OFFICE OF

PREVENTION, PESTICIDES, AND

TOXIC SUBSTANCES

UNITED STATES ENVIRONMENTAL PROTECTION AGENCY

WASHINGTO N, D.C. 20460

MEMORANDUM

TXR No. 0051358

Date: 21-November-2002

Subject: Alpha Olefin Sulfonate Sodium: (PC Code 000905). Section 3 Registration for

Application of EH-2001 for the Control of Richardson’s Ground Squirrels and Wyoming

Ground Squirrel. Human Health Risk Assessment. DP Barcodes: D275422, D275424,

D282331, and D286516. Submission: S598325, S598325, S608203, and S608203. Case

Number: 070054. 

From: PV Shah, Ph.D., Toxicologist

Guruva Reddy, D.V.M.; Ph.D., Toxicologist

Mark Dow, Ph.D., Biologist

Registration Action Branch 1; Health Effects Division (RAB1/HED; 7509C)

Through: G. Jeffrey Herndon, Branch Senior Scientist

RAB1/HED (7509C)

To: Meredith Laws/Geraldine McCann; PM Team 4

Registration Division (7505C)

And

Kathryn Boyle

Lower Toxicity Pesticide Chemical Focus Group

Registration Division (7505C)

ACTION REQUESTED: The Health Effects Division (HED) of the Office of Pesticide Programs

(OPP) is charged with estimating the risk to human health from exposure to pesticides. The

Registration Division (RD) of OPP has requested that HED evaluate toxicology and worker exposure 

as part of a joint review process between PMRA (Canada) and US EPA for the end-use product EH2001 (product containing yellow mustard seed [PC Code 014901] and sulfonic acids, C14-16-olefin

sulfonate [PC Code 000905]) for the control of Richardson’s Ground Squirrels and Wyoming Ground

Squirrel in rangeland, ornamental plantings, orchards, golf courses, parks, nurseries, and non-crop

rights-of-way. 
2

BACKGROUND: The Registration Action Branch 1 (RAB1) of the HED reviewed the data provided

in the MRID 45551102, 45551103, 45307616, and 45551101. In addition, RAB1 reviewed the

available toxicological studies on alpha olefin sulfate (C14-16) from the open literature. A decision

document on alpha olefin sulfate (C14-16) summarizing all available toxicity studies, has been

submitted to Kathryn Boyle (Lower Toxicity Pesticide Chemical Focus Group) by Dr. Guruva Reddy

of RAB1. This document has been reviewed by RAB1 toxicologists and Karen Whitby, Branch

Chief of RAB1. This document is also included in this memo in its entirety.

USE PATTERN - EH-2001

The product is diluted in a ratio of 4 L concentrate to 91 L water. It is delivered via some

modification of a hand-held spray wand with an aspirating nozzle to facilitate foaming action. 

The nozzle is inserted as far as possible into a burrow and the burrow is filled with foam.

The dilute is applied at 20-55 psi and it takes about 5 - 60 seconds (average 15 seconds) to treat one

burrow. Approximately 3.4 liters of foam are administered per burrow or about 0.9 gal. The number

of burrows treated per day is highly variable depending upon population density but is has been

estimated that 215 gallons of dilute could be used in an 8 hour work day. Upon administration of the

foam, a wire mesh basket is placed over the burrow opening. If no activity is observed after about

one minute, the burrow entrance is to be filled with earth and tamped firmly. Use of this product is

limited to areas in Canada of southern Alberta, Saskatchewan and the extreme Southwest of Manitoba

and in the U.S., of Idaho, Montana, North Dakota, Wyoming, South Dakota, Colorado, Minnesota

and Nevada. 

CONCLUSION: Based on available toxicity data and anticipated use pattern, RAB1 of the HED

concluded that a quantitative risk assessment is not required for the end-use product EH-2001 for the

control of Richardson’s ground squirrel and Wyoming ground squirrel. This conclusion is based on

the following:

• Based on the proposed use pattern and other information provided to HED for review, only short

term exposure via inhalation or dermal routes of exposure are anticipated. Exposure by the oral

route is not anticipated. HED expects that the material will be applied by some type of lowpressure hand-wand apparatus and that the volume of material per use/application will be relatively

small. 

• No significant toxicity was observed in the 28-day dermal toxicity study in the rabbit, 91-day

dermal toxicity study in the rat, 2-year chronic/carcinogenicity study in the rat, or the 92-week

chronic/carcinogenicity study in mice. No systemic toxicity via dermal route was observed in all

three species studied.

• The active ingredient is in Toxicity Category IV via inhalation. Therefore, no significant inhalation

toxicity is anticipated from the proposed use.

• Liver hypertrophy seen in the one-week feeding study in rats is due to adaptive changes and not

considered as a toxicologically significant effect. This effect was not observed in the subchronic

and carcinogenicity feeding studies in rats. No significant systemic toxicity was observed in the

subchronic feeding studies in rats at doses up to 1,000 mg/kg/day or in the carcinogenicity feeding

study in rats at doses up to 195 to 259 mg/kg/day.
3

• There was no evidence of carcinogenicity in mice or rats either by dermal or oral routes of

exposure.

• Mutagenicity studies indicate no conclusive evidence of mutagenic effects of alpha olefin.

• No evidence of qualitative or quantitative susceptibility was observed in the developmental toxicity

studies in mice, rats and rabbits.

• Developmental toxicity was observed at or above maternally toxic doses (300 mg/kg/day or above)

in mice, rats and rabbits. The effects of concern (i.e. maternal deaths) seen in these studies are

probably due to bolus administration (i.e. gavage) and/or surfactant property of the chemical. The

maternal toxicity is the most likely cause of fetal deaths and skeletal variations seen in these

studies. This is further supported by the fact that no significant systemic toxicity was observed in

feeding (oral) subchronic and chronic studies in rats. The maternal and fetal deaths would not be

expected to occur via the dermal route, which is the anticipated route of exposure because no

systemic toxicity were observed in subchronic and chronic dermal toxicity studies. In addition,

considering poor dermal absorption of alpha olefin (reported value of 0.6%), it would require

almost 30,000 mg/kg/day oral equivalent doses to produce developmental effects and these types of

exposure concentrations are not likely to occur in the work environment. 

• An available published peer reviewed dermal absorption study indicates poor dermal absorption

(0.6%) of alpha olefin through the skin of rats. Although, there are several reporting/deficiencies

in this study, the study adequately indicates very poor dermal absorption of alpha olefin in rats. In

this study, dermal absorption was measured following dermal application of radiolabelled alpha

olefin to the intact skin and also through the damaged skin in which the stratum corneum was

removed prior to application of the test material. It is generally believed that the primary barrier

property of the skin resides in the stratum corneum, the upper most layer of the skin. 

Approximately 0.6% of the applied dose was recovered following 24 hour exposure duration via

intact skin. Approximately 50.4% of the applied dose was recovered following 30 hour exposure

duration via damaged skin. In these sets of experiments, several guideline reporting/deficiencies

were noted. Despite all of the guideline deficiencies, the study was well conducted and does

provide valuable information on dermal absorption in rats that could be used for regulatory risk

assessment. If one looks at the results of intact and damaged skin (without barrier property), it

showed that only 0.6% of the applied dose was recovered through intact skin compared to 50.4% of

the applied dose through the damaged skin. One might expect 100% dermal absorption through the

damaged skin (barrier less skin), however, in this study only half of the applied dose was recovered

in various tissue examined. Based on this estimates, one could extrapolate approximately 1.2%

dermal absorption via intact skin. Therefore, this study does provide a useful calculated dermal

absorption value of 1.2% dermal absorption of alpha olefin through the skin. 

• The poor dermal absorption of alpha olefin is also supported by the results of dermal toxicity

studies in mice, rats and rabbits which indicate no systemic toxicity at doses up to 100 mg/kg/day

(HDT) in the subchronic toxicity studies and 38.9 mg/kg/day (HDT) in a dermal carcinogenicity

study. 

• The end-use-product is a severe eye irritant, however, the proposed labeling is explicit 

regarding possible ocular injury and directs handlers to use the appropriate Personal Protective 

Equipment to avoid injury.

• Based upon the method of delivery, the point of application, low application pressures, relatively

small volumes of material that might be used per day and upon the label directed Personal
4

Protective Equipment, HED expects that dermal, inhalation and ocular exposure to be negligible if

applied as directed by the proposed labeling. In addition, based on the specific sites of application

and the fact that the burrows are sealed with soil after the application is complete, HED believes

the post-application exposure to handlers or non-handlers is also negligible.

• Finally, only 6.91% of the alpha olefin is present in the product. Based on the low concentration of

alpha olefin in the product available for exposure, the low dermal absorption and the lack of

demonstrated systemic toxicity by the dermal route and oral feeding studies, RAB1 of the HED

has determined that no quantitative risk assessment is required.
5

Lower Toxicity Pesticide Chemical Focus Group

Decision Document

"-Olefin Sulfonate Sodium

EH-2001 (Alpha-Olefin Sulfonate Sodium and White Mustard Seed)

HAZARD CHARACTERIZATION

Sodium "-Olefin Sulfonates are long-chain sulfonic acids which are used in the Cosmetic Industry as

surfactants or cleansing agents. International J. of Toxicology, 17 (Suppl.5) 39 - 65, 1998, published

the findings of Cosmetic Ingredient Review Expert Panel on sodium "-olefin sulfonates. The review

compiled experimental data concerning the safety information of Sodium C14 - 16 Olefin Sulfonate,

Sodium C12 - 14 Olefin Sulfonate, Sodium C14 - 18 Olefin Sulfonate, and Sodium C16 - 18 Olefin

Sulfonate. Sodium olefin sulfonates are primarily the mixtures of sodium hydroxyalkane sulfonates

and sodium alkane sulfonates as shown in figures below. Since the registrant submitted the acute

studies with Sodium C14 - 16 Olefin Sulfonates, this review of the toxicity studies is primarily on

C14 -16 sulfonates only, with exceptions being the developmental and mutagenicity studies which

used other sulfonates.

 Sodium C14 - 16 "-Olefin Sulfonate has low acute toxicity (Tox. Cat. III) via oral, and dermal routes

of exposure. It is mild skin irritant (Tox. Cat. III) and not a skin sensitizer. It is a severe eye irritant

(Tox. Cat I) causing corneal opacity lasting 7 days post application. The inhalation LC50 is > 229

mg/L for powdered aerosol flakes (90% a.i.), which indicate low toxicity via inhalation route. No

subchronic, repeat dermal, developmental or mutagenicity data are submitted as required under Tier 1

data requirements for Non-food use chemical. However, the review by the Cosmetic Ingredient

Review Expert Panel published in the International J. of Toxicology, 17 (Suppl.5) 39 - 65, 1998, did

provide the necessary toxicity information to fulfill Tier 1 Data requirements. In subchronic feeding

studies in rats there was no systemic toxicity observed at doses up to 1000 mg/kg/day (Limit Dose). 

Systemic toxicity was not observed in 90-day dermal study in rabbit but resulted in mild to moderate

skin irritation at 100 mg/kg/day. In chronic toxicity/carcinogenicity study in rats decreased body

weight in males and females was observed at 195 and 259 mg/kg/day, respectively (LOAEL) and the

NOAEL was 96 and 132 mg/kg/day, respectively. Twice dermal application of 0.2 mL (30% a.i) for

92 weeks in rats did not elicit any systemic toxicity up to 38.9 mg/kg/day (NOAEL). The AOS was

not carcinogenic in both the feeding and dermal carcinogenicity studies. The AOS (C14 - 16, C14 -

18) were not mutagenic in in vitro mutagenicity assays in 4 separate trials. In a host-mediated assay,

the AOS (C14 - 16) caused positive response in salmonella typhimurium TA 1530. This positive

response was probably due to incomplete ether extraction and higher pH (11.1). When the pH of the

extract was adjusted with sulfuric acid to 8.5, the salmonella typhimurium TA 1530 test was negative. 

Developmental toxicity using C14 - 18 AOS in mice, rats and rabbits, did not cause developmental

toxicity below maternal toxicity. In Mice maternal toxicity NOAEL was 2 mg/kg/day based on

reduced body weights and litter loss observed at 300 mg/kg/day (LOAEL). The Developmental
6

NOAEL was 300 mg/kg/day based on decreased pup weight, major malformations (cleft palate) and

minor skeletal anomalies. In the rat there was no maternal or developmental toxicity was observed up

to 600 mg/kg/day (NOAEL; HDT). Rabbit appears to be sensitive species for developmental toxicity. 

Maternal toxicity was similar to mice i.e., the NOAELs (2 mg/kg/day) and LOAELs (300 mg/kg/day)

are same as mice. The developmental NOAEL is 2 mg/kg/day and LOAEL is 300 mg/kg/day based

on increased incidence of minor skeletal anomalies and extra ribs. The AOS was readily absorbed

from the GI tract, and rapidly excreted in the urine (72%) and feces (22%) within 24 hours. About

4.3% was excreted in the bile within 12 hours after oral administration. Blood levels peaked in 3

hours then declined. Less than 0.03% was retained in the tissues after 24 hours. Upon intravenous

administration, half of the administered dose was excreted within 1 hour and 90% was excreted 0 - 6

hours. The data suggest metabolites may be more polar than the parent and the intact AOS could bind

with proteins while metabolites can not. Dermal absorption study indicate about 0.6% was absorbed

from the intact skin. 

The End-Use formulation EH-2001 has low acute toxicity (Tox. Cat. IV) via oral, dermal and

inhalation routes of exposure. It is not a skin irritant (Tox. Cat. IV). It is a skin sensitizer. The skin

sensitization potential is probably due to impurities such as unsaturated and chloro-1,3-sultones. The

formulation is a severe eye irritant (Tox. Cat I) causing corneal opacity lasting 7 days post application.

File Symbol: 72919-R

Tolerance Reassessments ?: Yes [40 CFR 180.1001(c); sodium "-olefin sulfonate (sodium C14 -

C16)(olefin sulfonate)]

Chemical Name: Alpha-Olefin Sulfonate Sodium (AOS)

Chemical Category/Group: Alpha-Olefin Sulfonate Sodium - Surfactant

CAS. Reg. No.: 68439-57–6

PC Code (active): 000905

PC Code (inert): 900631 and 900905

1. Structure:

R-CH-CH2-CH2-SO2-Na

sodium 3-hydroxyalkane sulfonate

R-CH=CH-CH2

(OH)-SO2

-Na

sodium 2,3-alkane sulfonate
7

Sodium alkane sulfonates (AOS) are primarily a mixture of sodium hydroxyalkane sulfonate and

sodium alkane sulfonate. The sodium C14-C16AOS is also identified as tetradecenesulfonate or

sodium tetradecensulfonate.

2. Alpha-Olefin Sulfonate Sodium

Several types of studies were submitted to the Agency: (1) complete studies for review and

evaluation, (2) articles from open-leterature, (3) an assessment by a Cosmetic Ingredient Review

(CIR) expert panel, and (4) an assessment by the Soap and Detergent Association. The Agency does

not have access to the studies that support the assessments. A toxicity profile of the information

available for C14-C16 olefin sulfonate, sodium salt is below. Robust summaries of the submitted

studies and articles from open-literature and toxicity profile are listed below: 

Oral Toxicity

Species/Strain: Rat/Albino (HSD:SD)

Conclusion: LD50

Males = 2161 mg/kg

Females = 1895 mg/kg

Combined = 2000 mg/kg

Tox Cat. III.

Method: Groups of 5 young immature rats/sex/group were given a single oral dose of 1000,

2000 or 3000 mg/kg (purity unknown). Animals were observed for clinical signs of

toxicity for 2 weeks post-dosing.

GLP: Yes

Test Substance: Witconate AOK (Alpha-Olefin Sulfonate Sodium; Sodium c14-16), Lot # 3379,

purity - unknown

Results: No deaths in 1000 mg/kg group. Two males and 3 females in 2000 mg/kg group and

all animals in the 3000 mg/kg group died on day 1. Clinical signs of toxicity was

seen at all dose levels which included pilorection, salivation, polyuria, diarrhea,

respiratory chirp and gurgle, ptosis, decreased activity, nasal discharge, gasping,

aggressiveness, sensitivity to touch, exophthalmos and bloating were seen up to 10

days post dosing. Body weights of surviving animals were not effected by treatment

except one male at the 2000 mg/kg and one females at the 1000 mg/kg groups lost

weight. Necropsy of the surviving animals was unremarkable. Animals died on the

test exhibited discolored gastrointestinal contents and filled with gas, staining of

muzzle, and matting and staining of genital hair and ventral hair.
8

Reference: Kuhn, J. (1996) Acute Oral Toxicity of Witconate AOK in Rats. Stillmeadow, Inc.,

Sugar Land, Texas. Laboratory Study No.: 2830-96. July 1, 1996. MRID

45307612. Unpublished.

Reliability: (2) valid with restrictions. Purity was not provided.

Acute Dermal Toxicity

Species/Strain: Albino Rabbit/New Zealand White

Conclusion: LD50 > 2020 mg/kg

Severe dermal irritant; included erythema, edema, shallow lateral fissuring and

coriaceousness.

Tox Cat. III.

Method: Groups of 5 male and 5 female rabbits were applied a single dose of 2020 mg/kg of

Witconate AOK in 0.535 mL deionized water/g of substance, covered with surgical

gauze, secured with non-irritating adhesive tape and maintained in place orthopedic

stockinette. After 24 hours, the bandages were removed, the site was gently wiped

with clean cloth and dermal irritation was scored at approximately 60 minutes, and

on Days 4, 7, 10 and 14.

GLP: Yes

Test Substance: Witconate AOK (Alpha-Olefin Sulfonate Sodium; Sodium C14-16), Lot # 3379,

Purity - unknown

Results: There was no mortality during the study. There were no clinical signs of toxicity

were observed, except for a ruptured abscess in one animal which was considered

unrelated to treatment. One male lost < 3% of body weight during the study. Severe

skin irritation was observed which included erythema, edema, shallow lateral

fissuring and coriaceousness.

Reference: Kuhn, J. (1996) Acute Dermal Toxicity of Witconate AOK in Rabbits. Stillmeadow,

Inc., Sugar Land, Texas. Laboratory Study No.: 2829-96. June 7, 1996. MRID

45307613. Unpublished.

Reliability: (2) valid with restrictions. Purity was not provided.

Acute Inhalation Toxicity

Species/Strain: Rat/unknown
9

Conclusion: LC50 > 229 mg/L for C14 - 16 flakes (90% a.i.) 1 hour exposure

LC50 > 221 mg/L for C14 - 16 sprayed dried formulation (17% a.i.) 1 hour exposure.

Tox. Cat. Not determined

Method: No experimental details were provided. Groups of 10 rats were exposed for 1 hour

to a powdered aerosol of either C14-16 AOS flake (90% active) or a spray-dried

formulation containing 17% C14-16 AOS at concentrations of 229 mg/L and 221

mg/L, respectively. Five rats were sacrificed and examined grossly after the

exposure, but did not specify the time of sacrifice. The remaining rats were

necropsied 14 days after exposure.

GLP: No.

Test Substance: C14-16 AOS (Alpha-Olefin Sulfonate Sodium)

Results: There were no deaths. The rats appeared normal except for increased preening

behavior. Mild petechial hemorrhages in lungs were seen in 2 rats exposed to flakes

and in one rat exposed to spray-dried formulation. Necropsy of the rats killed after

14 days were unremarkable.

Reference: Little, D. (1993) Environmental and human safety of major surfactants. Volume I. 

Anionic surfactants. Part 4. Alpha Olefin Sulfonates. National Technical Service

(NTIS) report No. PB94-102423.

Reliability: Low. Guideline method was not used. 

Primary Eye Irritation

Species/Strain: Albino Rabbits/New Zealand White

Conclusions: Corneal opacity was present 7 days post application. 

Tox Cat. I.

Method: 0.1 ml undiluted sulframin AOS (Lot # U3541, purity - unknown) was instilled in the

one conjunctival sac of 3 male and 3 females albino rabbits and observed at 24, 48,

and 72 hours and at 4 and 7 days after treatment for eye irritation. 

GLP: No

Test Substance: Sulframin AOS (Lot # U3541, purity - unknown)
10

Results: Corneal opacity was present in 4/6 rabbits 7 days post treatment. Conjunctival

irritation was present in 5/6 rabbits 7 days post treatment. The maximum irritation

score was 26.8 at 48 hours and 15.5 at 7 days.

Reference: Sabol, E. (1980) Acute Eye Irritation Study of Sulframin AOS in Rabbits.

Stillmeadow, Inc., Houston, TX 77036. Laboratory Study No.: 1930-80. November

12, 1980. MRID 45307614. Unpublished.

Reliability: (2) valid with restrictions. Study not performed to guidelines and some experimental

details such as purity were not listed. 

Primary Dermal Irritation

Species/Strain: Albino Rabbits/New Zealand White

Conclusions: Sulframin AOS is moderately irritating to rabbit skin. Erythema and edema were

observed at both the intact and abraded skin sites post 72 hours.

Tox Cat. III.

Method: The backs of 3 male and 3 female albino rabbits were shaved 24 hours prior to

treatment. Two sites were identified on the backs, one intact and the other abraded.

The backs were abraded using a hypodermic needle. Each test site was applied with

0.5 mL of undiluted sulframin AOS, covered with gauze pad, secured with adhesive

tape. Twenty-four hours after treatment, dressing was removed, and the animals

were observed for erythema, edema, and eschar formation at 24, and 72 hours.

GLP: No

Test Substance: Sulframin AOS (Lot # U3541, purity - unknown)

Results: Sulframin AOS is moderately irritating to rabbit skin. Erythema and edema were

observed at both the intact and abraded skin sites post 72 hours. The mean score for

erythema and edema were 2.0 and 1.33, respectively. The primary irritation score is

3.33. There was no signs of ulceration, necrosis, or irritation (other than fissuring)

present 72 hours after treatment.

Reference: Sabol, E. (1980) Acute Dermal Irritation Study of Sulframin AOS in Rabbits.

Stillmeadow, Inc., Houston, TX 77036. Laboratory Study No.: 1931-80. November

19, 1980. MRID 45307615. Unpublished. 

Reliability: (2) valid with restrictions. Study not performed to guidelines and some experimental

details such as purity were not listed. 
1 Calculated based on the assumption that an 250 g adult rat would consume 20

g/day.

11

Dermal Sensitization

Species/Strain: Guinea Pig/Hartley

Conclusions: Sulframin AOS ( (Alpha-Olefin Sulfonate Sodium; Sodium C14-16) is not a dermal

sensitizer

Method: Delayed Contact Hypersensitivity method by Buehler was employed to evaluate the

sensitization potential of Sulframin AOS. Ten male and ten females guinea pigs

were induced with applications of 25%w/v sulframin formulation is distilled water

for 6 hours, once a week for a total of 3 applications. Following 2 weeks after last

induction exposure, the guinea pigs were challenged with 10% w/v sulframin AOS

in distilled water. Ten (5/sex; naive control) guinea pigs were also challenged with

10% formulation. All the treated guinea pigs and naive controls were rechallenged

with 5% formulation.

GLP: No

Test Substance: Sulframin AOS (EXP. 5113-161A), purity - unknown

Results: No animal showed positive signs of sensitization during the first, second, or third

challenges at either the 24 or 48 hour observations.

Reference: Kreuzmann, J. (1988) Skin Sensitization Study of C-14 to 16 AOS in Guinea Pigs.

Hill Top Biolabs, Inc., Miamiville, OH 45146. Laboratory Study No.: 88-3168-21. 

July 18, 1988. MRID 45357703. Unpublished.

Reliability: (2) valid with restrictions. Study not performed to guidelines and some experimental

details such as purity were not listed. 

Subchronic Oral Toxicity

a) Range-Finding Study

Species/Strain: Rat/unknown

Conclusion: NOAEL = 0.625% (125 mg/kg/day1

)

LOAEL = 1.25% (250 mg/kg/day), based on increased liver to body weight ratio.
12

Method: Groups of rats were fed diets containing 0.625, 1.25, or 1.5% AOS (70% C14: 30%

C16; 125, 250 or 300 mg/kg/day, respectively) for 7 days. No other details were

provided.

GLP: NO

Test Substance: AOS (Alpha-Olefin Sulfonate Sodium; 70% C14: 30% C16), unknown purity.

Results: No systemic toxicity was noted up to 125 mg/kg/day. A slight increase in liver to

body weight ratio was noted in males. At 300 mg/kg/day, a significant decreased in

body weights was noted for males on day 2 and day 7 in females.

Reference: Little, D. (1993) Environmental and human safety of major surfactants. Volume I. 

Anionic surfactants. Part 4. Alpha Olefin Sulfonates. National Technical Service

(NTIS) report No. PB94-102423.

Reliability: (2) valid with restrictions. Study not performed to guidelines and some experimental

details such as purity was not listed.

b) 90-day Feeding Study

Species/Strain: Rat/unknown

Conclusion: NOAEL $ 1000 mg/kg/day (HDT; Limit Dose)

LOAEL was not established

Method: Groups of rats (number not specified) were fed diets containing AOS (Alpha-Olefin

Sulfonate Sodium 89.7%) at doses of 40, 200 or 1,000 mg/kg/day for 90 days. No

other details were provided.

GLP: No

Test Substance: AOS (Alpha-Olefin Sulfonate Sodium 89.7%).

Results: No changes in hematology, clinical chemistry, feed consumption, gross or

histopathological lesions were not noted at the highest dose tested. A slight increase

in the liver to body weight ratio was noted. In the absence of histopathological and

liver enzyme changes, the increased ratio was considered as adaptive change.

Reference: Little, D. (1993) Environmental and human safety of major surfactants. Volume I. 

Anionic surfactants. Part 4. Alpha Olefin Sulfonates. National Technical Service

(NTIS) report No. PB94-102423.
13

Reliability: (2) valid with restrictions. Study not performed to guidelines and some experimental

details lacking.

c) 91-day Feeding study

Species/Strain: Rat/unknown

Conclusion: NOAEL $ 500 mg/kg/day (HDT)

LOAEL was not established

Method: Groups of rats (number not specified) were fed diets containing C14-16 AOS

(Alpha-Olefin Sulfonate Sodium34% a.i.) at doses of 50, 150 or 500 mg/kg/day for

91 days. No other details were provided.

GLP: No

Test Substance: C14-16 AOS (Alpha-Olefin Sulfonate 34% a.i.)

Results: No treatment-related toxicological or histopathological changes were noted at the

highest dose tested. Minor variations in hematology parameters were noted. No

details were provided.

Reference: Little, D. (1993) Environmental and human safety of major surfactants. Volume I. 

Anionic surfactants. Part 4. Alpha Olefin Sulfonates. National Technical Service

(NTIS) report No. PB94-102423.

Reliability: (2) valid with restrictions. Study not performed to guidelines and some experimental

details lacking.

Repeat Dermal Toxicity

a) 28-Day Dermal Toxicity

Species/Strain: Rabbits/unknown

Conclusion: NOAEL $2mg/animal i.e., 0.8 mg/kg (based on 2.5 kg average weight of a rabbit)

for 1%aqueous solution of C14-16 AOS or a 1% AOS formulation

LOAEL Was not established.

Method: Rabbits were dermally treated with 0.2 ml (2 mg assuming 90% purity) of a 1%

aqueous solution of C14-16 AOS or a 1% AOS formulation for 28 days. No other

details were provided.
14

GLP: No

Test Substance: C14-16 AOS or its formulation

Results: Not irritating to rabbit skin.

Reference: Little, D. (1993) Environmental and human safety of major surfactants. Volume I. 

Anionic surfactants. Part 4. Alpha Olefin Sulfonates. National Technical Service

(NTIS) report No. PB94-102423.

Reliability: (2) valid with restrictions. Study not performed to the guidelines method was not

used.

b) 91-day Dermal Toxicity

Species/Strain: Rat/unknown

Conclusion: NOAEL < 100 mg/kg/day (HDT)

LOAEL = 100 mg/kg/day, based on mild to moderate skin irritation.

Method: Six rabbits were dermally treated with 2 ml/kg/day of a 5% aqueous solution of AOS

(34% active) for 91 days. No other details were provided.

GLP: No

Test Substance: AOS (Alpha-Olefin Sulfonate Sodium 34%).

Results: Mild to moderate skin irritation.

Reference: Little, D. (1993) Environmental and human safety of major surfactants. Volume I. 

Anionic surfactants. Part 4. Alpha Olefin Sulfonates. National Technical Service

(NTIS) report No. PB94-102423.

Reliability: (2) valid with restrictions. Study not performed to guidelines and experimental

details were not provided. 

Chronic Oral Toxicity - Rat

Species/Strain: Rat/Sprague-Dawley

Conclusion: NOAEL = 2500 ppm (125 mg/kg/day). 

LOAEL= 5000 ppm (250 mg/kg/day), based on decreased body gains in males

and females.
15

Method: Alpha-Olefin Sulfonate (97.7% a.i., 60.4 Alkenyl sulphonate:39.6% hydroxyalkane

sulphonate w/w) was administered in diet to groups of 50 male and 50 females rats at

dose levels of 0, 1000, 2500, or 5000 ppm (0, 39, 96 or 195 mg/kg/day for males and

0, 57, 132, and 259 mg/kg/day for females) for 104 weeks. Food consumption and

body weights were recorded weekly. Urinalysis was conducted from 10 male and 10

female rats from the control and the high-dose group at 12, 25, 51, 77 and 103

weeks. Hematology and clinical chemistries were done 10/sex from the control and

the high-dose groups at 13, 25, 52, 78 and 103 weeks. At the end of the treatment

period all surviving animals were sacrificed, gross and histopathology of the organs

performed as recommended by the guidelines. Appropriate statistical analysis was

performed to assess tumor incidence.

GLP: No

Test Substance: Alpha-Olefin Sulfonate (97.7% pure)

Results: There was no treated-related effects on the clinical signs, food consumption,

hematology and clinical chemistries, organ weights and gross and histopathology. 

There was a slight depression in food consumption in females in 5000 ppm group,

but the animals recovered during the second year of treatment. Body weight gains

were significantly less in males (16%) and females (28%) during 14 - 26 weeks. 

Pancreatic islet tumors in 1000 ppm male rats (4), adrenal tumors in 2500 ppm males

(8), thyroid tumors in 2500 ppm females. These incidences considered not related to

treatment since there was no dose response. Mammary tumors were observed in

females in all treated groups. The tumor evidence did not support that AOS was

tumorogenic.

Reference : Hunter, B., Benson, H. (1976) Long-Term Toxicity of Surfactant Alpha Olefin

Sulphonate (AOS) in the Rat. Huntingdon Research Center, Huntingdon, UK. 

Toxicology 5:359-370.

Reliability: (2) valid with restrictions. Although the study was not performed as per the

guidelines, but the findings are peer reviewed. Therefore, the results are considered

valid and could be used for regulatory purposes. 

Carcinogenicity - Dermal

a) Rat

Species/Strain: Rat/Long-Evan

Conclusion: No evidence of carcinogenicity by application of 38.9 mg/kg C14-16 (38.9%) AOS

twice weekly for 2 years.
16

Method: 50 rats/sex were treated for 2 years as follows:

a) deionized water

b) hydrolyzed composite sample of C14-16 AOS and C16-18 AOS (30.0% active)

c) partially hydrolyzed sample of AOS (30.9%) identical to (ii) but containing a

residual level of sultone

d) commercial C14-16 AOS (38.9% active)

Each preparation was applied as a 10% active (v/v) aqueous solution twice weekly to the

clipped backs at a dose level of 1 mL/kg for 2 years. No other experimental details were

provided.

GLP: No

Test Substance: Commercial C14-16 AOS (38.9% active)

Results: Mean body weights, food consumption, hematology, urinalysis, mortality, and gross

and histopathology were comparable to controls. No increased incidence of tumors

were in treated animals were observed.

Reference: Little, D. (1993) Environmental and human safety of major surfactants. Volume I. 

Anionic surfactants. Part 4. Alpha Olefin Sulfonates. National Technical Service

(NTIS) report No. PB94-102423.

Reliability: (4) not assignable.

b) Mice

Species/Strain: Mice/Swiss Webster

Conclusion: No significant toxicity or histopathology

Method: 40/sex/group were treated three times a week for 92 weeks 0.02 mL of test material

applied shaved interscapular surface. The study is pertinent only for alpha olefins of

C14-16. The six treatment groups are:

C 20% C14-18 AOS

C 25% C14-18 AOS

C 20% C14-16 AOS

C 25% C14-16 AOS

C 6.7% C16-1,4-sultone 

C 8.3% C16-1,4-sultone

GLP: No
17

Test Substance: C14-16 AOS

Results: No significant toxicity or histopathology attributable to treatment was found.

Reference: Little, D. (1993) Environmental and human safety of major surfactants. Volume I. 

Anionic surfactants. Part 4. Alpha Olefin Sulfonates. National Technical Service

(NTIS) report No. PB94-102423.

Reliability: (4) not assignable.

Developmental Toxicity

Species/Strain: Mice/strain unknown

Rats/strain unknown

Rabbits/strain unknown

Conclusions: Mice

Maternal Toxicity NOAEL = 2 mg/kg/day

Maternal Toxicity LOAEL = 300 mg/kg/day, based reduced body weights, and litter

loss; deaths at 600 mg/kg/day

Developmental Toxicity NOAEL = 300 mg/kg/day

Developmental Toxicity LOAEL = 600 mg/kg/day, based on decreased pup weights

and major malformations (cleft palate) and minor skeletal anomalies

Rats

Maternal Toxicity NOAEL $ 600 mg/kg/day (HDT)

Maternal Toxicity LOAEL - not established

Developmental Toxicity NOAEL $ 600 mg/kg/day (HDT)

Developmental Toxicity LOAEL - not established

Rabbits

Maternal Toxicity NOAEL= 2 mg/kg/day

Maternal Toxicity LOAEL = 300 mg/kg/day, based reduced body weights, mortality

and litter loss; all rabbits died at 600 mg/kg/day

Developmental Toxicity NOAEL= 2 mg/kg/day

Developmental Toxicity LOAEL= 300 mg/kg/day, based on increased incidence of

minor skeletal anomalies and extra ribs. 

Method: Alpha Olefin Sulfonate was administered by gavage during gestation days 6 - 15 of

pregnancy in rats and mice and 6 - 18 days of pregnancy in rabbits at dose levels of

0, 0.2, 2, 300 and 600 mg/kg/day. Twenty rats and mice/group and 13 rabbits/group

were used in the study. No further experimental details were provided, but looking

at the data presented, maternal, litter and major formations, minor anomalies and
18

skeletal variations were evaluated which should be adequate to establish the endpoints for maternal and developmental toxicities.

GLP: No

Test Substance: Alpha Olefin Sulfonate ( 60.4% alkenyl sulfonate:39.6% hydroxy-alkane sulphonate

w/w)

Results: Rats

Systemic toxicity was not observed at any dose level tested.

Litter parameters (litter size, embryonic deaths, litter weight, mean pup weight), fetal

abnormalities, malformation and variations were unaffected at all dose levels.

Mice

At 0.2 and 2 mg/kg/day, body weights were reported slightly reduced and there was

no clinical signs of toxicity were exhibited. At 300 mg/kg pilo-erection, reduced

activity and weight loss was observed. At 600 mg/kg, there was mortality (6/20),

clinical signs and reduced body weights were reported. At 300 mg/kg no dams died,

but 5 dams their entire litter. The higher incidence litter loss resulted in the mean

high embryonic loss and low mean litter size in the 300 and 600 mg/kg/day group. 

Excluding the litter loss the litter size and embryonic loss is comparable to controls. 

Mean pup weights at all doses were significantly lower than the controls, however,

the decrease of 36% at the high dose at the maternally toxic doses appear treatmentrelated. At other doses there was a decrease of 10 - 15% pup body weights, however,

this decrease was due above laboratory concurrent pup body weights (1.23 vs 1.04). 

Similarly, the mean litter weights (8.95) at 600 mg/kg/day group considered

treatment-related. Cleft palate was observed in 4 pups (from 3 litters) in 600

mg/kg/day and in 2 pups (from litter) at 300 mg/kg/day. The incidence was 1.3, 0, 0,

1.4 and 5.4% in the control, 0.2, 2, 300 and 600 mg/kg/day groups.

Rabbit

All rabbits in 600 mg/kg/day group died and showed anorexia, diarrhea and weight

loss before death. At 300 mg/kg/day 1/13 rabbits died and survivors showed

anorexia and weight loss. Autopsy revealed gastrointestinal changes. There were no

treatment-related systemic toxicity/litter effects at 0.2 and 2 mg/kg/day. At a

maternal toxic dose of 300 mg/kg/day litter parameters were not affected, however,

litter and fetal weights decreased 24 and 20%, respectively, compared to the controls. 

The authors suggested that the fetal effects were probably due to maternal toxicity. 

No major malformations and minor visceral anomalies were affected, but incidences

minor skeletal anomalies (23 vs 6.7%) and proportion of pups extra ribs (86.9 vs

59%) significantly increased, compared to the controls. 
19

Reference: Palmer, A., Readshaw, M., Neuff, A. (1975) Assessment of the teratogenic potential

of Surfactants. Huntingdon Research Center, Huntingdon, UK. Toxicology (3): 107

- 113.

Reliability: (2) valid with restrictions. Although the study was not performed as per the

guidelines, but the findings are peer reviewed. Therefore, the results are considered

valid and could be used for regulatory purposes. 

Gene Mutation

Bacterial mutagenicity Testinga

AOS Concentration

(max)

Assay

(if indicated)

Strain Tested Result

In vitro

10,000 ppm Reversion plate Salmonella typhimurium TA

1535, 1536, 1537, 1538

Escherichia coli B/r WP try and

try her

Negative (strains 1536, and 1538

smaller colony size than His+

strains but were not revertants)

100 μg/plate Ames Salmonella typhimurium TA 98,

100, 1535, 1537, 1538

Negative

100 μg Salmonella typhimurium TA 98,

100

Negative

2 μg/plate Salmonella typhimurium TA

1535, 1536, 153 7, 1538 (7

preperations)

Salmonella typhimurium TA

1535 (4 AOS compounds)

Negative

In vivo

283 mg/kg

AOS C14-16 (28.4% active)

Host-mediated (rat) Salmonella typhimurium TA

1530, 1534

(+) in TA 1530

(-) in TA 1534

Positive response in TA1530

could be due to:

a) incomplete ether extraction

b) higher pH 11 .3; when adju sted

to pH 8.5 with H2SO4

 yielded

negative respons e.

AOS C14-16 (28.4% active)

i.m. 0.1 mL/1% solution

Host-mediated (mouse) Salmonella typhimurium TA

1535

Negative

a Copied from registrants response to Agency deficiency letter 7/19/2001. Toxicology Data. MRID 45551102. 

Metabolism

Species/Strain: Rat/Wistar

Conclusions: The metabolic fate of 14C-AOS (a mixture of sodium alkenyl sulfonate and 14Csodium-3-hydroxy alkane sulfonate) was readily absorbed from GI tract, and rapidly
20

excreted in urine (72%) and feces (22%) within 24 hours, while 4.3% was excreted

in the bile within 12 hours. By intravenous dosing 50% of the dose was excreted in

the urine within 1 hour and 90% in 6 hours. Results suggest no accumulation of

AOS occurs and it is rapidly absorbed, metabolized and excreted.

Method: Male Wistar rats were dosed with a single oral and intravenous dose of 100 mg/kg

(50 μCi) and 10 mg/kg (5 μCi) 14C-AOS (a 55:45 mixture of sodium-3-hydroxy

alkane sulfonate and sodium alkenyl sulfonate which was labeled 14C). The mass

balance, metabolic profile, and blood kinetics were evaluated.

GLP: No

Test Substance: 14C-AOS (a 55:45 mixture of sodium-3-hydroxy alkane sulfonate and sodium alkenyl

sulfonate which was labeled 14C). Greater than 98% radio chemical purity.

Results: The COS was readily absorbed (about 80%) from GI tract, and rapidly excreted in

the urine (72%) and feces (22%) within 24 hours, while 4.3% was excreted in the

bile within 12 hours after oral administration. Blood levels of radioactivity peaked 3

hours after dosing and then declined. Tissue residues in the cecum were 0.8% but it

was less than 0.03% in other tissues 24 after oral administration. Upon intravenous

administration, half of the administered dose was excreted within 1 hour and 90%

was excreted 0 - 6 hours in the urine. No intact labeled COS was found in the urine

after oral or intravenous administration. The data suggest metabolites may be more

polar than the parent and the intact COS could bind with proteins while the

metabolites cannot. 

Reference: Inoue, S., O’Grodnic, J., Tomizawa, S. (1982) Metabolism of alpha-Olefin 

sulfonate (AOS) in Rats. Biological Science Laboratory, Lion Corporation,

Odawara, Kanagawa 256, Japan. Fundamental and Applied Toxicology 2: 130 - 138. 

May/June, 1982.

Reliability: (2) valid with restrictions. Although the study was not performed as per the

guidelines, but the findings are peer reviewed. Therefore, the results are considered

valid and could be used for regulatory purposes. 

Dermal Absorption:

Species/Strain: Rats/Wistar

Conclusion: Dermal absorption 0.6%.

Method: Groups of 3 male rats were applied with 0.5 ml of a 0.2% solution of 14C-AOS

(specific activity 6.55 μCi/mg) to the dorsal skin as follows: (a) the intact skin dried
21

naturally after application, (b) the intact skin wiped off 0.5 hour after application, (c)

the intact skin wiped off 1 1⁄2 hour after application, (d) the intact skin with plastic

cup containing 14C-AOS solution and (e) the damaged skin without the stratum

corneum dried naturally after application. Bile and urine were collected at 1, 3, 6, 9,

12, and 24 hours in all groups and in addition in groups an 30 hour samples were

collected. The animals in groups (a) to (d) were sacrificed after 24 hours and group

(e) were sacrificed after 30 hours. The samples after appropriate preparation

radioactivity was analyzed using liquid scintillation counting. 

GLP: No

Test Substance: 14C-AOS ( (a 55:45 mixture of 3-hydroxytetradecane-1- 14C sulfonate and $-

tetradecane-1- 14C sulfonate).

Results: Under the experimental conditions (a), (b), and (c) the percutaneous absorption of

14C-AOS was almost completed by 1.5 hour after application. The excretion of

radioactivity in urine and bile was highest during 3 - 6 hours and gradually decreased

and continued even after 70 - 90 hours after application. Under the experimental

conditions (d) a small amount of radioactivity was continuously absorbed from the

skin. Under conditions (e) about 50% of the administered dose was excreted in the

urine and bile, and the recoveries were 36.26% in the urine and 1.83% in the bile and

12.28% in the organs after 30 hours after application. Based on the results the

calculated dermal absorption factor for intact skin is 0.6% 

Reference: Minegishi, K., Osawa, M., Yamaha, T. (1977) Percutaneous Absorption of "-Olefin

Sulfonate (AOS) in Rats. National Institute of Hygienic Sciences, Tokyo. Chem.

Pharm. Bull. 25: 821-825.

Reliability: (2) valid with restrictions. Although the study was not performed as per the

guidelines, but the findings are peer reviewed. Therefore, the results are considered

valid and could be used for regulatory purposes. 

4. EH-2001 End-Use Product

Oral Toxicity

Species/Strain: Albino Rat/Sprague-Dawley

Conclusion: LD50 > 5050 mg/kg in males and females

Tox. Cat. IV.

Method: Five and 5 female rats were administered by gavage single dose of 3.35 mL/kg

(density - 1.509 g/mL; 5050 mg/kg) EH-2001 (Batch # 20000209) and were
22

observed for clinical signs of toxicity at least three times a day on the day of dosing

and once daily for 14 days.

GLP: Yes

Test Substance: EH-2001 (batch # 20000209), purity - unknown

Results: There was no mortality during the study. Clinical signs included decreased activity,

diarrhea, polyurea, sensitivity to touch and withdrawn testes in males on the day of

dosing. Body weights were not effected. No unremarkable gross pathology was

observed.

Reference: Kuhn, J. (2000) Acute Oral Toxicity in Rats. Stillmeadow, Inc., Sugar Land, Texas

77478. Laboratory Study No.: 5627-00. March 28, 2000. MRID 45307603. 

Unpublished.

Reliability: 1. Guideline method was used.

 

Acute Dermal Toxicity

Species/Strain: Albino Rabbits/New Zealand White

Conclusions: LD50 > 2020 mg/kg in males and females

Tox. Cat. IV

Method: Groups of 5 male and 5 female rabbits were applied a single dose of 1.34 mL/kg

(density - 1.5090 g/mL; 2020 mg/kg) of EH-2001, covered with surgical gauze,

secured with non-irritating adhesive tape and maintained in place with orthopedic

stockinette. After 24 hours, the bandages were removed, the site was gently washed

with water and wiped clean with cloth and dermal irritation was scored at

approximately 60 minutes, and on Days 4, 7, 11 and 14.

GLP: Yes

Test Substance: EH-2001 (batch # 20000209), purity - unknown

Results: There were no deaths during the study. Soft feces was observed in two males on day

of dosing. Body weights were not effected. One male lost < 3% of body weight

during the study. Slight to moderate irritation was observed during the first 4 days of

treatment, subsided by Day 11, except one male and 2 females still had slight

irritation. By Day 14, desquamation (2 males and one female) and alopecia were

present (one male). Necropsy was unremarkable.
23

Reference: Kuhn, J. (2000) Acute Dermal Toxicity Study in Rabbits. Stillmeadow, Inc., Sugar

Land, Texas 77478. Laboratory Study No.: 5628-00. March 30, 2000. MRID

45307604. Unpublished.

Reliability: 1. Guideline method was used.

Acute Inhalation

Species/Strain: Rat/Sprague-Dawley

Conclusion: LC50 > 2.37 mg/L

Tox. Cat. IV

Method: In a 500 L nose-only stainless steel chamber, 5 males and 5 females rats were

exposed to EH-2001 (batch # 20000209) aerosol concentrations (gravimetric) of

2.37 mg/L for 4 hours. Concentration of test substance (from the breathing zone)

was determined gravimetrically twice per hour and nominally at the end of the

exposure. Particle size was determined twice during the exposure, using Cascade

impactor, at a rate of 7.3 L/min for a duration of 10 seconds. The MMAD and

particle size distributions were calculated. Observations for clinical signs of toxicity

were made frequently on the day of exposure and at least daily thereafter for 14 days. 

Body weights were determined just prior to the inhalation exposure and on Days 7

and 14.

GLP: Yes

Test Substance: EH-2001 (batch # 20000209), purity - unknown

Results: There was no mortality during the study. Decreased activity and pilorection were

observed in all males and females for 6 hours; and were asymptomatic by Day 7. 

Body weights were unaffected. Necropsy was unremarkable. The exposure

concentration in the chamber was 2.37 mg/L (gravimetric), the mean MMAD was

2.8 μm and the geometric standard deviation was 1.5.

Reference: Kuhn, J. (2000) Acute Inhalation Toxicity Study in Rats. Stillmeadow, Inc., Sugar

Land, Texas 77478. Laboratory Study No.: 5629-00. April 13, 2000. MRID

45307605. Unpublished.

Reliability: 1. Guideline method was used.

Primary Eye Irritation

Species/Strain: Albino Rabbits/New Zealand White
24

Conclusion: Corneal opacity did not resolve by Day 21. Conjunctival redness and chemosis was

present on Day 14 and redness on day 17. 

Tox. Cat. I

Method: 0.1 ml undiluted EH-2001 (Lot # 20000209, purity - unknown) was placed into the

conjunctival sac of right eye of 1 male and 5 females albino rabbits and observed at

1, 24, 48, and 72 hours and at 4, 7, 10, 14, 17 and 21 days after treatment for eye

irritation. All eye were washed after with deionized water after 24 observation. Left

eye acted as untreated control.

GLP: Yes

Test Substance: EH-2001 (batch # 20000209), purity - unknown

Results: The maximum average eye irritation score of 37.0 vs 80 (maximum score), obtained

at 24 hours after treatment. Corneal opacity was present in 6/6 at 24 hours, and 3/6

at 17 days and 1/6 at 21 day evaluation. Iritis was present 1/6 eyes at 48 and was

cleared by 72 hours. Conjunctival redness was seen in 5/6 rabbits at 72 hours and

1/6 at day 17 and was clear by day 21. Chemosis was present 6/6 at 24 hours and 1/6

at 14 days and was clear by 17 days. Therefore, EH-2001 is considered a moderate

eye irritant and is Tox. Cat. I.

Reference: Kuhn, J. (2000) Acute Eye Irritation Study in Rabbits. Stillmeadow, Inc., Sugar

Land, Texas 77478. Laboratory Study No.: 5630-00. April 5, 2000. MRID

45307606. Unpublished.

Reliability: 1. Guideline method was used.

Acute Dermal Irritation

Species/Strain: Albino Rabbits/New Zealand White

Conclusion: The Primary Irritation Index (PII) = 0.6

Tox. Cat. IV

Method: Three males and 3 females rabbits were tested at each site (right) with 0.5 mL

undiluted EH-2001. After application of the test material, the treatment site was

covered with semipermeable dressing and maintained in contact with skin. At the

end of 4 hours, the dressing was removed, washed with tap water, cleaned dry with

cloth and observed for erythema and edema formation at 1, 24, 48 and 72 hours and

on Day 7 after removal of the bandages. The skin reactions were evaluated as per

Draize (1994).
25

GLP: Yes

Test Substance: EH-2001 (batch # 20000209), purity - unknown

Results: Erythema was observed in five rabbits 1 hour post removal of bandages and cleared

of erythema by Day 7. The Primary Irritation Index score was 3.5 and the mean

score was 0.6. The Primary Irritation score was 1.75. There was no edema

observed. Therefore, EH-2001 was considered slightly irritating and has a Tox. Cat.

IV.

Reference: Kuhn, J. (2000) Acute Dermal Irritation Study in Rabbits. Stillmeadow, Inc., Sugar

Land, Texas 77478. Laboratory Study No.: 5631-00. March 16, 2000. MRID

45307607. Unpublished.

Reliability: 1. Guideline method was used.

Dermal Sensitization

Species/Strain: Guinea Pig/Hartley-Albino

Conclusion: Induced dermal sensitization. Also produced faint to moderate irritation.

Method: Modified Buehler method was employed. Male and female guinea pigs were

assigned into 2 groups, designated as Groups I (5/sex) and II (10/sex). Group I

remained untreated and acted as naive control group. Group II animals were induce

with 0.5 mL EH-2001 once a week for 3 weeks. The animals were rested for 2

weeks, then all animals (the treated animals along with naive controls) were

challenged with 0.4 mL of undiluted test substance. Observation of skin reactions

were made about 24 hours and 48 after each treatment. Observations for skin

reactions were made about 48 hours after challenge. DNCB (1-chloro-2,4-

dinitrobenze) was used as positive control.

GLP: Yes

Test Substance: EH-2001 (batch # 20000209), purity - unknown

Results: The test substance produced no irritation in the naive controls animals after a single

treatment at challenge. The test substance produced a very faint to moderate

irritation in the animals of test group. The average skin reaction score was 0.6 vs 0

for the naive control. The positive control DNCB produced a mean score of 0.4.
26

Reference: Kuhn, J. (2000) Skin Sensitization Study in Guinea Pigs. Stillmeadow, Inc., Sugar

Land, Texas 77478. Laboratory Study No.: 5632-00. May 3, 2000. MRID

45307608. Unpublished.

Reliability: 1. Guideline method was used.

3. Toxicity Profile

Guideline #/ Test

Substance

Study Type - Dose

Levels

Results

Alpha-Olefin Sulfonate Sodium

81-1

C14 -16 AOS

Acute Oral - Rat (1996)

MRID 45307612

Doses (mg/kg): 0, 1000,

2000, or 3000 for males

and females

Acceptable/guideline

LD50 males = 2161 mg/kg

LD50 females = 1895 mg/kg

Combined LD50 = 2000 mg/kg

Tox. Cat. III

81-2

C14 -16 AOS

Acute D ermal To xicity -

Rabbits (1996)

MRID 45307613

Doses (mg/kg): 2020

single dermal dose

Acceptable/guideline

Dermal LD50 males and females > 2020 mg/kg

Tox. Cat. III

81-3

C14 -16 AOS

Powdered aerosol flakes 

(90% a.i.) 

Sprayed dried formulation

(17% a.i.)

Acute Inha lation To xicity

- Rat (1993)

MRID - N/A

Doses (mg/L): 229

(powdered aerosol flakes

C14 - 16, 90% a.i.) or 221

mg/L sprayed dried

formulation (17% a.i. C14

- 16) for 1 hour 

Unacceptable/guideline

howe ver it is valu able

information for labeling

purposes.

No deaths. Mild petechial hemorrhages in lungs of 2/5 and

1/5 rats exposed to flakes an d spray-dried formulations,

respectively.

 LC50 > 229 mg/L for powdered aerosol flakes

 LC50 > 221 mg/L for sprayed dried formulation
27

81-4

C14 -16 AOS

Primary Eye Irritation -

Rabbit (1980)

MRID 45307614

Doses: 0.1 mL

Acceptable/guideline

Cornea l opacity was p resent in 4/6 ra bbits 7 days posttreatment. Co njunctival irritation was presen t in 5/6 rabb its

7 days post-treatment. The maximum irritation score was

26.8 at 48 hours and 15.5 at 7 d ays post-treatment. 

Tox. Ca t. I

81-5

C14 -16 AOS

Primary Dermal Irritation

- Rabbit (1980)

MRID 45307615

Dose: 0.5 mL

Acceptable/guideline

Moderate skin irritant. Erythema and edema was observed

at both the intact and abraded skin sites post 72 hours

application . The mea n erythema a nd edem a scores we re 2.0

and 1.33, respectively. There were no signs of ulceration,

necrosis, or irritation present 72 hours post application

Tox. Cat. III

81-6

C14 -16 AOS

Dermal Sensitization -

Guinea Pig (1988)

MRID 45357703

Doses: C14-16 at 25%

for induction and 10 and

5% for challenge

Acceptable/guideline

C14-16 A OS was not a dermal sensitizer.

NA

C14 -16 AOS (70% C14:

30% C16) 

One Week Feeding Study

- Rat (1993)

MRID N/A

Doses: 0, 0.625%, 1.25%

and 1.5% (0, 125, 250

and 300 mg/kg/day,

respectively) fo r 7 days

Acceptable/nonguideline

NOAEL = 125 mg/kg/day

LOAEL = 250 mg/kg/day based on increased liver to body

weight ratios

82-1a

AOS (8 9.7% a .i.)

90-day Fe eding Stud y -

Rats (1993)

MRID N/A

Doses: 0, 40, 200, or

1,000 mg/kg/day

Unacceptable/guideline

No changes in hematology, clinical chemistry, feed

consumption, gross or histopathological lesions were noted

at any dose le vel. There was slight increas e in the liver to

body weight ratios was noted in the absence of any

histopatho logical or en zymatic finding s. 

NOAEL $1000 mg/kg/day (Limit Dose)

LOAEL was not established.

82-1a

C14-16 (34% a .i.)

91-day Feeding Study

Rats (1993)

MRID N/A

Doses: 0, 50, 150, or 500

mg/kg/day

Unacceptable/guideline

NOAEL > 500 mg/kg/day

LOAEL was not established
28

82-2b

C14-16 AOS 

28-day D ermal To xicity -

Rabbit (1993)

MRID N/A

Doses: 1% aqueous C14-

16 AOS or formulation

containing 1% AOS for

28 days.

NOAEL > 2 mg/animal i.e., 0.8 mg/kg (based on 2.5 kg

average weight of the rabbit) for 1% aqueous solution of

C14-16 AOS o r formulation containing 1 % AO S for 28 d ays

produc ed no effec t on the intact rab bit skin. 

82-3b

AOS (3 4% a.i)

90-day D ermal To xicity -

Rabbit (1993)

MRID N/A

Doses: 2 mL/kg/day o f a

5% (10 0 mg/kg/da y)

aqueous solution of AOS

(34% a.i.) for 90 d ays.

Unacceptable/guideline

NOAEL < 100 mg/kg/day (HDT)

LOAE L = 100 mg/kg/day b ased on m ild to mod erate skin

irritation.

83-5

C14-16 AOS (9 7.9% a .i.,

alkenyl sulphonate 60.4:

39.6% hydroxyalkane

sulphonate w/w)

Chronic

toxicity/carcino genicity -

rat (1976)

MRID N/A

Doses: 0, 1000, 2500, or

5000 ppm (0, 39, 96, or

195 mg/kg/day for males

and 0, 57, 132, or 259

mg/kg/day for females)

for 104 weeks

Acceptable/guideline

NOAEL = 2500 ppm

LOAEL = 5 000 ppm, based on decreased body weight gains

in males and females.

No evidence of carcinoge nicity. 

83-2

Essentially hydrolyzed

C14-16 and C16-18 AOS

(30% a.i.)

Partially hydrolyzed AOS

(30.9% a.i.) + contains

residual levels of sultone

Commercial C14-16 AOS

(38.9%)

Carcinog enicity Derm al -

Rat (1993)

MRID N/A

Doses: 0.02 mL twice a

week for 92 weeks

Unacceptable/guideline

No treatm ent-related system ic toxicity was ob served. 

NOAEL > 38.9 mg/kg/twice a week.

No evidence of carcinoge nicity. 

83-2

20% C14-18 AOS

25% C14-18 AOS

20% C14-16 AOS

25% C14-16 AOS

6.7% C16-1,4-sultone

8.3% C16-1,4-sultone

Carcinog enicity Derm al -

Mice (1993)

MRID N/A

Doses: 0.02 mL twice a

week for 92 weeks

Unacceptable/guideline

No significan t toxicity/histopatho logy attributab le to

treatment was found.
29

83-3a

C14-18 AOS (60.4 %

alkenyl

sulfonate:39 .6%hyd roxyalkane sulphonate w/w)

Develo pmental T oxicity -

Mice (1975)

MRID N/A

Doses: 0, 0.2, 2, 300 and

600 mg/kg/day on

gestation days 6 - 15

Acceptable/nonguideline

Maternal Toxicity NOAEL = 2 mg/kg/day

Maternal Toxicity LOAEL = 300 mg/kg/day, based on

reduced body weights, and litter loss; deaths at 600

mg/kg/day.

Developmental Toxicity NOAEL = 300 mg/kg/day

Developmental Toxicity LOAEL = 600 mg/kg/day, based

on decre ased pup weights, and m ajor malfo rmations (cle ft

palate) and minor skeletal ano malies.

83-3a

C14-18 AOS (60.4 %

alkenyl

sulfonate:39 .6%hyd roxyalkane sulphonate w/w)

Develo pmental T oxicity -

Rats (1975)

MRID N/A

Doses: 0, 0.2, 2, 300 and

600 mg/kg/day on

gestation days 6 - 15

Acceptable/nonguideline

Maternal Toxicity NOAEL $ 600 mg/kg/day (HDT)

Maternal Toxicity LOAEL not established

Developmental Toxicity NOAEL $ 600 mg/kg/day (HDT)

Developmental Toxicity LOAEL not established

83-3b

C14-18 AOS (60.4 %

alkenyl

sulfonate:39 .6%hyd roxyalkane sulphonate w/w)

Develo pmental T oxicity -

Rabbits (1975)

MRID N/A

Doses: 0, 0.2, 2, 300 and

600 mg/kg/day on

gestation days 6 - 18

Acceptable/nonguideline

Maternal Toxicity NOAEL = 2 mg/kg/day

Maternal Toxicity LOAEL = 300 mg/kg/day, based on

reduced body weig hts, mortality and litter loss; all rabbits

died at 60 0 mg/kg/da y.

Developmental Toxicity NOAEL = 2 mg/kg/day

Developmental Toxicity LOAEL = 300 mg/kg/day, based

on increased incidence of minor skeletal anomalies and extra

ribs.

84-4

AOS products (21 - 38%

a.i.)

In in vitro mutagenicity 

MRID 45551102

Doses: 2, 100 and 100

μg/plate; 10 000 pp m in

reversion plate assay

Negative in 4 separate tests

84-4

C14-16 AOS (2 8.4% a .i.)

In in vivo mutagenicity

Host-mediated - rat

MRID 45551102

Doses: 283 mg/kg (28.4%

a.i. C14-16)

(+ ) in Salmonella typhimurium TA 1530

(-) in Salmonella typhimurium TA 1534

Positive response in TA1530 could be due to:

a) incomplete ether extraction

b) higher p H 11.3 ; when adju sted to pH 8.5 with sulfuric

acid yielded negative response.
30

85-1

C14-18 AOS (55%

mixture of sodium-3-

hydroxy alkane

sulfonate:45 sodium

alkenyl sulfonate)

Metabolism - Rat (1982)

MRID N/A

Single oral and

intravenous dose of 100

mg/kg (50 μCi) and 10

mg/kg (5 μCi) a 55:45

mixture of sodium-3-

hydroxy alka ne sulfonate

and sodium alkenyl

sulfonate which was C14

labeled.

Acceptable/nonguideline

The AO S was read ily absorbed from GI tra ct, and rapid ly

excreted in the urine (72%) and feces (22%) within 24

hours; 4.3% was excreted in bile within 12 hours after oral

administration. Blood levels peaked in 3 hours then

declined. Less than 0.03% was retained in the tissues after

24 hours. Upon intravenous administration, half of the

administered dose was excreted within 1 hour and 90% was

excreted 0 - 6 hours. The data suggest metabolites may be

more polar than the parent and the intact AOS could bind

with proteins w hile metabo lites can not. 

85-3

AOS (55% 3-

hydroxytetradecane-1-14C

sulfonate:45% $-

tetradecane-1-14C

sulfonate)

Dermal Absorption - Rat

(1977)

MRID N/A

Doses: 0.5 mL of a 0.2%

solution of 14C-AOS

(specific activity 6.55

μCi/mg) w as applied to

the dorsal skin and

various scenarios were

attempted.

Acceptable/nonguideline

Dermal absorption - 0.6%.

EH-2001 End-Use Product

81-1 Acute Oral Toxicity - Rat

(2000)

MRID 45307603

Groups of 5 male and 5

females rats were given a

single oral dose of 3.35

mL/kg (density - 1.509

g/mL; 5050 mg/kg) of

EH-2001 formulation.

Acceptable/guideline

Oral LD50 > 5050 mg/kg in males and females.

Tox. Cat. IV
31

81-2 Acute D ermal To xicity -

Rabbit (2000)

MRID 45307604

Doses: G roups of 5 male

and 5 female rabbits were

applied with a single dose

of 2020 mg/kg to 10%

clipped body surface area

for 24 hours.

Acceptable/guideline

Dermal LD50 > 2020 mg/kg (Limit Dose) in males and

females.

Tox. Cat. IV

81-3 Acute Inhalation - Rat

(2000)

MRID 45307605

Doses: G roups of 5 male

and 5 female rats were

exposed nose only to

aerosol concentration of

2.37 mg/L EH-2001 for 4

hours.

Acceptable/guideline

LC50 > 2.37 mg/L for m ales and females.

Tox. Cat. IV

81-4 Primary Eye Irritation -

Rabbit (2000)

MRID 45307606

Dose: 0.1 mL of EH-2001

was instilled into the

conjunctiv al sac of 1 ma le

and 5 female rabbits; eye

were washed 24 hours

after instillation.

Acceptable/guideline

Corneal opacity was not resolved by Day 21. Conjunctival

redness and chem osis were present on D ay 14 and redne ss

on Day 17.

Tox. Ca t. I

81-5 Primary Dermal Irritation

- Rabbit (2000)

MRID 45307607

Dose: Groups of (3 males

and 3 females) rabb its

were derm ally exposed to

0.5 mL of EH-2001 for 4

hours.

Acceptable/guideline

EH-2001 did not induce irritation and erythema at the 1 hour

post application. Irritation was resolved by 72 hours. The

mean prim ary irritation inde x (PII) was 0 .6 i.e., slightly

irritating.

Tox. Cat. IV
32

81-6 Dermal Sensitization -

Guinea Pig (2000)

MRID 45307608

Dose: In modified

Buehler method guinea

pigs were exposed to EH2001.

Acceptable/guideline

There w as a 100% incidence o f reaction to ch allenge with

EH-2001.

EH-2001 is a dermal sensitizer.