steel exposure chamber. Chamber airflow rates was approximately 

200 liters/minute with a chamber 99% equilibration 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 performed 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/m3

. 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 confirmed that the aerosol was respirable in the 

rat (estimated percent of particles <10 microns=93%). There was no test 

material exposure related mortality during 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 were 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; females: 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/m3

) 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/m3

 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) (approximately 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 control group was not included in the study. 

The test material was administered undiluted 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-Wallis 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 Guideline 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 was not used during this study. This is 

considered a minor deviation from the Guideline.) After approximately 6 hours 
108

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 termination 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 animals 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 treatment 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 findings were evident during recovery with 

a decreasing severity and incidence. These data did not exhibit a strong dose 

response. Erythema and desquamation were 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 females 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 increased 
109

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 dose 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 SGPT 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 dermal 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 treatment related morphologic changes in the 

skin, testes, epididymides and possibly the liver. Compound related 

microscopic lesions were seen in the treated skin of the high dose animals at 

termination of the treatment period (high dose recovery and low dose treatment 

and recovery animals were not examined). Treated skin findings included slight 

to moderately severe hyperkeratosis and epithelial hyperplasia. Findings in 

males and females were comparable. Possible treatment related liver findings 

were observed in the high dose group only. Findings present at termination of 

dosing but not following recovery included 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 hypoplasia of the epididymis accompanied the 

testicular changes in many animals at termination 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 termination and recovery blood values and 

organ weights. 

Dose rangefinding study Dose levels were selected based 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 wrapped 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 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. 
112

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 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-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 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 chemistry values were unremarkable in the 

treated animals at termination of the treatment period. Following recovery there 

were a number of small, but statistically significant differences observed in 

serum chemistry parameters of the satellite animals. These included a decrease 

in mean blood urea nitrogen (males), sodium (males) and chloride (females) and 

increases in phosphorus and billirubin (males) 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 weights 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 male 
113

and one female (1000 mg/kg) has epidermal acanthosis/hyperkeratosis. The 

male also exhibited slight focal epithelial 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, C14-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 duration 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 weight change, food consumption, functional 

observational battery observations, hematology 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-parametric 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 weeks 3 and 5. An abbreviated 

functional observation battery (FOB) was performed once prior to 
115

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 evaluated 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 reduced, 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, minimal 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 were 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 solutions 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.: 3580.1 

 
117

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 selected 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 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, 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-irritating tape and wrapped with an 

elastic sleeve. After at least 6 hours the test material residue was 
118

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 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-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 hemoglobin 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 lungs 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 incidental 
119

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 300 mg/kg females compared to control. This finding 

lacked a dose response and 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 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 organ 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 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 microscopic findings 

noted in any group. Livers from female 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 animals 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 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) 

 
120

4.3 Toxicity to Reproduction

Robust Summary 5-ReproTox-1 

Test Substance

CAS # CAS# 115733-09-0 

Chemical Name Benzenesulfonic acid, C14-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) IGS 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 F0 males- 70 days premating; mating period through completion of 

parturition. 

F0 females- 14 days premating; mating; 25 days of gestation and 20 

days of lactation. 

F1 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 F0 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 weight changes, food consumption, semen 

parameters, organ weights, number of days to mating, gestation length, 

pup viability data, total pups delivered, pup body weights and mean 

live litter size were analyzed by ANOVA followed, as needed, by 
121

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 gavage study conducted according to OECD Test 

Guideline 407.) 

Remarks field for test 

conditions 

F0 Generation: 

All F0 males were dosed for 70 days prior to mating and through the 

completion of parturition. All F0 females were dosed for 14 days prior 

to mating and through day 20 of lactation. All F0 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 F0 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 through nursing during lactation days 1-

21 until euthanization on post-natal day 21. The surviving F0 dams 

were necropsied on lactation day 21, following a minimum of 60 days 

of dosing. The surviving F0 males were necropsied at the conclusion of 

parturition following a minimum of 96 days of dosing. F0 females that 

failed to deliver were necropsied 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 surviving control and high dose F0 animals. 

Tissues examined microscopically included the liver, kidney, 

brain, right epididymides, cervix, coagulation gland, ovaries, 

pituitary, prostrate, seminal vesicles, testes, uterus, vagina and 

gross lesions. 

F0 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 F0 males and evaluated for 

sperm count, concentration, motility and morphology assessment.

F1 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 gross 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 analysis confirmed that the test 

article was at the appropriate concentration in the dosing solutions. 

F0 Generation: 

F0 males exhibited a dose related increase in post dosing salivation and 

dark material around the nose in the mid and high dose groups The 

remaining F0 male parameters were unremarkable 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 F0 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 F0 females at necropsy 

and no meaningful microscopic lesions were observed in any of the 

treated F0 females. 

F1 Generation: 

No treatment related findings were noted in the F1 pups during 

lactation. No treatment related gross necropsy findings were evident 

in any of the F1 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 F0 and F1 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: 4/26/2005 

 
124

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 variability in spontaneous frequency of micronucleated 

polychromatic erythrocytes were evaluated in vehicle controls. Statistically 

significant differences were evaluated in the frequency of micronucleated 

polychromatic erythrocytes between treated groups and vehicle controls. 

NCE/PCE (normochromatic erythrocytes/polychromatic erythrocytes) ratios in 

treated and control groups were compared. Tests included dispersion test of 

Amphlett and Delow, and Margolin, Fishers 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 conducted 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 control 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 were 
125

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 individual 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 positive control exhibited a statistically 

significant increase in micronuclei as expected. Chemical analysis confirmed 

that the dosing solution preparation procedure utilized for this study resulted in 

homogeneous solutions of appropriate 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; Cyclophosphamide 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-Smirnov statistic, Kruskal-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 examined 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. Bone 
127

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 observed since there were no statistically 

significant decreases in the percentage of polychromatic erythrocytes compared 

to the vehicle control. The positive control 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 nitrogen. Cultures were incubated at 37oC 

with shaking. Cultures were diluted daily to a cell density of approximately 3 x 

105

 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, 2000, 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 standard 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 were exposed to the test substance and 

incubated for approximately four hours, then washed and cultured for two days. 

Cell culture density was determined 24 and 48 hours post exposure. Treated 

cell suspension growth at each dose level 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 concentrations 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 percent 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 restrictive 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 37OC in 

a humidified 5% CO2 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 the solvent control; the negative control 

spontaneous mutation frequency should be in the range of 0.2 to 2.0/104

 cells; 

negative control plating efficiency should 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 mutagenicity 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 total growth of 10% or greater. Positive 

and vehicle control group responses were 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 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 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 concentrations up to 5000 ug/plate with 

and without metabolic activation. In the main study there were two treatment 

sets for each tester strain, with and without 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), a 
131

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 dose levels of 1000, 2000, 3000, 4000 and 

5000 ug/plate. After 2 days of incubation 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 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 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 significant increases in revertant colonies 

(equal to or greater than three times the THF 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 assay. The positive controls produced at 

least a three-fold increase in revertant colonies when compared with the DMSO 

control in each respective strain. The nontreated and vehicle controls responded 

appropriately. The 5000 ug/plate concentration of test substance in THF was 

evaluated analytically for concentration in both the initial and repeat assays. 

Analysis conformed that the test substance 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 the 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 McCoy's 5A Medium containing 10% fetal 

bovine serum and 2 mM L-glutamine at 37o

C, in 4-6% CO2 in air. Cultures 

were seeded at 1.2 x 106

 cells (16-hour harvest) and 0.8 x 106 (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 with 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 used 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 180 
133

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 controls 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. Cytotoxicity 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 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 MNNG (positive control) was tested without activation. Prepared cultures 

were treated with test substance or control 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 mitotic cells prior to harvest. 

The test substance treated groups, selected 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 following observations were recorded and 

excluded from the total aberration frequency: gaps, polyploid and 

endoreduplicated cells, pulverized chromosomes, Robertsonian translocations, 

translocations and abnormal monocentric chromosomes. The percent of 

aberrant cells and the frequency of aberration (%) per treatment group were 

determined. In order for a test substance 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 statistically significant response in at least one 

treatment group with a mean % of aberrant cells exceeding 5% was observed. 

Test substance concentration verification was performed on the highest stock 

concentration in both the initial and repeated assays. Results were within 8% of 
134

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 reduced 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 aberration 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 and without metabolic activation. In the 

initial 16-hour harvest without activation a statistically significant increase was 

observed with one dose level different from 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 the 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; cyclophosphamide (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 number 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 Multiple Range test and regression analysis. 

Residuals from the ANOVA were analyzed by Wilk’s Criterion or the 

Kolomogorov-Smirnov statistic. Nonparametric 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 study 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 polychromatic 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 sacrifice and were free of clinical signs. The 

responses of the vehicle control and positive 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 cytotoxicity. 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 desired 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 concentrations up to 5000 ug/plate with 

and without metabolic activation. In the 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 dosed 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 refrigerated. These plates were evaluated for 

gross toxic effects and total revertant colony numbers on the following day. In 

the repeat assay TA98, TA100 and TA1535 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 (notable 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 interference. 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 significant increases in revertant colonies 

(equal to or greater than three times the THF 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 inconsistent 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 produced at least a three-fold increase in 

revertant colonies compared with the DMSO control in their respective strains. 

Nontreated and vehicle controls were acceptable 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 substance 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 the 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/plate 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 strains TA98 and TA100, and a dose level of test 

material of 10 mg/plate and concentrations of S9 mix ranging from 25 to 250 ul 

S-9/plate. 

Remarks field for test 

conditions 

This study was conducted according to OECD 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 substance, 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 37o

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/plate 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 absence of metabolic activation. Positive 

control responses were 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-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 analog 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 WP2uvrA 

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 

WP2uvrA +S9 2-aminoanthracene 80.0 ug/plate 

WP2uvrA –S9 ICR-191 50.0 ug/plate 

Vehicle Control Pluronic F127 25% w/w in ethanol 

Statistical Analysis Mean revertant colony count and standard deviation 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 concentrations 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 concentrations of test substance, vehicle 

controls, and a positive control. Three plates/dose 

group/strain/treatment set were evaluated. 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 

37o

C. The condition of the bacterial 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 WP2uvrA 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) and 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 metabolic activation and in WP2uvrA 

with metabolic activation. However since these findings were not 

found during the first experiment they were not considered 

biologically significant. The positive 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 McCoy's 5A Medium containing 10% fetal 

bovine serum and 2 mM L-glutamine at 37o

C, in 4-6% CO2 in air. Cultures 

were seeded at 1.2 x 106

 cells (16-hour harvest) and 0.8 x 106 (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 with 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 used 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 180 
144

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 controls 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. Cytotoxicity 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 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 MNNG (positive control) was tested without activation. Prepared cultures 

were treated with test substance or control 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 mitotic cells prior to harvest. 

The test substance treated groups, selected 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 following observations were recorded and 

excluded from the total aberration frequency: gaps, polyploid and 

endoreduplicated cells, pulverized chromosomes, Robertsonian translocations, 

translocations and abnormal monocentric chromosomes. The percent of 

aberrant cells and the frequency of aberration (%) per treatment group were 

determined. In order for a test substance 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 statistically 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 were 
145

performed on the highest stock concentration in both the initial and/or repeated 

assays. Results were within 6% of nominal. 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 survival and/or mitotic index was not 

observed in either the initial or repeat assays. Precipitation was observed at 

concentrations greater than 40 ug/mL 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 test concentration to be evaluated. 

There were no statistically significant differences in the number of 

chromosomal aberrations between the treated 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 activation). In the initial 16-hour harvest, 

there were statistically significant increases with dose in the percent of aberrant 

cells for both the activated and nonactivated 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 have 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)