EPA/635/R-03/005 

www.epa.gov/iris 

TOXICOLOGICAL REVIEW

OF

HYDROGEN SULFIDE

(CAS No. 7783-06-4) 

In Support of Summary Information on the 

Integrated Risk Information System (IRIS) 

June 2003 

U.S. Environmental Protection Agency 

Washington, DC 
DISCLAIMER 

This document has been reviewed in accordance with U.S. Environmental Protection 

Agency policy and approved for publication. Mention of trade names or commercial products 

does not constitute endorsement or recommendation for use. Note: This document may undergo 

revisions in the future. The most up-to-date version will be made available electronically via the 

IRIS Home Page at http://www.epa.gov/iris. 

-ii­ 
-iiiCONTENTS — TOXICOLOGICAL REVIEW of HYDROGEN SULFIDE 

(CAS No. 7783-06-4) 

FOREWORD .................................................................v

AUTHORS, CONTRIBUTORS, AND REVIEWERS ................................ vi

1. INTRODUCTION ..........................................................1

2. CHEMICAL AND PHYSICAL INFORMATION

RELEVANT TO ASSESSMENTS ..........................................2

3. TOXICOKINETICS RELATIVE TO ASSESSMENTS .............................3

3.1. ABSORPTION .....................................................3

3.2. METABOLISM .....................................................4

3.3. DISTRIBUTION ....................................................9

3.4. ELIMINATION .....................................................9

4. HAZARD IDENTIFICATION ................................................10

4.1. STUDIES IN HUMANS—EPIDEMIOLOGY, CASE

REPORTS, CLINICAL CONTROLS ................................10

4.2. PRECHRONIC AND CHRONIC STUDIES IN ANIMALS .................18

4.2.1. Oral ......................................................18

4.2.2. Inhalation .................................................21

4.3. REPRODUCTIVE/DEVELOPMENTAL

STUDIES—ORAL AND INHALATION ..............................27

4.4. OTHER STUDIES ..................................................32

4.4.1. Respiratory Toxicity .........................................32

4.4.2. Neurotoxicity .............................................. 36

4.4.3. Cardiovascular Toxicity ...................................... 39

4.4.4. Ocular Toxicity .............................................40

4.4.5. Genotoxicity ............................................... 40

4.5. EVALUATION OF MODE OF ACTION FOR 

NONCANCER EFFECTS .........................................41

4.5.1. Mode of Action of Noncancer Effects ...........................41

4.5.2. Oral Exposure ..............................................43

4.6. WEIGHT-OF-EVIDENCE EVALUATION AND CANCER

CHARACTERIZATION—SYNTHESIS OF HUMAN, ANIMAL, AND OTHER

SUPPORTING EVIDENCE, CONCLUSIONS ABOUT HUMAN

CARCINOGENICITY, AND LIKELY MODE OF ACTION .............. 43

4.7. SUSCEPTIBLE POPULATIONS ......................................44

4.7.1. Possible Childhood Susceptibility ..............................44

4.7.2. Possible Gender Differences ...................................45

5. DOSE-RESPONSE ASSESSMENTS ..........................................45

iii 
-iv5.1. ORAL REFERENCE DOSE ..........................................45

5.2. INHALATION REFERENCE DOSE ................................... 45

5.2.1. Choice of Principal Study and Critical Effect with Rationale and

Justification ............................................... 45

5.2.2. Methods of Analysis—including Models (PBPK, BMD, etc.) ......... 47

5.2.3. RfC Derivation—including Application of Uncertainty Factors (UFs) and

Modifying Factors (MFs) ..................................... 49

5.3. CANCER ASSESSMENT ............................................ 49

6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION

OF HAZARD AND DOSE RESPONSE ..................................... 50

6.1. HAZARD IDENTIFICATION ........................................ 50

6.2. DOSE RESPONSE ................................................. 51

7. REFERENCES ............................................................ 53

APPENDIX A: EXTERNAL PEER REVIEW—SUMMARY OF 

COMMENTS AND DISPOSITION ........................................62

iv 
FOREWORD 

The purpose of this Toxicological Review is to provide scientific support and rationale 

for the hazard and dose-response assessment in IRIS pertaining to chronic exposure to hydrogen 

sulfide. It is not intended to be a comprehensive treatise on the chemical or toxicological nature 

of hydrogen sulfide. 

In Section 6, Environmental Protection Agency (EPA) has characterized its overall 

confidence in the quantitative and qualitative aspects of hazard and dose response. Matters 

considered in this characterization include knowledge gaps, uncertainties, quality of data, and 

scientific controversies. This characterization is presented in an effort to make apparent the 

limitations of the assessment and to aid and guide the risk assessor in the ensuing steps of the 

risk assessment process. 

For other general information about this assessment or other questions relating to IRIS, 

the reader is referred to EPA’s IRIS Hotline at 202-566-1676. 

-v­ 
-viAUTHORS, CONTRIBUTORS, AND REVIEWERS 

CHEMICAL MANAGER 

Gary L. Foureman, Ph.D.

National Center for Environmental Assessment

U.S. Environmental Protection Agency 

RTP, NC

AUTHORS 

Judy Strickland, Ph.D.

National Center for Environmental Assessment

U.S. Environmental Protection Agency 

RTP, NC

Audrey Cummings, Ph.D.

National Center for Environmental Assessment

U.S. Environmental Protection Agency

RTP, NC

Joseph A. Spinnato III, Ph.D.

Sciences International, Inc.

Alexandria, VA

John J. Liccione, Ph.D.

Sciences International, Inc.

Alexandria, VA

Gary L. Foureman, Ph.D.

National Center for Environmental Assessment

U.S. Environmental Protection Agency 

RTP, NC

This document and summary information on IRIS have received peer review both by 

EPA scientists and by independent scientists external to EPA. Subsequent to external review 

and incorporation of comments, this assessment has undergone an Agency-wide review process 

whereby the IRIS Program Director has achieved a consensus approval among the Office of 

Research and Development; Office of Air and Radiation; Office of Prevention, Pesticides, and 

Toxic Substances; Office of Solid Waste and Emergency Response; Office of Water; Office of 

Policy, Economics, and Innovation; Office of Children’s Health Protection; Office of 

Environmental Information; and the Regional Offices. 

vi 
-viiINTERNAL EPA REVIEWERS 

Susan Rieth

National Center for Environmental Assessment

U.S. Environmental Protection Agency

Washington, DC

Robert Beliles

National Center for Environmental Assessment

U.S. Environmental Protection Agency

Washington, DC

Mark Greenberg

National Center for Environmental Assessment

U.S. Environmental Protection Agency

RTP, NC

Karl Jensen

National Health and Environmental Effects Research Laboratory

U.S. Environmental Protection Agency

RTP, NC

EXTERNAL PEER REVIEWERS 

Kaye Kilburn, M.D.

University of Southern California

School of Medicine

Environmental Science Laboratory

Los Angeles, CA

Thomas Milby, M.D., MPH

Walnut Creek, CA 

Bonnie R. Stern, Ph.D., MPH

BR Stern Associates

Annandale, VA

Summaries of the external peer reviewers’ comments and the disposition of their 

recommendations are in Appendix A. 

-vii-
1. INTRODUCTION 

This document presents background and justification for the hazard and dose-response 

assessment summaries in EPA’s Integrated Risk Information System (IRIS). IRIS summaries 

may include an oral reference dose (RfD), inhalation reference concentration (RfC), and a 

carcinogenicity assessment. 

The RfD and RfC provide quantitative information for noncancer dose-response 

assessments. The RfD is based on the assumption that thresholds exist for certain toxic effects 

such as cellular necrosis but may not exist for other toxic effects such as some carcinogenic 

responses. It is expressed in units of mg/kg-day. In general, the RfD is an estimate (with 

uncertainty spanning perhaps an order of magnitude) of a daily exposure to the human 

population (including sensitive subgroups) that is likely to be without an appreciable risk of 

deleterious, noncancer effects during a lifetime. The inhalation RfC is analogous to the oral 

RfD, but provides a continuous inhalation exposure estimate. The inhalation RfC considers 

toxic effects for both the respiratory system (portal-of-entry) and for effects peripheral to the 

respiratory system (extrarespiratory or systemic effects). It is generally expressed in units of 

mg/m3

. 

The carcinogenicity assessment provides information on the carcinogenic hazard 

potential of the substance in question and quantitative estimates of risk from oral exposure and 

inhalation exposure. The information includes a weight-of-evidence judgment of the likelihood 

that the agent is a human carcinogen and the conditions under which the carcinogenic effects 

may be expressed. Quantitative risk estimates are presented in three ways. The slope factor is 

the result of application of a low-dose extrapolation procedure and is presented as the risk per 

mg/kg-day. The unit risk is the quantitative estimate in terms of either risk per μg/L drinking 

water or risk per μg/m3

 air breathed. Another form in which risk is presented is a drinking water 

or air concentration providing cancer risks of 1 in 10,000; 1 in 100,000; or 1 in 1,000,000. 

Development of these hazard identification and dose-response assessments for hydrogen 

sulfide has followed the general guidelines for risk assessment as set forth by the National 

Research Council (1983). EPA guidelines that were used in the development of this assessment 

may include the following: the Guidelines for the Health Risk Assessment of Chemical Mixtures 

(U.S. EPA, 1986a), Guidelines for Mutagenicity Risk Assessment (U.S. EPA, 1986b), Guidelines 

for Developmental Toxicity Risk Assessment (U.S. EPA, 1991), Draft Revised Guidelines for 

-1- 
Carcinogen Risk Assessment (1999), Guidelines for Reproductive Toxicity Risk Assessment (U.S. 

EPA, 1996), Guidelines for Neurotoxicity Risk Assessment (U.S. EPA, 1998); Recommend-ations 

for and Documentation of Biological Values for Use in Risk Assessment (U.S. EPA, 1988); 

(proposed) Interim Policy for Particle Size and Limit Concentration Issues in Inhalation Toxicity 

(U.S. EPA, 1994a); Methods for Derivation of Inhalation Reference Concentrations and 

Application of Inhalation Dosimetry (U.S. EPA, 1994b); Peer Review and Peer Involvement at 

the U.S. Environmental Protection Agency (U.S. EPA, 1994c); Use of the Benchmark Dose 

Approach in Health Risk Assessment (U.S. EPA, 1995b); Science Policy Council Handbook: 

Peer Review (U.S. EPA, 2000a); Memorandum from EPA Administrator, Carol Browner, dated 

March 21, 1995, Subject: Guidance on Risk Characterization; and Science Policy Council 

Handbook: Risk Characterization (U.S.EPA, 2000b). 

Literature search strategies employed for this compound were based on the CAS Registry 

Number and at least one common name. At a minimum, the following databases were searched: 

HSDB, TSCATS, CCRIS, GENETOX, EMIC, EMICBACK, DART, ETICBACK, TOXLINE, 

CANCERLINE, MEDLINE, and MEDLINE backfiles. The relevant literature was reviewed 

through 2002. Pertinent scientific information submitted by the public to the IRIS Submission 

Desk was also considered in the development of this document. 

Previous IRIS entries for hydrogen sulfide include an RfD listed as 3 x 10-3 mg/kg-day 

based on an oral pig study in which the critical endpoint was GI disturbance (Wetterau et al., 

1964). This figure is based on a NOAEL of 3.1 mg/kg-day and a LOAEL of 15 mg/kg-day with 

an uncertainty factor of 1,000. The RfC for H2S was previously listed as 1 x 10-3 mg/m3

 based 

on the mouse subchronic inhalation study by CIIT (1983a) using inflammation of the nasal 

mucosa as the critical endpoint. The derivation of the RfC used the NOAELHEC of 1 mg/m3

 and 

the LOAELHEC of 2.6 mg/m3, as well as an uncertainty factor of 1,000. The cancer assessment 

for hydrogen sulfide had not yet undergone a complete evaluation and determination under US 

EPA's IRIS program for evidence of human carcinogenic potential. 

2. CHEMICAL AND PHYSICAL INFORMATION 

RELEVANT TO ASSESSMENTS 

Hydrogen sulfide is known as dihydrogen monosulfide, dihydrogen sulfide, hydrosulfuric 

acid, sewer gas, stink damp, sulfureted hydrogen, and sulfur hydride. Some relevant chemical 

-2- 
and physical properties of hydrogen sulfide are listed below, National Library of Medicine,

Hazardous Substances Data Bank (HSDB, 1998).

CAS registry number: 7783-06-4

Molecular formula: H2S

Molecular weight: 34.08

Vapor pressure: 15,600 mm Hg at 25/C

Density: 1.5392 g/L at 0/C, 760 mm Hg

Boiling point: !60.33/C

Water solubility: 3980 mg/L at 20/C

Log Kow Not applicable

Dissociation constants: pKa1 = 7.04; pKa2 = 11.96

Conversion factor: 1 ppm = 1.39 mg/m3

Hydrogen sulfide is a colorless gas and has a strong odor of rotten eggs (HSDB, 1998). 

Hydrogen sulfide is soluble in certain polar organic solvents, notably methanol, acetone, 

propylene carbonate, sulfolane, tributyl phosphate, various glycols, and glycol ethers (HSDB, 

1998). It is also soluble in glycerol, gasoline, kerosene, carbon disulfide, and crude oil. 

Aqueous solutions of H2S are not stable; absorbed oxygen causes the formation of elemental 

sulfur and the solutions become turbid rapidly (HSDB, 1998). 

The primary uses of H2S include the production of elemental sulfur and sulfuric acid, the 

manufacture of heavy water and other chemicals; in metallurgy; and as an analytical reagent 

(HSDB, 1998). In agriculture, it is used as a disinfectant. Formulations include a technical 

grade (98.5%) and a purified grade (99.5% min.). Occupational exposure to H2S occurs 

primarily from its presence in petroleum, natural gas, soil, sewer gas and as a byproduct of 

chemical reactions, e.g., viscose rayon and certain leather tanning processes. 

Hydrogen sulfide gas is a natural product of decaying organic matter. In residential 

settings it is most commonly the result of decomposition in septic or sewer systems. 

3. TOXICOKINETICS RELATIVE TO ASSESSMENTS 

3.1. ABSORPTION 

No studies were identified which quantitatively determined the absorption of H2S in 

humans or animals. However, the inhalation of high concentrations of H2S (greater than 2000 

ppm or 2780 mg/m3

) can be fatal within seconds or minutes in both humans and animals, 

suggesting that it is absorbed rapidly through the lungs. Studies in pigs and rats measuring 

plasma sulfide levels suggested that H2S is absorbed following ingestion. Since H2S exists as a 

-3- 
gas, oral exposure is not likely to occur. At physiological pH 7.4 approximately one third of the 

H2S exists in the undissociated form and the remainder largely as the hydrosulfide anion 

(Beauchamp et al., 1984). Although hydrogen is reported elsewhere to be fairly water soluble, 

Milby and Baselt (1999a) indicated that H2S is only moderately water soluble and thus not wellabsorbed by the moist tissues of the upper respiratory tract. The percutaneous absorption of H2S 

has been described as slow (Laug and Draize, 1942). 

3.2. METABOLISM 

Hydrogen sulfide is metabolized by three pathways: oxidation, methylation, and reactions 

with metalloproteins. However, the major pathway is oxidation in the liver with methylation 

being a very minor pathway. The major oxidation product of H2S metabolism is sulfate 

(Beauchamp et al., 1984) although thiosulfate may also be isolated from tissues exposed to 

hydrogen sulfide (Kage et al., 1992). At physiological pH (pH-7.4), H2S is approximately 69% 

ionized (HS-

). The metabolic pathway for H2S is depicted in Figure 1. 

Bartholomew et al. (1980) perfused heparinized blood containing sodium [S35]sulfide 

through isolated rat lungs, liver, and kidney and determined the rate of oxidation for each organ. 

In the presence of water, sodium sulfide is rapidly converted to H2S (Haggard, 1921). 

Therefore, administration of sodium sulfide in the isolated perfused system would be similar to 

inhalation exposure to H2S. In the isolated perfused liver, approximately 70% sulfide was 

rapidly metabolized to sulfate within 15 minutes, and 82% of the detected radioactivity was 

sulfate after 2 hours perfusion. When unlabeled thiosulfate was added to the liver perfusion 

system, 54% of the radioactivity was thiosulfate and 22% was sulfate after 15 minutes of 

perfusion. After 2 hours, these proportions reversed. This would suggest that sulfide is oxidized 

to thiosulfate with further oxidation to sulfate. 

In the isolated perfused lung, 32% of the administered radioactivity was lost from the 

blood as volatile 35S after 15 minutes. However, after 15 minutes no further radioactivity was 

lost. Sulfide was oxidized slowly to thiosulfate, and only trace amounts of sulfate were present 

after 2 hours of perfusion. The small amount of detectable sulfate in the isolated perfused lung 

was thought to be due to the absence of sulfide oxidase in the lung. In the kidney, sulfide was 

slowly oxidized to sulfate possibly through thiosulfate as an intermediate metabolite. The 

investigators also reported that the rat liver mitochondria catalyzed the oxidation of sulfide to 

thiosulfate in vitro through an unknown mechanism. This oxidation was inhibited at high 

concentrations of sulfide (60:M) because of reduced oxygen consumption by the mitochondria 

-4- 
due to cytochrome c oxidase inhibition. When glutathione (GSH) was added to the rat liver 

mitochondria in vitro, thiosulfate was further metabolized to sulfate. 

Kage et al. (1992) exposed adult male Japanese white rabbits to 100-200 ppm (139-278 

mg/m3

) for 60 minutes and to 500-1000 ppm (695-1390 mg/m3) until fatal (22 minutes, approx.). 

In the low-level exposure group, blood and urine were collected immediately after exposure and 

1, 2, 4, 6, and 24 hours following exposure. Blood and organs were collected immediately after 

death in high-exposure animals. In the high-exposure animals, thiosulfate levels in the blood, 

lung, and brain were 2-7 times higher than sulfide levels. Thiosulfate and sulfide were absent or 

present only in trace amounts in the liver, kidney, and muscle. In the low-exposure animals, 

blood thiosulfate decreased rapidly to trace levels 2 hours after exposure and was not detected 4 

hours after exposure. Urinary thiosulfate levels exhibited a peak at one to two hours followed by 

a decline, but were still detectable 24 hours after exposure. Sulfide was not detected in the blood 

or urine of low-exposure animals. 

It appears that oxidation is also the major metabolic pathway for H2S in humans. 

Thiosulfate was found in the urine of volunteers exposed to 8, 18, or 30 ppm (11, 25, or 42 

mg/m3

) H2S for 30-45 minutes (Kangas and Savolainen, 1987). They also determined thiosulfate 

levels in maintenance workers in a pelt processing plant with known exposures to H2S, which 

were compared with thiosulfate levels in unexposed workers. Urinary thiosulfate concentration 

increased according to the gas concentration and exposure time. The highest concentration of 

urinary thiosulfate in the exposed workers was observed 15 hours after exposure. At 17 hours, 

urinary levels of thiosulfate had returned to control levels suggesting that all of the absorbed 

sulfide had been oxidized within the 15 hours after exposure. 

The relationship between sulfide concentration and cytochrome oxidase in target tissues 

was explored by exposing male CD rats (6/group) to H2S at 0, 10, 30, 80, 200, or 400 ppm (0, 

14, 42, 111, 278, or 556 mg/m3) once for 3 hr and for 70 days (Dorman et al., 2002). The only 

target organ that demonstrated a clear dose response relationship with increased sulfide levels 

and concomitant decreases in cytochrome oxidase was the lung examined at the end of the single 

3-hour exposure; statistical significance was achieved at 80 ppm (111 mg/m3

) and above for 

increases in sulfide and at 30 ppm (42 mg/m3

) and above for decreases in cytochrome oxidase. 

Sulfide concentrations in the lung decreased to preexposure levels within 15 min of the end of 

exposure. The response was not seen in the lung after the 70-day repeated exposure. Hindbrain 

sulfide concentrations and cytochrome oxidase activities were unaffected by single or repeated 

exposures. Cytochrome oxidase concentrations were decreased (p#0.05) in both the nasal 

respiratory and olfactory epithelium at levels of 30 ppm (42 mg/m3

) although sulfide levels did 

-5- 
not appear to be decreased until 400 ppm (556 mg/m3

) H2S. These results indicate that 

cytochrome oxidase is a sensitive biomarker of effect for H2S exposure (more so than sulfide 

levels) for both lung and nasal epithelial tissues, but only for short term exposures. 

Methylation has also been reported as a possible metabolic pathway for H2S. Hydrogen 

sulfide was reported to be methylated to methanethiol in vitro by the intestinal mucosa of 

Sprague-Dawley rats (Weisiger et al., 1980). Methanethiol can be further methylated, although 

much more slowly, to dimethylsulfide. Thiol S-methyltransferase was reported to catalyze the 

two reactions. It is a ubiquitous enzyme, and the investigators reported the highest activity in the 

colonic and cecal mucosa (10-13 mol/min/mg of protein). High activities were also reported in 

the liver, lung, and kidney. 

Levitt et al. (1999) also studied the metabolism of H2S in the cecal mucosa of SpragueDawley rats and concluded that oxidation, rather than methylation, was the primary metabolic 

route in the cecal mucosa. When incubated with H2S, cecal tissues did not produce methanethiol 

or dimethylsulfide even when a methyl donor, S-adenosylmethionine, was added to the reaction 

mixture. The rates of metabolism for cecal and liver homogenates were calculated to be 21 and 

2.5 nmol/min/mg of protein, respectively, and the metabolic products of the cecal and liver 

homogenate were reported to be sulfate and thiosulfate, the primary metabolite. The oxidation 

rate is approximately 10,000 times the methylation rate reported by Weisiger et al. (1980), which 

would suggest that methylation is not an important pathway for H2S detoxification. 

Another possible pathway of H2S metabolism is conjugation with GSH. Smith and 

Abbanat (1966) reported that exogenously-oxidized, but not reduced, GSH protected mice from 

lethal intraperitoneal (i.p.) injections of sodium sulfide. They also stated that endogenous 

oxidized GSH, and other endogenous compounds containing disulfide bridges might provide an 

important detoxification process following H2S exposure. 

Hydrogen sulfide has also been reported to bind to heme proteins such as CytOx and 

methemoglobin. While H2S interaction with the former has been implicated in its toxicity, the 

interaction between methemoglobin and H2S is a possible detoxification pathway. Smith and 

Gosselin (1964) pretreated rabbits and armadillos with sodium nitrite, an inducer of 

-6- 
methemoglobin, and reported increased survival following intracardiac and intravenous (i.v.) 

injection with lethal doses of sodium sulfide. The investigators also reported that female Charles 

River CD1 mice pretreated with sodium nitrite and p-aminopropiophenone, also an inducer of 

methemoglobin, had increased survival following i.p. injection with sodium sulfide (60 mg/kg). 

Survival time also significantly increased in mice exposed to 722 and 985 ppm (1003 and 1369 

mg/m3

) H2S and pretreated with sodium nitrite and p-aminopropiophenone. 

-7- 
Figure 1. Metabolic pathways of hydrogen sulfide. 

Source: adapted from Beauchamp et al., 1984. 

-8-
3.3. DISTRIBUTION 

Forensic autopsies of three men who were overcome by H2S (estimated concentration 

767-906 mg/m3 or 552-652 ppm) demonstrated that the gas is widely distributed in exposed 

humans (Kimura et al., 1994). Blood sulfide concentrations of the three victims were 0.1, 0.2, 

and 0.8 :g/g 2-3 hours after death. At 24-hours after death, blood sulfide was measured at 0.5, 

0.23 :g/g, and not detected, respectively. Sulfide was also detected in the brain (0.2-1.06 :g/g), 

lung (0.21-0.68 :g/g), liver (1.30-1.56 :g/g), kidney (0.47-1.50 :g/g), and spleen (0.32-0.62 

:g/g) 24 hours after exposure. The investigators state that postmortem generation of H2S by the 

blood, liver, and kidney was extremely remarkable making the values from these three victims 

unreliable. However, sulfide is not normally detected in the brain and lung of control 

postmortem specimens, and sulfide detected in these tissues is considered a reliable indicator of 

exposure. Imamura et al. (1996) also reported detectable concentrations of sulfide in the blood, 

lung, brain, liver, and kidney of a worker overcome by H2S. The investigators also reported that 

sulfide was detected in the skeletal muscles of the abdomen and leg. 

Distribution of H2S in animals is very similar to humans. Kohno et al. (1991) exposed 

male Wistar rats to 75 ppm (104 mg/m3) for 20, 40, or 60 min. Following exposure, animals 

were sacrificed and blood and tissues (brain, lung, heart, liver, spleen, and kidney) were 

collected within five min of sacrifice. After 20 minutes of exposure, the concentration of sulfide 

in the blood, brain, lung, heart, liver, spleen, and kidney was approximately 10, 25, 22, 38, 23, 

27, and 30 :g/g of tissue, respectively. The concentration of sulfide in these tissues was 

relatively constant regardless of the exposure duration. 

3.4. ELIMINATION 

Urine is the primary route of elimination following H2S exposure. Gunina (1959) 

reported that following exposure to sodium sulfide via i.v. and subcutaneous (s.c.) routes or 

exposure to H2S by inhalation routes in dogs and rats the majority of the dose (70-99%) was 

eliminated in the urine by 24 hours postexposure. Kage et al. (1992) detected thiosulfate but not 

sulfide in the urine of rabbits exposed to 100-200 ppm (139-278 mg/m3) H2S by inhalation up to 

24 hr after exposure with the highest levels being detected 2 hr after exposure. Thiosulfate was 

also found in the urine of volunteers exposed to 8, 18, or 30 ppm (11, 25, or 42 mg/m3) H2S for 

30-45 minutes and maintenance workers in a pelt processing plant with known exposures to H2S 

(Kangas and Savolainen, 1987). 

-9- 
-10-

4. HAZARD IDENTIFICATION 

4.1. STUDIES IN HUMANS—EPIDEMIOLOGY, CASE REPORTS, CLINICAL 

CONTROLS 

On December 14, 2001, after internal peer review of this document, the Agency 

articulated its interim policy on the use of third-party studies submitted by regulated entities 

(U.S. EPA, 2001). For these purposes, EPA is considering “third party studies” as studies that 

have not been conducted or funded by a federal agency pursuant to regulations that protect 

human subjects. Under the interim policy, the Agency will not consider or rely on any such 

human studies (third-party studies involving deliberate exposure of human subjects when used to 

identify or quantify toxic endpoints such as those submitted to establish a NOAEL or NOEL for 

systemic toxicity of pesticides) in its regulatory decision making, whether previously or newly 

submitted. Some of the supporting studies discussed in this Toxicological Review are thirdparty studies. The studies cited provide data which suggests and inform a public health concern 

for hydrogen sulfide, but were not designed or used as principal studies in the derivation of any 

quantitative value for hydrogen sulfide based on NOAELs or LOAELs. The Agency is 

requesting that the National Academy of Sciences conduct an expeditious review of the complex 

scientific and ethical issues posed by EPA’s possible use of third-party studies that intentionally 

dose human subjects with toxicants to identify or quantify their effects. 

Recent reviews of the health hazards associated with H2S exposure and subsequent 

treatment include Milby and Baselt (1999a) and Guidotti (1996). Earlier reviews of the health 

effects were provided by Glass (1990), Reiffenstein et al. (1992), and Mehlman (1994). 

Exposure to H2S has been reported to be an important cause of morbidity and mortality in the 

workplace (Snyder et al., 1995) and olfactory dysfunction (Hirsch and Zavala, 1999). These 

reviews indicate that the typical “rotten-egg odor” of H2S is an inadequate warning indicator of 

exposure since levels in the range of 100-200 ppm (140-280 mg/m3) can lead to loss of smell 

followed by olfactory paralysis (Reiffenstein et al., 1992). These authors estimate the odor 

threshold of H2S in the range of 3 to 20 ppb (0.004 to 0.03 mg/m3

). The case reports cited below 

involving overexposure have documented a variety of central nervous system (CNS) transitory 

symptoms, such as dizziness, nausea, headache and others more long-acting such as abrupt 

physical collapse or “knockdown,” all of which have been attributed to direct effects of H2S on 

the brain (Milby and Baselt, 1999b). Prolonged unconsciousness can lead to respiratory failure, 

hypoxia, and death. In the reviews cited above, levels associated with “knockdown”have been 

estimated to be in the range of 500-1,000 ppm (695-1,390 mg/m3

). Levels associated with 

pulmonary edema have been estimated to be in the range of 250-500 ppm (348-695 mg/m3

). A 

rather extensive list of acute symptoms of intoxication resulting from an accidental release of 

H2S in an industrial setting was provided by Kleinfeld et al. (1964). 

10
-11-

The following case studies describe exposures to a number of volatile and semivolatile 

compounds in addition to hydrogen sulfide (e.g., liquid manure) whereby the actual toxic agent 

is unclear. The point of these descriptions is that exposure to high concentrations of hydrogen 

sulfide, even for very brief periods, can have life-threatening consequences involving respiratory 

arrest and secondary CNS effects as well as persistent neurological sequelae. 

Case Studies: 

A 19-year-old oil rig worker was exposed to unspecified concentrations of H2S (Burnett 

et al., 1977) rendering him unconscious for an indeterminate amount of time. Upon 

resuscitation, he exhibited malaise, anterior chest pain, dyspnea, headache, nausea and vomiting, 

tearing of the eyes and photophobia and coughed up blood. Upon arrival at the hospital for 

further treatment, his vital signs were normal and he was no longer in respiratory distress. He 

had severe photophobia and blepharospasms, but no signs of conjunctivitis. He also possessed a 

cough and some motor weakness of his right arm and leg. A neurologic examination and a chest 

x-ray revealed no abnormalities. After a 3-day stay in the hospital, he was discharged. 

Two fatalities were due to massive aspiration of liquid manure and one fatality was due 

to severe pulmonary edema with no aspiration of the manure (Osbern and Crapo, 1981). 

Another 41-year-old individual fell unconscious into liquid manure during a rescue attempt. 

After resuscitation, he had difficulty breathing and was agitated, but he exhibited no focal 

neurologic deficits. His initial chest radiograph showed a five-lobed alveolar infiltrate. After 

two weeks in the hospital, his chest radiograph showed improvement and his lung function was 

normal except for a slightly reduced maximum midexpiratory flow rate. Six months after the 

accident, he had a normal chest radiograph and was asymptomatic. Blood sulfide levels of the 

two individuals who had fatally aspirated manure were 5.0 and 3.6 mg/L. The individual who 

had died by pulmonary edema had a blood sulfide level of 0.8 mg/L. Control blood samples 

exhibited sulfide levels below 0.05 mg/L while blood samples from random autopsy cases of 

badly decomposed subjects did not exceed 0.4 mg/L. No blood sulfide measurements were 

performed on the 41-year-old survivor. Eight days following the accident, air analyzed from the 

liquid manure storage tank contained 6,360 ppm methane, 400 ppm carbon monoxide, 1.5 ppm 

ammonia, 2% carbon dioxide, 18% oxygen, and 76 ppm (106 mg/m3

) H2S. 

Another case study illustrates the possible long-term sequelae of H2S exposure. A 30-

year-old man displayed dyspnea, chest tightness, and haemoptysis following exposure to a toxic 

gas in a lavatory facility at his place of work (Parra et al., 1991). The facility was connected to a 

manure pit; no measurements of H2S were performed. Physical examination and routine 

laboratory studies revealed no abnormalities. However, a chest radiograph detected a mild 

bilateral interstitial pattern. Bronchoscopy showed a widespread reddish mucosa. Pulmonary 

function tests showed a mild restrictive disease. After five months, the patient possessed 

residual exertion-dyspnea but was otherwise asymptomatic. The diagnosis was pneumonitis 

11
-12-

caused by the inhalation of a toxic gas. Other exposed workers exhibited nausea, vomiting, 

dizziness, dyspnea, and eye and nose irritation. One of the exposed workers died a few hours 

after exposure. An autopsy revealed haemorrhagic bronchitis and the cause of death was 

asphyxia attributed to the inhalation of a “toxic gas,” with H2S suspected as a major component. 

Several case studies report on the rapid toxicity following exposure to high levels of H2S. 

A 14-year-old boy found a discarded cylinder containing H2S in a rubbish dump and 

immediately died when he opened the tank (Allyn, 1931). His father also died during a rescue 

attempt. Both bodies were deeply cyanosed. A fatality, apparently due to release of H2S from a 

load of refinery waste was described by Simson and Simpson (1971). This report also mentioned 

eyewitness accounts of intense central cyanosis in the victim. 

At a poultry feather fertilizer plant, a worker was exposed to H2S while attempting to 

repair a leak and was killed (Breysse, 1961). Hydrogen sulfide was created as a byproduct of the 

putrefaction of the feathers, and was eliminated through a pipe leading to a sawmill log pond 

where it was discarded. In the lungs of the victim, the alveolar spaces were filled with edema 

fluid and numerous pigment-filled macrophages. The diagnosis was pulmonary edema, and the 

cause of death was H2S inhalation. Measurements of H2S concentrations at various locations in 

the fertilizer plant revealed levels as high as 4,000 ppm (5,560 mg/m3) during the cooking and 

putrefaction of feathers. 

A 16-year-old boy suffered fatal H2S exposure during transport of liquid manure (Hagley 

and South, 1983). He was found at the bottom of the manure tank, pale, unconscious and apneic. 

There was no evidence that he had aspirated any manure. The boy began breathing following 

resuscitation and his color returned to normal. His heart rate and blood pressure were normal 

though he did not respond to painful stimuli. Over the following hour, he became responsive to 

stimuli, but then developed extensor spasms and began to hyperventilate. A chest radiograph 

and a CT scan of the head were normal. The patient developed pneumonia. However, his 

neurological condition deteriorated, and he suffered partial seizures and exhibited a decerebrate 

response to painful stimuli. The patient died five days after the accident with clinical signs of 

brain stem damage. Postmortem examination revealed that he had cerebral edema. A week 

later, H2S was measured 30 cm below the manhole of the tank and was found to exceed the upper 

limit of detection of the monitoring equipment at 150 ppm (208 mg/m3

). 

By contrast, Milby (1962) has reported two cases of severe, but non-fatal H2S poisonings. 

While disposing of a H2S-filled gas cylinder and the subsequent release of its contents, two men 

that were exposed to the gas collapsed immediately and underwent convulsions while 

unconscious. Upon being rescued from the gas, the men required artificial respiration to begin 

breathing on their own. Both men were hospitalized in an unconscious state and were later 

revived. At the conclusion of their hospital stay, both men were without symptoms. The author 

12
-13-

states that they “remained well to the present,” but it is unclear how long an interval that 

represents. 

A mass exposure to H2S took place among workers laying the foundation of a municipal 

sewage pumping station, leading to the death of a police officer attempting to rescue an 

unconscious worker and persistent neurological sequelae in at least one other individual (Snyder 

et al., 1995). Complications included: decreased ability to communicate, slow speech, marked 

visual memory impairment with poor acquisition, and difficulty retaining and recalling new 

information. The findings were reported as essentially unchanged 12-18 months after exposure. 

Physical findings in affected individuals were limited to pharyngeal and conjunctival erythema 

and corneal abrasions. Additional reports associated H2S exposure (via sewer gas) with lung 

function and neurological decrements and, in certain studies, deaths were reported by Watt et al. 

(1997) and Hall and Rumack (1997). 

Tvedt et al. (1991a) described the follow-up of six H2S-exposed patients who lost 

consciousness and experienced signs of long-term neurological sequelae associated with hypoxic 

brain damage. Immediate symptoms included cyanosis, pulmonary edema, seizures, and coma. 

Delayed symptoms (5-10 year follow-up) ranged from worker disability due to neurological 

symptoms to brain damage severe enough to qualify as dementia in one individual. Five patients 

who had been unconscious in an H2S atmosphere for 5-20 minutes showed persistent impairment 

with memory and motor function most affected. The two patients with the most serious 

symptoms developed pulmonary edema. No measures of H2S in air, blood or urine were 

reported. The investigators concluded that the outcome of acute exposures can be variable and 

that neurological symptoms may not become apparent until a considerable period of time has 

elapsed. 

Tvedt et al. (1991b) also described delayed neuropsychiatric symptoms in an individual 

who became unconscious for 15-20 minutes during an H2S exposure. He regained consciousness 

after two days, but then deteriorated again and remained comatose for a month. Upon waking up, 

the individual was amnesiac, nearly blind, had reduced hearing and ataxia. At follow-up (five 

years later) he had not been able to resume work and evidenced slight motor, memory and visual 

impairments and clinical indications of slight cerebral atrophy. Electroencephalogram and 

evoked responses were normal at this time. 

Symptoms similar to those described by Tvedt et al. (1991b) were also reported by 

Kilburn (1993) in an oil well tester rendered semiconscious from exposure to high levels of H2S. 

Follow-up (39 and 49 months) symptoms suggested damage to the brain stem, basal ganglia, 

vestibular apparatus, cortex, and other brain structures. There was profound impairment of 

cognitive function, memory, visual perception and coordination, intelligence and 

neurophysiologic functions. Malingering was ruled out by the reproducibility of performance 

during testing sessions. 

13
-14-

Persistent neurocognitive impairment from H2S poisoning was also reported by Wasch et 

al. (1989). Three cases are described with 2/3 individuals experiencing unconsciousness during 

exposure. All three individuals had moderate to severe attention and concentration deficits, as 

well as varying degrees and types of memory, learning, and perceptual deficits and prolonged 

abnormal event-related potentials (P-300 latency). In one individual, symptoms persisted at 

follow-up three years after exposure. 

In another study, a 20-month-old child developed intermittent paroxysmal tonic deviation 

of the eyes (Gaitonde et al., 1987). After a few months the abnormal eye movements resolved, 

progressive involuntary movements of the entire body developed, and the child fell frequently. 

The child was admitted to the hospital with gross truncal ataxia, choreoathetosis, dystonia, and 

an inability to stand. The child was dysarthic but had normal eye movements. Computer 

tomography revealed bilateral areas of low attenuation in the basal ganglia and some of the 

surrounding white matter. Virology was negative and there was no evidence of streptococcal 

infection. The brain scan suggested toxic encephalopathy. Shortly after admission, the child’s 

condition improved spontaneously. Ten weeks after admission, the ataxia had resolved and 

choreathetoid movements were reduced. A repeat brain scan was normal. The child’s illness 

was attributed to H2S exposure. The family lived next to a coal mine where a burning tip emitted 

H2S for nearly one year. Three months prior to the child’s admission, H2S emissions were 

monitored for four months, and the maximum recorded level in family’s housing scheme was 0.6 

ppm (0.8 mg/m3

). Local authorities admitted that H2S levels may have been many times higher 

prior to monitoring. The burning tip had been extinguished just prior to the child’s admission to 

the hospital. This study is discussed at length in Section 4.7.1., Possible Childhood 

Susceptibility. 

Epidemiological Studies: 

Chronic low-level health effects in two communities exposed to industrial sources of H2S 

were assessed by means of a symptom-survey using trained interviewers (Legator et al., 2001). 

Results of the survey were compared to health effects in three reference communities not 

exposed to H2S. Symptoms were elevated in both exposed communities compared to the referent 

populations in: CNS effects, respiratory effects, and blood (e.g., anemia, clotting, etc.). Although 

the odds ratios were elevated over controls, they fell within the confidence intervals. In addition, 

respondents from the two exposed communities had long-standing involvement with H2S 

exposure issues which may have contributed to study bias. Subjects in one of the exposed 

communities were also assessed by means of several objective psychoneurological tests, the 

results of which were consistent with results of the symptom survey (Borda, 1997; cited by 

Legator et al., 2001). 

A mortality study was undertaken among Finnish sulfate mill workers exposed to H2S 

and organic sulfides (Jappinen and Tola, 1990). Workers had been employed for at least one 

14
-15-

year between 1945 and 1961 at three pulp and paper mills owned by the same company. No 

exposure data were presented. Deaths from all causes were not increased. However, workers 

exposed to H2S and organic sulfides exhibited an increase in cardiovascular-related deaths 

compared to national death rates (37 observed compared to 24.7 expected). Cardiovascular 

mortality was higher in workers employed for $ 5 years compared to workers exposed for 1-4 

years. The investigators state that increased mortality could not be explained by common risk 

factors and that differences in smoking habits did not explain the findings. They suggest that 

increased mortality may have been associated with H2S and organic sulfide exposure. 

A cross-sectional study investigated the effects of presumed H2S exposure in sewer 

workers to determine if chronic exposure to the gas was associated with decreased lung function 

(Richardson, 1995). Sixty-eight sewer workers performed spirometric tests and results were 

compared to 60 nonexposed water treatment workers. Job titles were used to categorize sewer 

workers according to presumed H2S exposure levels. There was a statistically significant 

decrease in mean FEV1/FVC in sewer workers compared to water treatment workers. The effect 

was greater in sewer workers presumed to have higher exposures to H2S and longer exposure 

histories. In nonsmoking subjects, sewer workers were only able to attain 89% of the predicted 

FEV1/FVC value compared to 98% in water treatment workers. Although the study author states 

that chronic low level exposure to H2S may be associated with decreased lung function, no 

measurements of H2S were made so any quantitative relationship between “low levels” of 

exposure and effect are speculative. 

The pulmonary effects resulting from H2S exposure were assessed in 175 workers who 

extracted and processed oil and gas (Hessel et al., 1997). The workers received a questionnaire 

concerning sour gas exposures that caused symptoms or loss of consciousness. Thirty-four 

percent of the respondents indicated that they had exposures serious enough to cause symptoms, 

and 8% of the workers stated they had experienced a loss of consciousness due to sour gas 

exposure. In workers that experienced symptoms, no decrease in spirometric values or excess 

symptoms were noted. While spirometric values were also not affected in workers that lost 

consciousness, the workers experienced shortness of breath during physical activity, wheezing 

with tightness in the chest, and wheezing attacks. The investigators stated that these symptoms 

are consistent with bronchial hyperresponsiveness. The data also demonstrate the refractiveness 

of the lung to the effects of H2S, with lung function parameters being unchanged despite 

unconsciousness from H2S exposure. 

A cross-sectional pulmonary function study was performed in males that worked in 

viscose rayon plants (Higashi et al., 1983). A one-workday study was performed in which 

workers were monitored for H2S exposure for 8 hr and had a forced expiratory flow-volume test 

performed. Hydrogen sulfide levels were determined using passive diffusion dosimeters worn 

by the workers (n = 30 in exposed and matched control workers). The workers had been 

15
-16-

exposed to H2S for an average of 12.3 years. Occupational exposure levels in the exposed 

workers ranged between 0.3 and 7.8 ppm (2.9 ppm average; 4 mg/m3

) compared with < 0.1 ppm 

(0.1 mg/m3

) in matched control workers. No significant differences in the results from the 

pulmonary function tests were observed in exposed compared to nonexposed workers. Also, no 

significant differences were noted in the pulmonary function tests taken at the beginning and 

ending of work shifts. 

In addition to the one-workday study, a cross-sectional study was undertaken on 115 

exposed workers and 209 nonexposed workers. The subjects underwent forced expiratory flowvolume tests, but were not monitored for exposure. The investigators (Higashi et al., 1983) 

concluded that chronic exposure to low levels of H2S would not cause adverse pulmonary health 

effects. 

A survey of 123 male viscose rayon workers exposed to H2S and/or carbon disulfide 

(CS2) and 67 nonexposed metal, plastic, and starch workers was conducted in which subjects 

were questioned about eye irritation complaints (Vanhoorne et al., 1995). Exposed and control 

workers were employed for at least one year by their employer. Exposure to H2S and CS2 was 

determined using personal monitoring pumps. Exposure to H2S ranged between 0.2 and 8.9 

mg/m3. Workers exposed to H2S also tended to have high exposure to CS2. No workers were 

exposed to H2S only. Several types of eye complaints were reported by workers exposed to CS2 

and H2S including: pain, tension, burning, hazy sight, photophobia, and irritation. While the 

percentage of complaints increased with exposure, the frequency of complaints was higher in 

workers exposed to H2S and CS2 compared to workers exposed to CS2 alone. The investigators 

conclude, however, that deciding which of the two gases was responsible for eye irritation could 

not be determined, but argue that a priori of evidence suggests that H2S is responsible. 

Considering all of the evidence, the investigators further suggested that CS2 enhances the eye 

irritation properties of H2S. 

A study by Kilburn and Warshaw (1995) included 13 former workers and 22 near-plant 

residents of a desulfurization unit within a refinery processing “sour’ crude oil. Air monitoring 

for H2S and other chemicals at street level near the subjects’ homes during one week in July 

1990 revealed that the H2S concentration was 0.01 ppm with periodic peaks of 0.1 ppm along 

with mercaptans (0.002 ppm), ethane (0.5 ppm), propane (0.5 ppm) and unquantified levels of 

vanadium and thiodiglycolic acid. In addition, dimethylsulfide, and vanadium (as vanadium 

pentoxide) were detected in air. Reduced sulfur gases measured outside the facility showed 24-

hr averages of up to 21 ppm mercaptans, 0 to 8.8 ppm H2S, carbon oxide sulfide (COS: 6 to 52 

ppm) and for total reduced sulfur 6 to 71 ppm. 

Questionnaires were completed by subjects and controls. Neurobehavioral functions and 

a profile of mood states were compared to 32 controls, matched by age and educational level. 

Neuropsychological functions were also evaluated, including: two-choice reaction time, balance, 

16
-17-

color discrimination, digit symbol, trail-making A and B, and immediate recall. Abnormal mean 

values of exposed subjects compared to controls were determined to be statistically significant 

for these functions. Mood state scores were found to be much higher in the exposed subjects than 

the controls. However, many of the neurophysiological findings had large standard deviations 

and consequently were highly-variable. While alcohol use was excluded as a confounder, all of 

the ex-workers had been exposed to solvents. Although the authors concluded that long-term 

exposure to “low doses” of H2S is a plausible cause of the neurological function deficits, they 

acknowledged that other effects were attributable to other chemicals that were monitored (e.g., 

asthma and dermatitis in workers were attributed to sulfur dioxide and vanadium pentoxide). 

Given the complexity of the exposure environment - in terms of the numbers of chemicals 

monitored, the few numbers of individuals studied and the very limited exposure measurements -

it is premature to attribute the deficits cited with H2S exposure. 

Neurobehavioral effects of H2S were studied in 16 individuals (male and female, ages 21 

to 68 years) 2 to 22 years after exposure (Kilburn, 1997). Five individuals had been unconscious 

after exposure and four had downwind exposures. The exposure level was not reported in any of 

the cases. Five of the 16 individuals were categorized in the “chronic” exposure group, exposed 

for 11-22 years. Five were in the “acute” exposure category having been exposed for minutes 

and the remaining six were in the “hours” category. Twelve of the 16 individuals were plaintiffs 

in lawsuits. Results from a battery of tests were compared to those from 353 national referents, 

matched by age, sex, and years of education. Smoking status was not a significant factor. 

Neurophysiologic and neuropsychologic tests, the latter adjusted for educational level, were 

employed. Permanent neurobehavioral impairment was apparent in all 16 subjects. Brief high 

doses were “devastating,” whereas protracted low doses of individuals in the chronic category 

showed effects (p<0.05) on the more sensitive neurobehavioral tests, such as balance, visual 

fields, and choice reaction time. There was no indication of bias from the lawsuit and it was 

considered unlikely that the symptoms could be attributed to other causes. Kilburn (1997) 

concluded that moderate occupational exposure and downwind environmental exposure can 

cause permanent impairment. This finding was assessed in a subsequent study. 

Neurobehavioral deficits attributed to low-level environmental exposure of 103 

individuals to H2S were reported by Kilburn (1999). The neurobehavioral battery included 28 

tests. One group of 24 homeowners was exposed to H2S that had collected in crawlspaces and 

under concrete foundations. Concentrations ranged from 0.1 to 1.0 ppm with several peaks up to 

5 ppm during a two-month period in 1996. The homeowners were assessed about one year later. 

They exhibited abnormal balance, color discrimination, grip strength and delayed verbal recall. It 

was not stated if the exposure was ongoing at the time of the neurobehavioral assessment. In 

another group, 48 adults were exposed to measured peak levels ranging from 1 to 20 ppm at 

street level during a refinery explosion and fire in 1992. They were assessed for 

17
-18-

neurobehavioral deficits four years later in 1996. The subjects had 11-12 abnormalities 

compared to 4 in the first group. A third group consisted of 16 patients with a wide range of 

exposure who were assessed after latent periods of 1.7 to 22 years [these were the same 

individuals discussed in the preceding paragraph by Kilburn (1997)]. A fourth group consisted 

of 13 workers and 22 residents living at or near stack gas emissions from a refinery and 

desulfurization plant. These individuals were also exposed carbon oxide sulfide (2.6 - 52.1 ppm), 

mercaptans (1.0 - 21.1 ppm), and 24-hr levels of H2S at 0 to 8.8 ppm. Referents (357) were 

selected from four towns (in four states) free of known chemical contamination. Analysis of 

variance and covariance adjusted for differences in age, education, gender, and height. Kilburn 

(1997) concluded that peak dose rather than duration of exposure is predictive of effects, and 

those exposed to nonlethal levels do not recover completely from H2S. Given the small number 

(24) of homeowners in the first group exposed to low levels and the type of deficits observed, it 

is premature to ascribe cause and effect to H2S. 

A five-year retrospective study (Arnold et al., 1985) of 250 workers exposed to H2S 

considered compensation claims submitted from 1979-1983. Fifty-four percent of the workers 

became unconscious after exposure. Of the clinical findings, neurologic, respiratory and 

opthalmologic ailments predominated. The overall fatality rate was 2.8%. 

In conclusion, H2S exposure levels that result in temporary unconsciousness (e.g., 15-30 

minutes) can cause profound neurophysiological, neurobehavioral, neurocognitive, 

neurophysical, respiratory, and opthalmologic deficits that are persistent. While some of these 

deficits can occur when the exposed individual remains conscious, it is less clearly established if 

intermittent or continuous exposure to “low levels” of H2S cause similar persistent symptoms. 

4.2. PRECHRONIC AND CHRONIC STUDIES IN ANIMALS 

4.2.1. Oral 

Wetterau et al. (1964) examined the effects of H2S in chickens and pigs that were fed 

dried green animal fodder. The fodder had been dried by sulfur-containing brown coal or other 

fuels containing H2S. The fodder was analyzed for H2S content by acidification with 

hydrochloric acid and iodometric determination prior to animal administration. The H2S content 

in all of the experiments ranged from 0.035-0.121%. In chickens (50 per group), no adverse 

effects were observed in feed intake, body weight, or survival in animals fed H2S-treated alfalfa 

(2-12% of total feed) and H2S-treated wheat bran (1-12% of total feed) daily for up to 70 days. 

Adult pigs (numbers not given) were fed diets containing 100, 200, or 400 grams of H2S-treated 

dried alfalfa (4, 8, and 24% of the diet, respectively) daily for 105 days. Pigs fed 200 or 400 

grams of H2S-treated dried alfalfa had decreased weight gains of 4 and 22%, respectively, 

compared with the control group. No statistical analysis was presented. Food intake decreased 

18
-19-

by 33% in animals fed 400 grams of H2S-treated dried feed per day. Diarrhea was reported in 

adult animals that consumed diets containing no treated dried feed that was suddenly changed to 

diets containing 24% treated dried feed. 

In a separate experiment, weaned pigs (numbers not provide) were fed diets containing 

100, 200, or 400 grams of H2S-treated dried alfalfa (4, 8, and 24% of the diet, respectively) daily 

for 105 days. In weaned animals previously exposed to treated dried feed, the animals did not 

develop diarrhea when the amount of treated dried feed was increased to 24% of total feed 

intake. The percent of H2S in the diet containing 24% dried feed was 0.121%. The investigators 

concluded that there was no indication that diets containing a high amount of H2S could cause 

adverse health effects in animals. The diarrhea observed in naive adult pigs may have been 

caused by the sudden change in diet rather than the presence of H2S, since increasing the feed 

containing H2S in weaned pigs previously exposed to a similar diet had no adverse effects. 

Decreased weight gain observed in the second study is apparently due to a decrease in feed 

intake. The pigs in this study did not develop diarrhea, but there was a decrease in food intake. 

The investigators reported that previous data suggested that animals refused to eat food 

containing H2S, which may explain the decreased feed intake and the subsequent decreased 

weight gain observed in the study. Results of the study were likely caused by food avoidance 

from odor rather than toxicity. 

A study by Wetterau et al. (1964) was used previously on IRIS as the basis for deriving 

an RfD. However, the low concentration of H2S in the feed, estimated at only about 15 mg/kgday, coupled with the investigators’ conclusions that the diets did not cause adverse or 

reproducible health effects in pigs precludes the use of this study in the derivation of an RfD in 

this current assessment. 

Male and female Sprague-Dawley rats (20/sex/dose group) were administered 0, 1, 3.5, 

or 7 mg/kg-day H2S by gavage daily for 89 days (Anderson, 1987). Dose-levels were 

determined after a 14-day range-finding study. Hydrogen sulfide doses were prepared by purging 

deoxygenated deionized water with pure H2S gas. Prepared H2S solutions were stored in amber 

vials with Teflon seals and fresh vials were used at each dose administration. Dosing solutions 

were prepared weekly and solution stability was determined. Animals were examined for 

mortality at the beginning and end of each working day, and clinical signs were monitored 

immediately after dose administration. Food consumption of 10 rats per dose group was 

determined weekly. Animals were weighed weekly during the treatment period and at necropsy. 

Blood samples were collected from the suborbital sinus of animals prior to study initiation, days 

27 to 31 of treatment, and before necropsy. Blood and platelet counts were determined, and 

clinical chemistry was performed. At study termination, animals were sacrificed by carbon 

dioxide asphyxiation. Sacrificed animals and animals that died during the treatment period were 

subjected to a full necropsy. In control and high-dose animals, liver, kidney, spleen, gonad, 

19
-20-

brain, heart, adrenal, and gross lesions were examined microscopically. In the low- and mid-dose 

groups, only lung, kidney, liver, and gross lesions were examined microscopically. Mortality 

was 50% in the high-dose males compared to 5% in control males. No deaths were observed in 

high-dose females. Mortality in mid-dose males and females was 10 and 5%, respectively, and 

no deaths were observed in low-dose animals or control females. Weekly mortality is reported 

in Table 1. 

Table 1. Cumulative weekly mortality in male and female Sprague-Dawley rats 

administered H2S orally 

Dose 

(mg/kg-day) 

Males 0 

1 

3.5 

7 

Females 0 

1 

3.5 

7 

Source: Anderson, 1987 

Weeks 

1 2 3 4 5 6 7 8 9 10 11 12 13 % 

0 

0 

0 

0 

5 

0 

1 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 1 1 1 1 1 1 1 1 1 

0 0 0 0 0 0 0 0 0 0 

0 0 0 0 1 1 1 1 1 1 1 1 2 10 

0 0 0 0 2 2 4 4 6 6 7 9 10 50 

0 0 0 0 0 0 0 0 0 0 

0 0 0 0 0 0 0 0 0 0 

0 0 0 0 0 0 0 1 1 1 1 1 1 5 

0 0 0 0 0 0 0 0 0 0 0 0 0 0 

Compound-related neuromuscular and behavioral signs were observed in high-dose male 

and female animals, with the males displaying a higher incidence of these effects (Anderson, 

1987). High dose-males exhibited abnormal posture, convulsions, fist clutching, sedation, 

unusual vocalization, piloerection, labored breathing, and increased respiratory rates. High-dose 

females exhibited convulsion, sedation, and labored respiration. Although restlessness was 

observed in all treated males, and increased salivation in all treated females, these clinical signs 

generally occurred sporadically, and the incidence of these findings did not show a clear doseresponse. Therefore, the toxicological significance of restlessness in treated males, and 

salivation in treated females, is not clear. 

No significant differences in body weights were noted in any of the treated animals 

(Anderson, 1987). In addition, no differences in food consumption were noted between treated 

and control groups. There were no treatment-related effects in hematology or clinical chemistry. 

20
-21-

Ophthalmological findings prior to sacrifice were normal. The most common findings at 

necropsy were pink and red mottled lungs, which occurred in all groups. In high-dose males that 

died prior to sacrifice, pink-red discoloration of the lungs, nasal discharge, penile discharge, 

reddening of the scrotal sac, and darkening of the spleen were noted. Although absolute organ 

weights were not affected by treatment, relative heart weights increased (p < 0.05) in high-dose 

males, and relative kidney weights increased (p < 0.05) in high-dose females. No gross lesions 

could be attributed to compound administration and all microscopic findings were considered 

incidental to compound administration. 

It should be noted that there are a number of problems with this study that prevent its use 

in the derivation of an RfD. For example, a 75 to 100% incidence of pneumonia was reported in 

the vehicle control and treated groups, however, a 0% incidence of pneumonia was reported for 

the nongavaged, rack control group. This information indicates procedural problems with the 

dosing of the animals. Also, there was one group where mortality was 50%, the high dose male 

group. However, the cause of death was not determined. In addition, a malady diagnosed as 

ulcerative dermatitis was common across all groups. Good Laboratory Practice would preclude 

the acceptance of data from such diseased animals for drawing conclusions regarding the effects 

of H2S. 

4.2.2. Inhalation 

The Chemical Industry Institute of Toxicology (CIIT, 1983a) performed a 90-day 

inhalation toxicity study in B6C3F1/CrlBr mice using H2S. Animals were individually housed 

and exposed to 0, 10.1, 30.5, or 80 ppm (0, 14, 42, or 111 mg/m3

) H2S for 6 hr/day, 5 days/week 

for 90 days (10 mice/sex/group). Animals were observed twice daily for mortality and clinical 

signs. Body weight and feed consumption were determined weekly beginning just before the 

first exposure. A final fasted body weight was determined for all animals just before necropsy. 

Each animal also received an ophthalmological exam before the first day of exposure and within 

seven days of necropsy. A neurological exam was performed before necropsy in which posture, 

gait, facial muscle tone, and pupillary, palpebral, extensor thrust, and crossed-extensor thrust 

reflexes were assessed. Animals were housed in metabolism cages for 12 hr prior to necropsy. 

Urine samples were collected for all animals and volume, appearance, occult blood, specific 

gravity, protein, pH, ketone, and glucose were determined. On the day of necropsy, animals 

were anesthetized with ether and blood was drawn from the suborbital sinus (hematology) and 

abdominal aorta (chemistry). Animals were sacrificed on study day 91, 92, 93, 94, 95, and 99. 

The brain, kidney, spleen, liver, heart, and ovaries/testes were removed, weighed, and examined 

for gross and histopathologically observable abnormalities. In addition, the following tissues 

were examined microscopically: cerebellum, cerebrum, medulla, optic nerve, spinal cord, sciatic 

and anterior tibial nerves, eyes, pituitary, thyroid, parathyroid, salivary glands, heart, lungs, 

21
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spleen, liver, pancreas, adrenals, mesenteric and mandibular lymph nodes, kidneys, bladder, 

lacrimal glands, ovaries, uterus, oviducts, vagina, cervix, stomach, duodenum, ileum, jejunum, 

large and small colon, caecum, skeletal muscle, skin, mammary glands (both sexes), femur, bone 

marrow, aorta, ear canal with zymbal gland, nasal turbinates (four levels), trachea, testes, 

epididymis, esophagus, thymus, prostate, seminal vesicle, and gross lesions. 

Body weights were significantly depressed in both males and females in the highexposure groups with sporadic significant decreases in the lower male exposure groups (CIIT, 

1983a). Decreases in body weights tended to be more pronounced in the male and female high 

exposure groups beginning in the 6th week of exposure. Feed consumption was significantly 

reduced in the high-exposure animals. There were no toxicologically- or statistically-significant 

differences in hematology, serum chemistry, urinalysis, ophthalmology, or neurological function 

except for a 30.5 ppm (42 mg/m3

) female and 80 ppm (111 mg/m3) male that did not respond to 

artificial light stimulus. 

Two female controls and one 30.5 ppm (42 mg/m3

) male died prior to study termination. 

Both control animals exhibited multiple focal black discoloration of the glandular stomach. The 

30.5 ppm (42 mg/m3

) male exhibited a skull fracture with red discolorations and trauma 

apparently due to a feeding accident. A female and male in the 80 ppm (111 mg/m3) exposure 

group were sacrificed in extremis on study days five and six, respectively. Both sacrificed 

animals exhibited prostration and/or hypoactivity prior to sacrifice. Both animals also had the 

same black discoloration of the glandular stomach as those in the control group. A low 

incidence of alopecia and emaciation was observed in control and exposed animals. In addition, 

a 10.1 ppm (14 mg/m3

) male lost the use of anterior appendage with the appearance of paralysis 

while one 30.5 ppm (42 mg/m3

) female and a 80 ppm (111 mg/m3) female were observed to have 

a missing front appendage. The investigators stated that it was unknown how the male lost its 

appendage but that the female’s loss was apparently congenital. The investigators considered 

none of the above findings to be compound-related. Gross pathology of surviving animals also 

revealed no gross lesions that were considered compound-related (CIIT, 1983a). 

The mean absolute weights of the heart, liver, and spleen from male mice exposed to 80 

ppm (111 mg/m3

) H2S were significantly depressed. In addition, the mean absolute kidney 

weight of 80 ppm (111 mg/m3

) female mice was also significantly reduced by approximately 

20%. However, relative weights were not significantly different and clinical pathology and 

histology were negative in both sexes. Histological examination of surviving animals revealed 

the nasal tract as the only site where lesions were considered compound-related. Male (8/9) and 

female (7/9) mice exposed to 80 ppm (111 mg/m3) H2S exhibited minimal to mild inflammation 

of the anterior portion of the nasal mucosa (section I; section I and II in two mice). The lesion 

was primarily located in the squamous portion of the nasal mucosa but extended into the 

respiratory type epithelium (ciliated) in some animals. In one female, the lesion was suppurative 

22
-23-

and severe involving the entire nasal passage and associated structures. The lesion was also 

observed in two 80 ppm (111 mg/m3

) mice that were exposed in extremis. No other histological 

findings were considered compound-related (CIIT, 1983a). 

The critical effect in mice was inflammation of the nasal mucosa. This effect was present 

in male (8/9) and female (7/9) animals exposed to 80 ppm (111 mg/m3

) H2S. The lesions, judged 

as minimal to mild in severity, were localized primarily in the squamous portion of the nasal 

mucosa but extended to regions of ciliated respiratory-type epithelium. Since H2S is an irritant 

gas and other researchers (Brenneman et al., 2000; Dorman et al., 2000; Lopez et al., 1987) have 

reported nasal inflammation following H2S exposure, the critical effect in mice was considered 

to be inflammation of the nasal mucosa. The LOAEL for this effect in mice was 80 ppm (111 

mg/m3

) and the NOAEL was 30.5 ppm (42 mg/m3) (CIIT, 1983a). 

With the following exception, the same methods described in the CIIT (1983a) mouse 

study were used in a similar study using Fischer 344 rats (CIIT, 1983b). Following a 90-day 

exposure period in rats, 10 males and females from each group were selected for clinical 

pathology and histology, while the remaining 5 males and females from each group were used 

for special neuropathologic studies. In the neuropathologic studies, rats were anesthetized with 

sodium pentobarbital containing 200 units of heparin. Rats were perfused via the left ventricle 

with a 4% phosphate buffered glutaraldehyde solution. The perfused animals were then 

refrigerated at 4°C overnight. On the following day, the right and left sciatic nerves and the 

cervical and lumbar portions of the spinal cord were dissected and placed in 4% glutaraldehyde. 

The left sural nerve and large muscle branch of the tibial nerve were oscimated and placed in 

cedarwood oil for two weeks. Nerve fibers in the oil were teased and individual fibers mounted 

to glass slides. After staining, specimens were examined by light microscopy for pathological 

changes. 

In Fischer 344 rats, no mortality was observed during the 90-day study (CIIT, 1983b). 

Body weights were significantly reduced from weeks 1 to 13 in males and females exposed to 80 

ppm (111 mg/m3

) H2S. Feed consumption was also significantly depressed in males and females 

exposed to 80 ppm (111 mg/m3) H2S. No abnormalities in ophthalmology, neurological 

function, serum chemistry, or urinalysis were reported. Sulfhemoglobin levels were significantly 

increased in males exposed to 80 ppm (111 mg/m3

) H2S. Although elevated sulfhemoglobin 

levels were also observed in females exposed to 30.5 or 80 ppm (42 mg/m3 or 111 mg/m3) H2S, 

the levels were not statistically significant. The investigators state that assigning biological 

significance to the sulfhemoglobin values should be done with caution because of sensitivity of 

the method and the size of the groups investigated. Also, there were no H2S-related lesions in 

animals selected for special neuropathologic studies. Gross pathology revealed a low incidence 

of lesions that were not compound-related. Organ weights were statistically similar between 

control and exposure weights except for the relative brain weight of males exposed to 80 ppm 

23
-24-

(111 mg/m3

) H2S. Relative, but not absolute, brain weights were significantly increased in these 

animals. Minimal multifocal peribronchial lymphocytic infiltrate was observed in the lungs of all 

rats. Minimal to mild mononuclear cellular infiltration of sections III and IV of the nasal mucosa 

and nasolacrimal duct was observed in all rats including the controls. There was also minimal 

to moderate inflammation of sections I and II of the nasal mucosa, but this effect was not 

dependent on dose with 45, 40, 75, and 55% of animals affected in the 0, 10.1 (14 mg/m3

), 30.5 

(42 mg/m3) and 80 ppm (111 mg/m3) exposure groups, respectively. None of the histopathologic 

changes were considered treatment-related by the investigators. The critical effect was depressed 

body weight gain in female rats. The LOAEL for this effect was 80 ppm (111 mg/m3) and the 

NOAEL was 30.5 ppm (42 mg/m3

). 

The same methods described above (CIIT, 1983a,b), including the special 

neuropathologic studies in CIIT (1983b) were used in the following study which used SpragueDawley rats (CIIT, 1983c). In the Sprague-Dawley rats, 15 males and 15 females per group, no 

mortality was observed during the 90-day study. Statistically significant decreases in body 

weights were observed in males exposed to 80 ppm (111 mg/m3

) H2S during weeks 1 through 3, 

and in 80 ppm-treated females exposed throughout the 90-day study. Feed consumption was 

also significantly depressed in males and females exposed to 80 ppm (111 mg/m3) H2S. No 

abnormalities in ophthalmology, neurological function, hematology, serum chemistry, or 

urinalysis were reported, and there were no H2S-related lesions in animals selected for the 

special neuropathologic studies. Gross pathology revealed a low incidence of lesions that were 

not compound-related. The black discoloration noted in the glandular stomach of mice was also 

present in 5 treated rats. However, this effect was not dose-dependent. Organ weights were 

statistically similar between control and exposure weights, excluding the absolute brain weight 

of males exposed to 80 ppm (111 mg/m3) H2S. Absolute, but not relative, brain weights were 

significantly reduced in these animals; a finding concordant with the results of the CIIT (1983b) 

study with Fisher 344 rats. The investigators also suggested that the decreased brain weight was 

due to the chemical nature of H2S, e.g., low molecular weight and high degree of lipophilicity 

allowing passage through the blood-brain barrier. Minimal to mild multifocal peribronchial 

lymphocytic infiltrate was observed in the lungs of all rats including controls. Minimal to mild 

mononuclear cellular infiltration in sections III and IV of the nasal mucosa and nasolacrimal duct 

was observed in all rats, including the controls. Although there was also minimal to moderate 

inflammation in sections I and II of the nasal mucosa, the effect was not related to dose with 35, 

35, 30, and 50% of animals affected in the 0, 10.1 (14 mg/m3

), 30.5 (42 mg/m3) and 80 ppm (111 

mg/m3) exposure groups, respectively. None of the histopathologic changes were considered 

treatment-related by the investigators. The critical effect was decreased absolute brain weight in 

males. The LOAEL for this effect was 80 ppm (111 mg/m3

) and the NOAEL was 30.5 ppm (42 

mg/m3

). 

24
-25-

The following study was actually performed in 1943 by Haskell Laboratory and 

submitted by Dupont to the Office of Pollution Prevention and Toxics, U.S. EPA (Haskell 

Laboratory, 1994). Four dogs (sex and species not reported) were exposed to 15 ppm (21 

mg/m3

) H2S 6 hr/day, 5 days/week for 7 weeks. At the start of the week 8, dogs were coexposed 

to 15 ppm (20.8 mg/m3) H2S and 10 ppm carbon disulfide. During week 9, dogs were only 

exposed to 15 ppm (20.8 mg/m3

) H2S which was increased to 30 ppm (42 mg/m3) in weeks 10-

12. Blood pressure and heart rate were monitored in the morning and afternoon of exposure 

days. In addition, blood morphology (not described by the investigators), and arterial and 

venous CO2 and O2 were determined. The frequency with which the investigators determined 

these parameters was described as “time to time.” All measured endpoints were compared to 

control values that were established during a 4-week preexposure period. The dogs exhibited 

normal health and behavior during the exposure period. Appetites and weights were maintained 

satisfactorily, and no blood abnormalities were noted. Abnormal pulse and blood pressure 

determinations increased in frequency during the course of the experiment (weeks 1-7). The 

investigators did not state what constituted an abnormal blood pressure reading. However, the 

investigators suggested a cumulative effect from low exposure. Coexposure with carbon 

disulfide increased the number of abnormal blood pressure readings. Increasing the 

concentration to 30 ppm (42 mg/m3

) H2S doubled the number of abnormal blood pressure 

examinations when compared to a two-week 15 ppm (21 mg/m3) exposure. 

Brenneman et al. (2000) exposed 10-week-old male Sprague-Dawley CD rats 

(12/exposure group) to 0, 10, 30, or 80 ppm (0, 14, 42.7, or 111 mg/m3

) H2S for 6 hr/day, 7 

days/week for 10 weeks. At the end of the 10-week exposure period, animals were euthanized 

with CO2 and the noses of the animals were dissected free. The nasal cavities were sectioned at 

6 different levels such that sections 3-6 each allowed for histological evaluation of transitional 

/respiratory and olfactory epithelium. The lesions noted were limited to olfactory tissue, i.e., 

olfactory neuron loss and basal cell hyperplasia at the olfactory mucosa, and were graded in 

severity by a subjective scale where 0 = normal, 1 = mild, 2 = moderate, 3 = marked, and 4 = 

severe. 

Table 2 shows that no effects were observed in the control animals or in animals exposed 

to 10 ppm (14 mg/m3

) H2S (Brenneman et al., 2000). Lesions, limited to the olfactory mucosa, 

were observed in animals exposed to either 30 (427 mg/m3) or 80 ppm (111 mg/m3) H2S. These 

olfactory lesions consisted of multifocal, bilaterally-symmetrical olfactory neuron loss and basal 

cell hyperplasia affecting the lining of the dorsal medial meatus and dorsal and medial region of 

the ethmoid recess. The incidence, mean severity, and distribution of the exposure-related 

lesions increased in a concentration-dependent manner. 

25
-26-

Table 2. Incidence (Inc) and average severity (Sev) of nasal lesions in male CD rats 

exposed to H2S 

Lesion 

Nasal Cavity 

Level 

0 10 ppm 30 ppm 80 ppm 

Inc Sev Inc Sev Inc Sev Inc Seva 

Olfactory 3 0/9 — 0/8 — 0/6 — 8/8 2.4 

Neuron Loss 4 0/12 — 0/12 — 11/12 1.4 12/12 2.4 

5 0/12 — 0/12 — 9/12 1.1 11/12 1.5 

6 0/12 — 0/12 — 0/12 — 5/12 1.2 

Basal Cell 

Hyperplasia 

3b — — — — — 

4 0/12 — 0/12 — 10/12 1.8 12/12 1.2 

5 0/12 — 0/12 — 7/12 1.3 11/12 1.3 

6 0/12 — 0/12 — 0/12 — 6/12 1.0 

— — — 

a

Average severity of the lesion in affected rats in the exposure group: 1 = mild, 2 = moderate, 3 = 

severe. 

b

Rats were not evaluated at level 3 because of the common occurrence of basal cell hyperplasia 

at background levels. 

Source: adapted from Brenneman et al. (2000). 

Respiratory/transition epithelium largely comprises level 3 of the nose, the olfactory 

mucosa being only a minor component limited to the most caudal portion of the section. 

Olfactory neuron loss was only observed in 80 ppm (111 mg/m3

) exposure animals at this level 

of the nose. Basal cell hyperplasia was not evaluated due to the common background occurrence 

of this lesion at this level. 

Olfactory mucosa comprises a slightly larger portion of level 4 of the nasal cavity. This 

level had lesion distribution restricted to the olfactory tissue in the dorsal medial meatus and the 

most rostral projection of the third ethmoturbinate. Olfactory neuron loss in the 30 ppm (42 

mg/m3

) exposure animals was mild (grade 1) to moderate (grade 2), but olfactory neuron loss in 

the 80 ppm (111 mg/m3) exposure group increased to an average severity between moderate 

(grade 3) and severe (grade 4). Basal cell hyperplasia was observed in both exposure groups at 

this level of the nasal cavity but was slightly more severe in the 30 ppm (42 mg/m3

) exposure 

group. 

Olfactory mucosa lines most of the nasal cavity at levels 5 and 6 while the ventral meatus 

and lateral walls of the nasal cavity are lined with respiratory epithelium. Although the olfactory 

mucosa was widely distributed, lesions in this tissue were found at select sites. At level 5, mild 

26
-27-

to moderate olfactory neuron loss and mild basal cell hyperplasia mainly affecting the nasal 

septum, dorsal nasal cavity, and marginal ethmoturbinate were observed in both exposure 

groups. The nasal septum was not affected in the 30 ppm (42 mg/m3

) exposure group. The same 

pattern and severity of lesions were observed at level 6 in the 80 ppm (111 mg/m3) exposure 

group. 

The critical effects in this study are nasal lesions of the olfactory mucosa, 30 ppm (42 

mg/m3

) is the LOAEL and 10 ppm (14 mg/m3) the NOAEL (Brenneman et al., 2000). 

It should be noted that lesions in the nasal tract reported in other studies are not 

completely in agreement with those reported by Brenneman et al. (2000) where lesions were 

clearly related to dose and localized in the olfactory epithelium. In mice exposed to similar 

levels of H2S (CIIT, 1983a), inflammation of the nasal mucosa was reported and described as 

occurring primarily in the squamous nasal mucosa and extending into the respiratory epithelium, 

with no specific mention of olfactory mucosal involvement. Sectioning of the nasal epithelium 

was through 4- rather than 6-areas examined by Brenneman et al. (2000) explaining, in part, this 

difference. Effects in the nasal cavity reported in Sprague-Dawley rats in an earlier study (CIIT, 

1983c) differ in severity and location from those reported by Brenneman et al. (2000). Reasons 

for such a discrepancy are unknown. 

4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES—ORAL AND INHALATION 

Dorman et al. (2000) attempted to determine if perinatal exposure by inhalation to H2S 

had an adverse effect on reproductive performance and pregnancy outcomes. Virgin male and 

female Sprague-Dawley rats (12/sex/group) were exposed to 0, 10, 30, or 80 ppm (0, 14, 42, or 

111 mg/m3) H2S 6 hr/day, 7 days/week for two weeks prior to breeding. Exposure continued 

during a 2-week mating period and throughout gestational days 0-19 (GD 0-19). Evidence of 

copulation (vaginal plug or sperm in vaginal lavage fluid) during the 2-week mating period was 

considered GD 0. On postnatal day (PND) 4, litters were randomly reduced to 4 animals per sex 

when possible. Remaining pups were euthanized and discarded without being examined. Dams 

and pups were then exposed PND 5-18. Nonpregnant adult females were exposed for an 

additional 23-24 days following the 2-week breeding period. Adult males were exposed to H2S 

for 70 consecutive days. 

Clinical examinations were performed on all animals before and after each exposure 

(Dorman et al., 2000). The body weights of the F0 males and females were determined weekly 

throughout the study, except that female body weights were not determined weekly once 

evidence of mating was present. Presumed pregnant females were weighed on GD 0, 7, 14, and 

20, and dams were weighed on PND 0, 4, 7, 14, and 21. Feed consumption was determined 

weekly in F0 males and prebreeding females. Feed consumption in presumed pregnant females 

27
-28-

was recorded for GD 0-7, 7-14, and 14-20. Dam feed consumption was recorded for PND 0-4, 

4-7, 7-14, and 14-21. At the end of exposure, adult rats were euthanized and a complete 

necropsy was performed with emphasis on reproductive and accessory sex organs. Postparturient animals were necropsied the day of or the day after weaning. At necropsy, the right 

testis from each F0 male was examined for sperm number, production, motility, and morphology. 

No deaths or adverse clinical signs were observed in F0 males and females for any 

exposure group. There was a statistically significant decrease in feed consumption in male rats 

exposed to 80 ppm (111 mg/m3

) H2S during the first week of the study. There was a small, but 

not statistically significant, decrease in body weight (5-6%) observed in F0 males and females 

exposed to 80 ppm (111 mg/m3

) H2S that was present throughout entire exposure period. The 

only significant difference in organ weights was an increase in absolute and relative adrenal 

gland weights observed in F0 males exposed to 10 ppm (14 mg/m3) and 80 ppm (111 mg/m3) but 

not in the mid-dose of H2S and a decrease in the relative ovary weight of females in the 10 ppm 

(14 mg/m3

) exposure group. 

There were no statistically significant effects on reproductive performance (mating index, 

fertility index, postimplantation loss per litter, and number of late resorptions or stillbirths) in F0 

animals. Also, the number of live pups, litter size, average length of gestation, and average 

number of implants per female were not affected. There were no statistically significant effects 

on sperm production or morphology noted in F0 males; however, a large percentage of abnormal 

sperm was observed in one F0 male from both the 30 (42 mg/m3

) and 80 ppm (111 mg/m3) 

exposure groups (29 and 73%, respectively). In comparing high-exposure and control animals, 

there were a few histologic diagnoses with a higher incidence in the high-exposure group, 

including: testicular tubular degeneration, intratubular sperm stasis, tubular mineralization, 

sperm granulomas, and multinucleated giant cells, degenerate sperm forms in the lumen, 

aspermia, and oligospermia. While the incidence of testicular tubular degeneration in highexposures was higher (42%) than the controls (17%), statistical significance was not achieved. 

One incidence each of sperm granuloma and unilateral necrosis of the cauda was present in the 

80 ppm (111 mg/m3

) exposure group. Notable histological findings in females included one 

each of ovarian cysts and squamous metaplasia of the endometrium in the 10 ppm (14 mg/m3

) 

and 30 ppm (42 mg/m3

) exposure groups. The olfactory neuron loss and basal cell hyperplasia 

observed in the male rats in this study are reported elsewhere (Brenneman et al., 2000). The 

assignment of any effect level for reproductive toxicity is problematic since the study considered 

only 12 rats/sex/group and the study also lacked any statistical significance in either function or 

histopathology related to reproduction endpoints. In summary, the reproductive segment of this 

study did not demonstrate reproductive toxicity in either male or female rats following relatively 

high H2S exposure of 80 ppm (111 mg/m3

) under repeated conditions (up to 70 days for males). 

28
-29-

With regards to developmental effects from H2S exposure, there were no statistically 

significant increases in structural malformations. Observed malformations included kinked tail, 

agenesis of the tail, anophthalmia, small rear legs and body, frontal bone defects, hypognathia, 

and skin lesion characterized by detachment of the skin and dermis. However, none of these 

effects was dose-related. There were no significant differences in pup weight gain or 

development (pinnae detachment, surface righting, incisor eruption and negative geotaxis, 

vaginal patency, preputial separation, and eyelid separation). Surface righting was also 

equivalent across exposure groups. There were no treatment-related effects on motor activity, 

acoustic startle response, passive avoidance observed, or FOB. Terminal body and organ 

weights in all exposure groups were comparable to controls. Although a wide variety of gross 

observations were noted, they were not considered treatment-related by the investigators. 

Microscopic examination of six levels of the brain from pups from the control and high-dose 

group failed to demonstrate any histologic abnormalities. 

Saillenfait et al. (1989) reported that the exposure of pregnant Sprague-Dawley rats to 0, 

70, 140 or 210 mg/m3

 (0, 50, 100, or 150 ppm) H2S 6 hr/day during gestational days 6-20 

resulted in no signs of maternal toxicity or adverse effects on the developing fetus. While there 

was a slight but significant decrease in fetal weight in all treated groups, the biological 

significance of this finding is questionable since the dams also lost weight and there were greater 

numbers of live fetuses per litter in those groups. 

Significant elevations in maternal blood glucose levels were observed in Sprague-Dawley 

rats exposed to 0, 28, 70, or 105 mg/m3

 (0, 20, 50, or 75 ppm) H2S, 7 hr/day from gestation day 1 

through postnatal day 21 (Hayden et al., 1990a). There were no changes in serum protein, LDH, 

SGOT, or alkaline phosphatase activities in the treated dams or pups. Pups exhibited decreased 

serum triglycerides in the 70 mg/m3 (50 ppm) dose group. In a similarly-designed study using 

the same exposure levels (Hayden et al., 1990b), there was a dose-dependent increase in 

parturition time of approximately 10, 20, and 40% over matched controls at 0, 28, 70, or 105 

mg/m3

 (0, 20, 50, or 75 ppm), respectively. The biological significance of this latter effect is 

questionable since parturition time was variable among control groups and was not examined 

statistically. Maternal liver cholesterol content was significantly elevated on day 21 postpartum 

in the high-dose dams. 

Although no studies have reported effects that prenatal exposure to H2S may eventually 

have upon the adult nervous system, early postnatal measurements provide indications of 

possible neurotoxic effects that may occur in adulthood. Hannah et al. (1989) exposed timedpregnant Sprague-Dawley rats (number not reported) to 105 mg/m3

 (75 ppm) H2S, 7 hr/day from 

day 5 postcoitus until PND day 21. Groups of 8 randomly-selected offspring, exposed in utero 

through PND 21, were sacrificed on PNDs 7, 14, and 21. The levels of putative neurotransmitter 

aspartate and gamma amino-butyric acid (GABA) were all significantly (p<0.05) depressed in 

29
-30-

the cerebellum on PND 21. Glycine was unaltered. GABA is an inhibitory neurotransmitter of 

the central nervous system that appears in Purkinje cells such that decreases in GABA could be 

due to a decrease in the population of Purkinje cells. Analysis of cerebellum (in the 75 ppm 

animals only), however, established that this was not the case. There were no significant 

differences in the brain weights of treated pups compared to controls. The significance of these 

amino acid changes to neurobehavioral development is uncertain. 

In a similarly designed study using the same exposure protocol, Hannah and colleagues 

(Skrajny et al., 1992) exposed groups of 20 pregnant Sprague-Dawley rats to 0, 28, or 105 mg/m3 

(0, 20, or 75 ppm) H2S. Ten randomly-selected pups, exposed in utero through PND 21, were 

sacrificed on PND 7, 14, and 21. Significantly increased (p<0.05) serotonin levels were 

observed in the frontal cortex on postpartum day 21 in pups exposed to 28 mg/m3

 (20 ppm) H2S, 

while increased (p<0.01) serotonin levels were observed in the cerebellum and frontal cortex on 

postpartum days 14 and 21 in pups exposed to 105 mg/m3 (75 ppm) H2S. Norepinephrine levels 

increased (p<0.05) at 105 mg/m3

 (75 ppm) in the cerebellum on PNDs 7, 14, and 21, and in the 

frontal cortex on PND 21. At 28 mg/m3

 (20 ppm), frontal cortex norepinephrine levels were 

decreased compared to controls on days 14 and 21. 

In a study designed to evaluate the perinatal effect of H2S on developing cerebellar 

Purkinje cells, Sprague-Dawley dams were exposed to 28 or 70 mg/m3 H2S (20 or 50 ppm) for 7 

hr/day from postcoital day 5 until PND 21 (Hannah and Roth, 1991). Control animals were 

exposed similarly, but without H2S. Pups born to treated dams were also exposed perinatally up 

to PND 21 (Roth, 2002), at which time one pup was randomly selected from each litter for 

analysis, resulting in a total of 10 pups for each concentration and 10 pups from each of the 

control groups. Cerebella were processed using the Golgi method. One Purkinje cell from the 

pyramis of each cerebellum was digitized for morphometric analysis. Quantitative analysis of 

dendritic morphology is generally regarded as a traditional morphometric method appropriate to 

assess the effect of environmental influences, including chemical insult, on the developing brain. 

The authors used a vertex analysis (after Berry and Flinn, 1984) that can be used on a small 

number of nodal vertex types. This method leads to a determination of random versus 

nonrandom growth. Statistical evaluation was carried out using the Student’s t-test. Exposure to 

both 20 and 50 ppm (28 or 70 mg/m3

) produced significantly longer segment length than controls 

over the low and middle branching orders, but not the higher orders. It was stated that segment 

lengths are typically increased when afferent fibers are experimentally reduced in number. The 

mean vertex path length (defined as a measure of the mean distance from any one equivalent 

order back to the axon hillock) was found to increase (p<0.001) in incremental steps in both 

treated groups compared to controls. When considered over the entire dendritic tree, these 

changes could alter the capacity of Purkinje cells to integrate afferent impulses and may be 

associated with an increased likelihood of an abnormal physiological response. Segment 

30
-31-

branching was also found to be different in cells from the treated animals. There was a 

significant increase in branch number of the middle orders and a significant decrease in numbers 

over the higher orders (considered to represent the ‘growth front’). 

The investigators considered this to be a compensatory response when there is restriction 

on the ‘growth front’. The question of random versus nonrandom growth patterns was evaluated 

by examining the symmetry of the branching. It was found that vertex analysis indicated that the 

treated cells were undergoing a significant amount of nonsymmetrical or nonrandom growth, 

suggesting that H2S affected the branching pattern. The resulting alterations in dendritic 

arborization appeared to be in response to H2S exposure at the 20 and 50 ppm (28 or 70 mg/m3

) 

levels such that the 20 ppm (28 mg/m3

) level may be considered a low-effect rather than a noeffect level. 

The significance of the findings in this and the Skrajny et al. (1992) studies to humans is 

unclear. Since alterations may constitute a chemically-induced change in the growth or 

organization of structural (or neurochemical) elements, they are to be regarded as possible 

indicators of a neurotoxic effect in accordance with guidance in EPA’s neurotoxicity risk 

assessment guidelines (U.S. EPA, 1998). The question as to whether the alterations are adverse, 

however, remains unclear for several reasons. The effects reported by Hannah and Roth (1991), 

for example, are highly selective and could be due to environmental factors not directly related 

to exposure including variability resulting from the restricted sampling technique (i.e., one 

Purkinje cell/pup). Also, the relationship of these alterations to a functional impairment is 

unclear and cerebellar neurological sequelae are not the most obvious symptom associated with 

H2S exposure. It is acknowledged, however, that predicting functional impairments from 

specific structural alterations such as reported by Hannah and Roth (1991) is difficult due to the 

selective nature of the observed alterations and the dynamic self-organizing response of the 

developing brain to injury. 

The effect of H2S on spermatogenesis in the rat was assessed by Andrew et al. (1980). A 

group of 10 14-week-old male Wistar rats were exposed to 220 ppm (306 mg/m3) H2S for 3 

hr/day for 7 days, compared to a control or nonexposed group. Following the last exposure, each 

male was caged with two unexposed virgin females for 10 consecutive weeks. Four male rats 

were administered triethylenemelamine and used as positive controls. The females were 

sacrificed after 18 days cohabitation. Parameters evaluated included fertility, corpora lutea, total 

implants, and dead implants. There were no adverse effects of exposure on these parameters. 

Subsequently, the investigators evaluated exposure on prenatal development by exposing 

pregnant Wistar rats to 220 ppm (306 mg/m3

) for 3 hr/day, for 5 days either days 1-18 of 

gestation, or days 7-11 or 12-16 (5 days total). All animals were sacrificed at day 21 and 

examined for dead implants, fetal malformations, and growth retardation. There was no evidence 

of embryotoxicity or external malformations. 

31
-32-

4.4. OTHER STUDIES 

4.4.1. Respiratory Toxicity 

Cytotoxic effects of H2S exposure in the respiratory tract of rats was investigated under 

acute exposure conditions in a series of studies by Lopez et al. (1987). Fischer 344 rats were 

exposed to 0, 10, 200, or 400 ppm (0, 14, 278, or 556 mg/m3

) H2S for 4 hours and then sacrificed 

at 1, 20, or 44 hours postexposure (Lopez et al., 1987). Bronchoalveolar lavage (BAL) and nasal 

lavage (NL) were performed on all animals. Lavage protein, lactate dehydrogenase (LDH), 

alkaline phosphatase (AP), cellular composition, (-glutamyl transpeptidase (GGT), and 

cytopathology were determined. Animals exposed to 400 ppm (556 mg/m3

) H2S exhibited a 

significant, but transient increase (800%) in NL protein at 1 hour postexposure that returned to 

normal at 20 hours postexposure. There was also a transient increase (320%) in LDH from NL 

fluid in the 400 ppm (556 mg/m3

) exposure group. The cellularity in the NL fluid was increased 

by 139 and 483% in the 10 and 200 ppm (14 and 278 mg/m3

) 1 hour postexposure groups, but 

these values returned to baseline by 20 hr postexposure. Cellularity was increased by 817, 501, 

and 73% at 1, 20, and 44 hours postexposure, respectively, for animals exposed to 400 ppm (556 

mg/m3

) H2S. Differential cell counts indicated that the increase in cellularity was initially due to 

desquamation of nasal epithelial cells (1 hour postexposure) followed by exudation of 

polymorphonuclear lymphocytes (PMN). Cytopathology revealed that the desquamated 

epithelial cells showed stages of ciliocytophthoria, cytoplasmic vacuolation, pyknosis, 

karyorrhexis, cytoplasmic constriction between the basal region and terminal plate, and 

separation of ciliated tufts. In the 10 ppm (14 mg/m3

) and 200 ppm (278 mg/m3) animals, no 

other effects in NL fluid were observed. 

Similar to NL fluid, the BAL fluid of rats exposed to 400 ppm (556 mg/m3

) had an 

increase in protein content of approximately 3000, 1400, and 450% at 1, 20, and 44 hours 

postexposure. There was no increase in BAL fluid proteins in the other exposure groups. In 

animals exposed to 400 ppm (556 mg/m3

) H2S, LDH significantly increased at all postexposure 

times, but LDH decreased toward baseline at the 20 and 44 hour postexposure times. In the 200 

ppm (278 mg/m3

) exposure group, LDH activity in the BAL fluid was significantly decreased in 

the 44 hr postexposure group. In animals exposed to 400 ppm (556 mg/m3

) H2S, AP initially 

significantly increased in the 1 hour postexposure group, but significantly decreased below 

baseline at 20 and 44 hours postexposure. Animals exposed to 200 ppm (278 mg/m3

) H2S had 

AP levels below baseline at 20 and 44 hours postexposure. GGT was elevated (900%) in the 400 

ppm (556 mg/m3

) 1 hour postexposure animals. Animals exposed to 400 ppm (556 mg/m3) H2S 

had a 42% reduction in nucleated cells in the BAL fluid at 1 hour postexposure, but which 

returned to normal at 20 hours and increased to 30% over baseline at 44 hours postexposure. 

32
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Pulmonary alveolar macrophages were significantly reduced at 1 hour postexposure in animals 

exposed to 400 ppm (556 mg/m3

) H2S, but returned to normal by 20 hours postexposure. PMN 

increased in all postexposure times (highest at 20 hours) in animals exposed to 400 ppm (556 

mg/m3

) H2S. In the 10 ppm (14 mg/m3) and 200 ppm (278 mg/m3) animals, no other effects in 

BAL were observed. 

The investigators concluded that vacuolization, ciliocytophthoria (a cellular degenerative 

change often from accompanying viral infections), and nasal sloughing is evidence of 

cytotoxicity of the nasal epithelium. The investigators also cautioned that although cell count in 

the NL fluid was elevated in animals exposed to 10 ppm (14 mg/m3

) H2S, dramatic changes can 

rapidly be restored due to the remarkable repair capacity of the respiratory epithelium. In 

addition, the cell counts in rats exposed to 10 ppm (14 mg/m3

) returned to baseline values by 

postexposure hour 20. Exudation of protein into the nasal passages is a sign of vascular 

permeability, and the investigators suggested that increased LDH in the NL fluid is a sign of 

damage to the epithelium. The investigators also suggested that increased LDH and AP 

observed 1 hour postexposure in BAL fluid is suggestive of damage to the pulmonary 

epithelium. However, the investigators stressed that H2S appears to be a weaker toxicant to the 

respiratory epithelium than other pneumotoxicants. The authors stated that the olfactory 

epithelium appears to be more sensitive to the toxic effects of H2S than the respiratory 

epithelium. In addition, the investigators considered the large increase in protein content in the 

BAL to be consistent with the observed edematous properties of the gas. The LOAEL in this 

study is 200 ppm (278 mg/m3

). The NOAEL is 10 ppm (14 mg/m3). 

Histologic and ultrastructural alterations in the lungs of rats were reported in a similar 

study in which male Fischer 344 rats were exposed for 4 hours to either 82 or 440 ppm (116 or 

613 mg/m3

; n = 12 rats per dose level) H2S followed by sacrifice at 1, 18, or 42 hours 

postexposure (Lopez et al., 1988a). Histologic changes were transient and mainly present in rats 

exposed to 440 ppm (613 mg/m3) H2S. While some histologic changes were noted at 82 ppm 

(116 mg/m3), no pathologic changes were reported at this exposure level. In rats exposed to 440 

ppm (613 mg/m3) H2S, bronchiolar ciliated cells developed necrosis, but necrotic damage was 

rapidly repaired through mitosis. Examination of the lung demonstrated the notable 

edematogenic effect following exposure to 440 ppm (613 mg/m3

) H2S. Based on findings from 

the postexposure groups, the investigators suggested a chronology to the edematogenic effect in 

which fluid first accumulates around the blood vessels, then the interstitium, and finally in the 

alveoli. Perivascular edema without involvement of the alveoli in animals exposed to 82 ppm 

(116 mg/m3) H2S suggested that fluid that accumulated in the interstitium was reabsorbed before 

it entered the bronchoalveolar spaces. This may explain the finding by Lopez et al. (1987) that 

protein was detected in BAL fluid from animals exposed to 400 ppm (556 mg/m3

) H2S, but not 

200 ppm (278 mg/m3

) H2S. The investigators also found a lack of structural changes in the 

33
-34-

alveolar endothelium, basement membrane, or type I pneumocytes, suggesting that H2S exposure 

to concentrations as high as 440 ppm (613 mg/m3

) does not compromise the air-blood barrier. In 

addition, there was no adverse effect on the mast cells. If damage to the air-blood barrier and 

mast cell degranulation is not responsible for the observed pulmonary edema following H2S 

exposure, then the investigators suggested that the edema may be due to an outflow of liquid 

from the peribronchovascular connective tissue into the lumen of small airways via high 

conductance pathways that fill the alveolar spaces in a retrograde manner. 

Lopez et al. (1988b) examined the effect of acute H2S exposure on nasal epithelial cells. 

Male Fischer 344 rats were exposed to 0, 14, 280, or 560 mg/m3

 (0, 10, 200, or 400 ppm) H2S for 

4 hours. Animals were sacrificed at 1, 18, or 44 hours postexposure. No animals died, but 

clinical signs of lethargy and epiphora were present in animals exposed to 560 mg/m3

 H2S. 

Nasal lesions were present only in the high-dose group and manifested as necrosis and 

exfoliation of the respiratory and olfactory epithelium. Of the four different sections of the nasal 

cavity, sections two and three (mid-nasal cavity) were the most severely affected. The rostral 

section (section 1) was not affected, and section 4 was only slightly affected. These results are 

similar to those reported by Brenneman et al. (2000). At 44 hours postexposure, the respiratory 

mucosa was essentially repaired, but the olfactory mucosa continued to exfoliate. Effects of H2S 

on nasal parameters were much less severe in the Sprague-Dawley rat study reported by CIIT 

(1983c). 

Green et al. (1991) examined the effects of H2S from the BAL of rats under the 

hypothesis that the hemorrhagic pulmonary edema observed from exposure to high levels of H2S 

is associated with changes in pulmonary surfactant. Male Fischer 344 rats were exposed to 0, 

200, or 300 ppm (0, 278, or 417 mg/m3) H2S for 4 hours, and at 1 hour postexposure BAL fluid 

was obtained. Marked abnormalities in surfactant activity were demonstrated in the lavagates 

from rats exposed to the highest concentration only 300 ppm (417 mg/m3

) and not in the lavagate 

from the lower dose 200 ppm (278 mg/m3

) or the controls. The lungs of high-dose animals 

showed areas of red atelectasis, patchy alveolar edema, and perivascular edema. These results 

are suggestive of a threshold for this surfactant response and subsequent histopathological 

progression. 

The effects of H2S on lung bacterial defense has been investigated. Rogers and Ferin 

(1981) exposed male Long-Evans rats to 45 ppm (636 mg/m3) H2S for 2, 4, or 6 hr followed by 

bacterial challenge to Staphylococcus epidermidis. In control animals, most of the bacteria was 

inactivated by the 6-hour postchallenge sacrifice time. Rats exposed to H2S for 2 hours 

responded similarly to controls. However, rats exposed to H2S for 4 and 6 hours had 6.5- and 

52-fold greater percent bacteria remaining, respectively, compared to controls. The investigators 

suggest that an H2S-induced absence of bacterial inactivation may explain secondary 

pneumonias in humans subsequent to acute or subacute H2S exposure. The effect of bacterial 

34
-35-

inactivation was hypothesized by the investigators to be due to alveolar macrophage inactivation. 

The hypothesis is supported by Robinson (1982) who demonstrated that rabbit alveolar 

macrophages lost the phagocytic ability in vitro when exposed to 54 ppm (75 mg/m3

) H2S for 24 

hours. 

Although the above studies are acute in nature (i.e., mostly 4 hr) and are not appropriate 

for the establishment of a chronic guideline value, the results may offer insights into the 

processes underlying affects observed in the respiratory tract tissues in animals exposed in a 

more protracted manner to H2S such as by Brenneman et al. (2002). 

Nasal tissues were examined for histology and cytochrome oxidase immunoreactivity in 

10-week-old male CD rats 24 hours after exposure to 0, 30, 80, 200, or 400 ppm (0, 42, 112, 280, 

or 560 mg/m3) H2S for 3 hr/day for 1 day or for 5 consecutive days (Brenneman et al., 2002). 

The nose was histologically examined 24 hours after exposure, and lesion recovery was assessed 

at 2 and 6 weeks following the 5-day exposure. The single 3-hour exposure to $ 80 ppm H2S 

resulted in regeneration of the respiratory mucosa and full thickness necrosis of the olfactory 

mucosa localized to the ventral and dorsal meatus, respectively. Repeated exposure to the same 

concentrations caused necrosis of the olfactory mucosa with early mucosal regeneration that 

extended from the dorsal medial meatus to the caudal regions of the ethmoid recess. Acute 

exposure to 400 ppm H2S induced severe mitochondrial swelling in sustentacular cells and 

olfactory neurons, which progressed to olfactory epithelial necrosis and sloughing. Cytochrome 

oxidase immunoreactive cells were more frequently observed in regions of the olfactory mucosa 

commonly affected by H2S than in regions that were not. These findings demonstrate that acute 

exposure to >80 ppm H2S resulted in reversible lesions in the respiratory and olfactory mucosae 

of the CD rat and, further, that cytochrome oxidase activity (as judged from cytochrome oxidase 

immunoreactivity) may be a susceptibility factor for H2S-induced olfactory toxicity in the CD 

rat. 

Transmission electron microscopy of cultured rabbit alveolar macrophage (AM) cells 

exposed in vitro to 54 ppm (75 mg/m3) for 24 hours revealed extensive damage to interior 

organelles and membrane resulting in a decreased percentage of AM capable of phagocytosis 

(Richardson, 1995). Khan et al. (1991) found evidence that H2S exposure of F344 rats 

decreased in vitro respiratory rates of collected AM cells (zymosan-stimulated) and at the 

highest concentration decreased cell viability. Rats had been exposed to 0, 70, 280, or 560 

mg/m3 (0, 50, 200 or 400 ppm) for 4 hours before cell collection. 

Moulin et al. (2002) correlated nasal pathology in H2S-exposed rats with regions of high 

flux in the rat nasal cavity generated from anatomically accurate computational fluid dynamics 

(CFD). The specific location of lesions from nasal tract sections approximating sections 4 and 5 

of a study of Brenneman et al. (2000) were noted and the predominately olfactory-lined portions 

of the perimeter of these 2 levels were divided into roughly similar size regions. CFD flux of 

35
-36-

H2S at these regions and the incidence of nasal pathologies within these regions were then 

calculated and correlated. The analysis showed that H2S-related lesions were observed only at 

specific sites within the olfactory epithelium and that areas of respiratory epithelium were not 

affected. Further, the incidence, severity and distribution of lesions seen within the olfactory 

epithelium increased with concentration (approximately 50% of the olfactory epithelium affected 

at 30 ppm [42 mg/m3] and 70% at 80 ppm [111 mg/m3]) whereas the olfactory epithelium of rats 

exposed to 10 ppm (14 mg/m3) and controls were unaffected. In contrast to the localization of 

nasal lesions in the olfactory epithelium, CFD simulations predicted higher flux (and therefore a 

higher dose) to those regions lined by respiratory epithelium where no H2S-related lesions were 

observed. Region by region ranking of lesion incidence in the olfactory-lined epithelium with 

predicted flux revealed a high degree of correlation (Spearman’s rank correlation coefficient, p < 

0.005 for level 5 section) for the 30 and 80 ppm exposure groups. 

Results from Moulin et al. (2002) suggest that olfactory epithelium is specifically 

sensitive to the effects of H2S while respiratory epithelium is resistant. These results therefore 

lend credence to considering nasal toxicity from H2S as relevant to humans having olfactory 

epithelium that can be impinged upon by inhaled air streams containing H2S. The high 

correlation between predicted H2S flux (a surrogate of dose) in the olfactory epithelium and 

nasal response (histopathology) indicate dose-response analysis should consider such 

information. Such procedures could be used, for example, to extrapolate dose-response 

relationships between animal models and humans to accommodate the differences between these 

species, i.e., the relatively large area of olfactory epithelium in areas of high flux in rats and mice 

(Kimbell et al., 1997) compared to the relatively small area of olfactory epithelium in areas of 

low flux in humans (Subramaniam et al., 1998). In considering this possibility, however, the 

authors caution that more refined estimates of necessary parameters such as nasal extraction 

efficiency would be required before such a procedure could be applied in a quantitative manner. 

4.4.2. Neurotoxicity 

Kombian et al. (1988) examined the acute effects of H2S on amino acid levels in various 

brain regions of the rat following i.p. administration of 10 or 30 mg/kg doses. Hydrogen sulfide 

administration was associated with alterations in several amino acid transmitters, in particular, 

enhanced levels of aspartate, glutamate, glutamine, GABA, glycine, taurine and alanine in the 

brainstem. Amino acid transmitters may play a role in the neuronal control of breathing. 

Kombian et al. (1988) postulated that the observed changes in neurotransmitter amino acid levels 

in the brainstem may at least partially contribute to the acute toxicity of H2S through 

mechanisms involving respiratory arrest. Hannah et al. (1989; see Section 4.3) also reported 

depressed levels of these same neurotransmitters (save for glycine) in the cerebella of 21-day-old 

rats that had been exposed in utero and up to postnatal day 21 to airborne levels of H2S at 105 

mg/m3 for 7 hr/day. 

36
-37-

Elovaara et al. (1978) demonstrated a marked reduction in the incorporation of labeled 

leucine in cerebral protein and myelin in adult female mice exposed to 100 ppm (139 mg/m3

) 

H2S for 2 hours. The results indicate decreased brain protein synthesis. The authors suggested 

that the changes may have resulted from the inhibition of cerebral CytOx activity. The authors 

also noted that 100 ppm (139 mg/m3) is 5 to 20 times lower than exposure levels associated with 

human fatalities. 

Utilizing in vitro whole brain preparations, Roth et al. (1997) showed that the addition of 

0.13μM Na2S can inhibit CytOx and carbonic anhydrase. Also, KCN, a specific inhibitor of 

cytochrome oxidase, altered the rhythmical activity of rat hippocampal neurons in a manner 

similar to Na2S or H2S. The authors speculated that the inhibition of the respiratory enzyme 

CytOx may be involved in the sulfide-induced alteration of neuronal function in brain regions 

such as the hippocampus. 

Skrajny et al. (1996) investigated the effects of low H2S levels on the hippocampus and 

neocortex of freely moving rats using electroencephalographic activity (EEG) as a measurement 

of neuronal function. The hippocampus was a focus of this study because this exposure of 

humans to high levels of H2S (i.e., approximately 500 ppm or 695 mg/m3) has been associated 

with memory loss and varying degrees of learning and perceptual deficits (Kilburn, 1993; Tvedt 

et al., 1991a,b; Wasch et al., 1989). The hippocampus plays a role in processing between 

cortical structures involved in cognitive behavior, and is very susceptible to toxic insult. Rats 

were exposed to 25, 50, 75 or 100 ppm (35, 69, 104, or 139 mg/m3) H2S for 3 hr/day for 5 days. 

Results, recorded during the last 10 minutes of the exposures, indicated that repeated exposure to 

H2S produced a dose-related increase in the amplitude of hippocampal theta activity the total of 

which was cumulative with exposure duration. The effect of H2S on hippocampal EEG was 

dose-dependent and highly significant at all concentrations. The authors postulated that elevated 

catecholamine and/or serotonin levels from the inhibition of MAO may be involved in the 

sulfide-induced alteration of hippocampal theta activity, since the time for reactivation of MAO 

activity correlated with the time required for the recovery of the total power of hippocampal 

EEG activity following exposure to H2S. The LOAEL for this study is 35 mg/m3

 (25 ppm). The 

authors also noted that exposure to 100 ppm (139 mg/m3

) H2S produced no significant effects on 

rat EEG activity recorded from the neocortex. 

In an earlier study, Skrajny et al. (1992) examined the effect of H2S exposure on 

serotonin and norepinephrine levels in the developing cerebellum and frontal cortex of SpragueDawley rats. Timed-pregnant rats were exposed to 20 or 75 ppm (28 or 104 mg/m3

) for 7 hr/day 

from day 5 postcoitus until PND 21. At parturition, treated and control animals were culled to 6 

dams, with a maximum of 12 pups per litter with 10 pups in each group randomly selected for 

euthanization at PND 7, 14, and 21. The number of litters in each group of dams was not stated 

nor was there an indication if pups were selected from randomly selected litters. Exposure to 75 

37
-38-

ppm (104 mg/m3

) resulted in a statistically significant (p<0.01) increase in serotonin in the 

cerebellum from PND 7 to 14, compared to controls. At PND 21, the value remained unchanged 

from PND 14. The increase was even more pronounced in the frontal cortex from PND 7 through 

21 (p<0.01). The increase at 20 ppm (28 mg/m3) in the frontal cortex was less pronounced than 

at 75 ppm (104 mg/m3) and reached statistical significance only at PND 21 (p<0.05). There was 

no significant increase in the level in the cerebellum. Changes in norepinephrine were more 

difficult to interpret as there was no clear pattern of change. The investigators concluded that the 

effects of exposure on serotonin and norepinephrine, coupled with the laboratory’s prior 

evidence demonstrating possible morphological changes in Purkinje cell development and the 

known influence of monoamines on neural development, suggest a mode of action for H2S on the 

CNS. 

Higuchi and Fukamachi (1977) examined the effects of inhalation exposure to H2S on 

avoidance behavior in rats. Rats were exposed to 100–500 ppm (139-695 mg/m3) H2S. 

Hydrogen sulfide was generated from the reaction of iron sulfide and hydrochloric acid. The 

exposure duration was 2 hours. Both the Sidman-type conditioned avoidance and discriminated 

avoidance behavior were assessed. A rapid and significant decrease in discriminated avoidance 

response was observed at concentrations $ 200 ppm (278 mg/m3

). At concentrations $300ppm 

(417 mg/m3) , the Sidman-type conditioned avoidance response was also decreased. The 

behavioral effects were reversed following ventilation with clean air or when exposure was 

terminated. 

In an attempt to demonstrate that the neurological sequelae following exposure to high 

concentrations of H2S is due to neuronal necrosis, Baldelli et al. (1993) administered sodium 

sulfide (Na2S) to unventilated and anesthetized-ventilated (to compensate for the loss of central 

respiratory drive) male Wistar rats by i.p. injection. Doses in unventilated rats ranged from 84 to 

200 mg/kg, while ventilated rats were administered 120, 150, or 200 mg/kg. In unventilated 

animals, all animals that were administered doses greater than 120 mg/kg (n=11) sulfide died in 

# 10 minutes. Doses of 120, 108, 100, 96, and 84 mg/kg were lethal to 7/10, 3/3, 1⁄2, 2/5, and 0/1 

animals, respectively. An LD50 of 94 mg/kg was calculated for 32 unventilated rats by the 

investigators. Of the 8 surviving unventilated animals, only one 120 mg/kg rat demonstrated 

histopathological signs of neuronal necrosis of the cerebral cortex. In ventilated animals (5 

rats/group), an LD50 of 190 mg/kg was calculated. None of the 120 or 150 mg/kg group animals 

died during compound administration. However, 4/5 of the 200 mg/kg group animals died 

within minutes of compound administration, and the sole-surviving 200 mg/kg animal and 4/5 

of the 150 mg/kg group animals died within one week of exposure. Blood pressure and arterial 

blood gases were monitored only in ventilated animals. None of the ventilated animals exhibited 

a decrease in arterial pO2 or alteration in pH or pCO2. Mean arterial blood pressure decreased to 

42.8, 34.2, and 16.8 mm Hg for ventilated animals within 2-5 minutes of compound 

38
-39-

administration. Mean arterial blood pressure recovered within 15- 20 minutes for the 120 and 

150 mg/kg dose groups, but blood pressure did not return to baseline for 50 minutes in the lone 

surviving 200 mg/kg ventilated animal. There were significant deceases in EEG activities that 

were also dose-dependent. After doses of $ 150 mg/kg, EEG activity decreased to near 

isoelectric levels. EEG activity returned to normal in the two lowest doses and showed some 

recovery at the highest dose. Neuronal necrosis of the cerebral cortex, caudate, and brain stem 

was observed in the surviving 200 mg/kg animals. The investigators concluded that neuronal 

necrosis is not directly related to H2S exposure. Rather, neuronal necrosis is indirectly caused by 

hypotension induced by H2S exposure. The profound hypotension observed in the surviving 200 

mg/kg ventilated animal probably resulted in cerebral ischemia. 

Three reports describe effects of H2S on neurological function in rats (Partlo et al., 2001; 

Struve et al., 2001). Partlo et al. (2001) exposed adult rats to125 ppm (174 mg/m3) H2S for 4 

hr/day for 5 days/week for 5 weeks. Controls were exposed to a nitrogen/air mixture. Overt 

signs of eye irritation, respiratory distress, behavioral dysfunction or impaired consciousness 

were not observed at any time during the experiment. Testing of the animals revealed that this 

level of exposure did not affect memory retention or acquisition, but did impair one measure of 

performance during the reacquisition of a reversed contingency (radial arm maze task). These 

data provide evidence of mild brain dysfunction following a prolonged period of repeated 

hydrogen sulfide at this exposure level. The authors discuss the prefrontal cortex as a potential 

target site. Struve et al. (2001) exposed adult rats to the inhalation of 0, 30, 80, 200, or 400 ppm 

(0, 43, 111, 278, or 556 mg/m3

) H2S for 3 hr/day for 5 consecutive days. When exposure levels 

equaled or exceeded 80 ppm (111 mg/m3

), the animals exhibited significant reductions in motor 

activity, body temperature, and water maze performance (spatial learning); it is to be noted, 

however, that the latter result may have been confounded by the former. Pregnant SpragueDawley rats (12/group ) were exposed to either 0, 10, 30, or 80 ppm (0, 14, 42, or 111mg/m3

) 

H2S throughout gestation and postnatally to both dams and pups (8 rats/litter after culling) PND 

5 - 18. No decrement in behavioral performance, as judged by testing for motor activity 

(conducted on PNDs 13, 17, 21, and 60), passive avoidance (conducted on PND 22 and 62), 

functional observational battery (conducted blind on PND 60), and acoustic startle response 

(conducted on PND 21 and 62), was noted in the offspring at any exposure concentration 

(Dorman et al., 2000). 

4.4.3. Cardiovascular Toxicity 

In a series of experiments, Kosmider et al. (1967) exposed 10 rabbits to 100 mg/m3

 (72 

ppm) H2S for 1.5 hours and 17 rabbits to 100 mg/m3

 for 0.5 hours for 5 days. 

Electrocardiograms were taken after exposure and histochemical studies of the heart were 

performed. Ten rabbits served as controls for the histochemical studies and control 

39
-40-

electrocardiograms were made on all animals 10 days prior to exposure. Nine of ten animals 

exposed for a single 1.5 hour exposure exhibited mainly disorders in ventrical repolarization (T 

wave depression or inversion) without cardiac arrhythmia. However, animals exposed for 5 days 

exhibited arrhythmias such as ventricular extrasystoles and bigeminal rhythm or contractions 

elicited from several pacemakers. These effects were noted in 15 of 17 rabbits, and two of the 

affected animals exhibited atrial fibrillation. The arrhythmias lasted for several days after 

exposure. In addition, multiply exposed animals also exhibited T wave depression. 

Histochemical studies revealed a decrease in ATP phosphohydrolase and NADPH2 

oxidoreductase activity, which suggests a direct effect on myocardial cells. A direct effect on 

ATP phosphohydrolase could alter Na+ and K+

 transport to the myocardial cell and could result 

in arrhythmia. Administration of sodium citrate, a calcium chelator, attenuated the arrhythmias. 

Electrocardiogram effects have also been reported in rats. Kohno et al. (1991) exposed 

male Wistar rats to 75 ppm (105 mg/m3

) H2S for 20, 40, or 60 minutes. Heart rate, blood 

pressure, and electrocardiogram were monitored during exposure and for one hour 

postexposure. At 20, 40, and 60 minutes, heart rates decreased by 10, 17, and 27%, respectively, 

and continued to decrease by 29, 35, and 38%, respectively, during the postexposure monitoring 

period. Electrocardiograms revealed that the P-Q intervals were longer during the exposure and 

postexposure periods, which suggests some disorder in stimulus transmission. No changes were 

noted in blood pressure either during or after exposure. 

4.4.4. Ocular Toxicity 

Hydrogen sulfide has previously been reported to affect the eyes in a condition called 

“sore eye” or “gas eye”. Lefebvre et al. (1991) examined this effect by exposing male Fischer 

344 rats to 560 mg/m3

 (403 ppm) H2S for 4 hours or 2100 mg/m3 (1511 ppm) H2S for 4 min. 

Ocular lavages were performed and cells were examined. There was a significant increase 

(approximately 2 times) in exfoliated ocular cells associated with both H2S exposures. In all 

animals, the majority of recovered cells were corneal epithelial cells. However, a greater 

percentage of conjuctival epithelial cells was detected in exposed animals. The study 

demonstrates that H2S is an ocular irritant at very high concentrations. 

4.4.5. Genotoxicity 

The mutagenic potential of H2S was investigated using the Ames Salmonella 

typhimurium mutagenicity assay with and without Aroclor-induced hamster and rat liver S9 

fractions. Hydrogen sulfide vapor (17-1750 :g/plate) was not mutagenic in S. typhimurium 

strains TA 97, TA 98, or TA 100 with and without metabolic activation (Hughes et al., 1984). 

Although H2S is not mutagenic, Berglin and Carlsson (1986) reported that H2S gas potentiated 

the mutagenicity of hydrogen peroxide in S. typhimurium strain TA 102. The investigators 

40
-41-

believed that the increased mutagenicity was due to H2S-mediated formation of iron sulfide, 

which converts hydrogen peroxide to hydroxyl radicals more efficiently than ferrous iron. 

4.5. EVALUATION OF MODE OF ACTION FOR NONCANCER EFFECTS 

4.5.1. Mode of Action of Noncancer Effects 

At low levels of exposure H2S is an irritant such that effects on tissues-of-contact would 

be anticipated, either in laboratory animals or humans (Shusterman, 1992). 

At high levels, neurotoxic effects have been reported. Neurotoxicity is thought to occur 

via the ventilatory centers of the brain that lead to secondary effects including respiratory 

paralysis, pulmonary edema and death. Some investigators interpret the potential mode(s) of 

action for H2S very differently, whether it be via a neurotoxic pathway or effects on the 

respiratory system. 

The potential mode-of-action of the neurotoxicity of H2S has been investigated. Studies 

included neurochemical, neuroelectrophysiological, and behavioral approaches to address this 

issue. Baldelli et al. (1993) concluded from studies using H2S with or without simultaneous 

ventilation that the neuronal necrosis seen following H2S exposure is not a direct result of H2S, 

but an effect of hypotension and ischemia. Warenycia et al. (1989a) demonstrated a selective 

uptake of sulfide by the brainstem of rats that, in part, may explain the lethal effect of sulfide on 

respiratory function as a consequence of an interaction with the brainstem respiratory center. 

Most of the catecholaminergic innervation of the brain originates from within the brainstem, 

while catecholamines and serotonin affect respiratory rhythm (Warenycia et al., 1989b). To 

explore this possibility further, experiments on monoamine oxidase (MAO) enzyme activity and 

neurotransmitter levels in various regions of the brain have been performed in rats (Warenycia et 

al., 1989b). It was concluded that the inhibition of MAO activities and increased 

neurotransmitter levels in the brainstem may be important in the loss of central respiratory drive 

after H2S exposure. 

On the other hand, some investigators believe that pulmonary edema might be a primary 

cause of death following exposure to high concentrations of H2S (Lopez et al., 1989), possibly 

by alterations in pulmonary surfactants (Green et al., 1991). Other investigators, using dogs, 

have suggested that sulfide was an irritant to the afferent endings of the pulmonary vagi, and that 

high amounts of sulfide tend to paralyze the ventilatory center (Haggard and Henderson, 1922). 

Almeida and Guidotti (1999), on the basis of rat studies, suggest that the lung may be the 

primary locus of HS ion action that can lead to vagal paralysis. Khan et al. (1990) investigated 

the action of H2S on a molecular level in the lung, in which lung mitochondria incubated with 

H2S exhibited a dose-dependent inhibition of cytochrome oxidase (CytOx) activity. Exposure to 

41
-42-

50 ppm (70 mg/m3

) H2S and higher significantly reduced CytOx activity in lung mitochondria in 

a dose-dependent manner . Since CytOx is the terminal step in aerobic oxidative metabolism, 

inhibition by H2S would stop tissue respiration and lead to the development of hypoxia. 

Case reports involving individuals in accidental overexposure situations indicate that H2S 

can cause direct neurotoxicity leading to respiratory distress and a variety of secondary adverse 

effects such as pulmonary edema, ataxia, motor weakness, headache, nausea, dyspnea, chest pain 

and dizziness. In some instances, death resulted from hypoxia. The concentrations causing these 

effects are largely unknown. Concentrations estimated to cause loss of smell are in the range of 

100-200 ppm (140-280 mg/m3

). Those associated with immediate physical collapse or 

“knockdown”have been estimated to be in the range of 500-1,000 ppm (695-1,390 mg/m3

). 

Levels associated with secondary pulmonary edema have been estimated to be in the range of 

250-500 ppm (348-695 mg/m3) (Milby and Baselt, 1999a,b; Reiffenstein et al., 1992). 

Limited epidemiological studies (Richardson, 1995; Hessel et al., 1997; Vanhoorne et al., 

1995), supported by findings in laboratory animals, have reported cardiovascular, pulmonary, 

and ocular effects attributable to H2S. High concentrations of H2S, such as in the Breysse (1961) 

report “as high as 4,000 ppm (5,560 mg/m3) H2S,” can result in pulmonary edema and death. 

Chronic exposure to low concentrations on the order of 0.3 to 7.8 ppm (0.4 to 11 mg/m3) 

(Higashi et al., 1983) appears to result in no change in pulmonary function in humans. 

Inhalation studies in laboratory animals (rat and mouse) have been subchronic in nature 

and have identified the olfactory nasal mucosa as the principal target site affected (Dorman et al., 

2000; Brenneman et al., 2000), most likely as a consequence of the irritant properties of H2S. In 

these two subchronic studies, nasal lesions of the olfactory mucosa were observed in animals 

exposed to 30 or 80 ppm (42 or 111 mg/m3) and consisted of olfactory neuron loss and basal cell 

hyperplasia. No effects were observed at 10 ppm (14 mg/m3). Male and female B6C3F1/CrlBr 

mice exhibited minimal to mild inflammation of the anterior portion of the nasal mucosa when 

exposed to 80 ppm (111 mg/m3

) H2S for 6 hr/day, 5 days/week for 90 days (CIIT, 1983a). Only 

minimal lesions were found in Sprague-Dawley or Fischer 344 rats (CIIT 1983 b,c). While 

strain and species differences may explain the discrepancy in response between the SpragueDawley rats and the Fischer-344 rats and B6C3F1 mice, it is not known why contrasting effects 

of H2S on nasal lesions were seen by Dorman et al. (2000) vs. CIIT (1983c) since SpragueDawley rats were used in both cases. This discrepancy may relate to the use of computational 

fluid dynamics models (e.g., Kimbell and Subramaniam, 2001) to determine specifically where 

to look for lesions. This methodology was not available at the time of the earlier CIIT 

(1983a,b,c) studies. 

It has been made clear that exposure to H2S can elicit neurologic alterations in animals 

that are to be regarded as possible indicators of a neurotoxic effect. These alterations are elicited 

by exposure to H2S near to those at which nasal lesions of Brenneman et al. (2000) are observed. 

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Neurotransmitter levels were significantly altered in the brains of postpartum rats pups exposed 

in utero and postpartum to 28 mg/m3

 (20 ppm) H2S (Skrajny et al., 1992). Altered morphology 

of the cerebellar Purkinje cells was noted by Hannah and Roth (1991), also at 28 mg/m3

 H2S. 

Evidence for mechanisms by which neurotoxic effects may occur implicates ischemia mediated 

by H2S inhibition of respiratory chain enzyme activity, in particular cytochrome oxidase. Khan 

et al. (1990) reported significant decreases in several respiratory chain enzymes during 

exposures at H2S above but not at 10 ppm (14 mg/m3

). This closeness in exposures eliciting 

these various effects indicates that this exposure range is in the critical transitional areas in the 

exposure-dose-severity continuum for a variety of endpoints, with data available in some 

instances to discriminate the mechanistic transition from being a nonadverse- to an adverseresponse as are the olfactory lesions noted by Brenneman et al. (2000) at a NOAEL of 10 ppm 

(14 mg/m3

). 

4.5.2. Oral Exposure 

In a subchronic oral study in rats, the only one that was found, mortality and 

neuromuscular and behavioral signs have been reported in animals gavaged with 7 mg/kg-day 

H2S in deionized/deoxygenated water for 89 days (Anderson, 1987). Effects included labored 

breathing, convulsions, and sedation. Mortality was also noted in some males and females 

exposed to 3.5 mg/kg-day H2S for 89 days. Although restlessness was observed in all treated 

males and increased salivation in all treated females, these clinical signs generally occurred 

sporadically, and the incidence of these findings did not show a clear dose-response. The 

toxicological significance of restlessness in treated males, and salivation in treated females, is 

not clear. No histopathological lesions attributable to H2S were found. However, this study has 

serious problems with execution that prevent it from being used for derivation of an RfD. These 

problems include a prevalence of pneumonia and ulcerative dermatitis in vehicle control and 

treated groups, that fact that the study is not peer-reviewed, and that although 50% of the high 

dose males died, the cause of death could not be determined. 

4.6. WEIGHT-OF-EVIDENCE EVALUATION AND CANCER 

CHARACTERIZATION—SYNTHESIS OF HUMAN, ANIMAL, AND OTHER 

SUPPORTING EVIDENCE, CONCLUSIONS ABOUT HUMAN CARCINOGENICITY, 

AND LIKELY MODE OF ACTION 

Under the Draft Revised Guidelines for Carcinogen Risk Assessment (U.S. EPA, 1999), 

data are inadequate for an assessment of the carcinogenic potential of hydrogen sulfide. No 

human or animal studies assessing the potential carcinogenicity of H2S were located. 

Genotoxicity studies also indicate that H2S was not mutagenic in S. typhimurium strains TA97, 

TA98 or TA100 with or without metabolic activation (Hughes et al., 1984). Although H2S does 

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not appear to be mutagenic, H2S gas potentiated the mutagenicity of hydrogen peroxide in S. 

typhimurium strain TA102 (Berglin and Carlsson, 1986). The investigators reported that the 

increased mutagenicity was due to H2S-mediated formation of iron sulfide, which converts 

hydrogen peroxide to hydroxyl radicals more efficiently than ferrous iron alone. 

4.7. SUSCEPTIBLE POPULATIONS 

Data reported by Hannah and Roth (1991) showed that in utero and perinatal exposure of 

Sprague-Dawley rats to 20 or 50 ppm (28 or 70 mg/m3) H2S resulted in abnormal growth and 

morphology of developing cerebellar Purkinje cells and changes in neurotransmitter levels 

(Skrajny et al., 1992; Hannah et al., 1989). Therefore pregnant women might be considered as a 

susceptible subpopulation with the hazard, if any, to the offspring of these women. 

4.7.1. Possible Childhood Susceptibility 

Conclusions about childhood susceptibility are extremely limited because of poor 

exposure data reported in existing case reports and lack of supporting data in the literature. 

There is some anecdotal evidence (following) that children may be more susceptible than adults 

to the adverse effects H2S exposure. 

A case study reported that a 20-month-old child developed intermittent paroxysmal tonic 

deviation of the eyes, choreoathetosis and dystonia with progressive involuntary movements of 

the entire body following exposure to at least 0.6 ppm (0.8 mg/m3

) H2S over a period of up to 

“nearly a year” (Gaitonde et al., 1987). From the text within the report, coexposure to the same 

levels of H2S can be inferred for the parents and at least one other child; this is evidence, 

although anecdotal, that children and possibly children of a certain age may be selectively 

susceptible to the neurological effects of H2S at least at relatively high levels, > 600 ppb. 

Ascribing relevance of this apparent susceptibility to environmental levels of H2S would be, 

however, conjectural. 

Based on the findings of the effects of H2S on developing brain cells in rats treated both 

pre- and postnatally (Hannah and Roth, 1991; see Section 4.3) and the fact that in humans the 

period of synaptogenesis extends from the sixth month of gestation to several years after birth 

(Ikonomidou et al., 2000), a possibility of children’s vulnerability to effects from high levels of 

exposure to H2S may exist. 

4.7.2. Possible Gender Differences 

Dorman et al. (2000) examined effects on reproductive performance from inhalation 

exposure to H2S (see Section 4.3). There were no statistically significant effects on reproductive 

performance (mating index, fertility index, postimplantation loss per litter, and number of late 

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resorptions or stillbirths) in F0 female animals. Also, the number of live pups, litter size, 

average length of gestation, and average number of implants per female were not affected. In 

comparing high-exposure and control male animals, a few histologic diagnoses related to 

testicular tubular degeneration and changes in the epididymis, with a higher incidence noted in 

the high-exposure group. In none of these cases, however, was statistical significance achieved. 

5. DOSE-RESPONSE ASSESSMENTS 

5.1. ORAL REFERENCE DOSE 

Relevant quantitative human subchronic and chronic oral toxicity data are not available. 

Only one subchronic oral animal study was found (Anderson, 1987). A number of problems 

with that study precluded the derivation of an oral RfD. Among the problems with that study are 

the following: (1) it is unpublished and therefore not peer-reviewed, (2) a 75 to 100% incidence 

of pneumonia was reported in vehicle controls and all treatment groups. However, a 0% 

incidence of pneumonia was reported for the rack control group (which was not gavaged). 

Major problems were evident in the execution of this study. The prevalence of pneumonia across 

all gavaged groups indicates a prevalence of disease that precludes drawing conclusions from 

this study regarding the effect of H2S, (3) there was a 50% death rate in the high dose male 

group, the only group with significant mortality, but the cause of death “could not be 

determined.” This also inspired a lack of confidence in the data, (4) alopecia was common in all 

groups, and the problem was diagnosed by a veterinarian as ulcerative dermatitis. This disease 

plus the pneumonia prevents one from calling this group “normal” and thus appropriate for the 

production of reliable data. 

5.2. INHALATION REFERENCE DOSE 

5.2.1. Choice of Principal Study and Critical Effect with Rationale and Justification 

It is clear from the clinical findings of numerous case reports of overexposure situations 

and occupational studies that neurotoxicity is the principal concern at high exposure levels of 

H2S. Several groups of investigators (Tvedt et al., 1991a,b; Kilburn, 1993, 1997; Wasch et al., 

1989) have reported long-term adverse neurological sequelae of H2S-induced unconsciousness in 

humans during occupational, accidental, and chronic exposures including neuropsychological 

and neurobehavorial decrements, brain damage, and, in one instance, dementia. 

As reported and analyzed in prior Sections (see 4.3.3, 4.4.1, and 4.5.2), both nasal tract 

lesions and neurologic effects occur in the same concentration range at lower levels of H2S 

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exposure, and both are clearly of relevance to humans. Based on the relative clarity of the 

mechanistic basis, the dose-response information at lower levels of H2S exposure, and adverse 

nature of the endpoint for nasal tract lesions as compared to the neurologic endpoints, the former 

is chosen as the critical effect on which to base the noncancer dose-response assessments. That 

the levels of H2S associated with these effects appear to be similar for either endpoint (e.g., the 

possible indicator of neurotoxic effects reported by Hannah and Roth (1991) to occur at 28 mg 

H2S /m3

 and the no-effect level of nasal tract lesions reported by Brenneman et al. (2000) at 14 

mg H2S /m3

) is some indication that consideration for the one will also address the other. On the 

other hand, the same judgments made here concerning clarity of mechanism or dose-response or 

adversity could serve as the basis for a reevaluation of the critical endpoint if these matters were 

further resolved for neurologic effects. 

The Purkinje-cell alterations noted by Hannah and Roth (1991) are a chemically induced 

change in the structure of the nervous system. While such alterations may constitute a possible 

indicator of a neurotoxic effect defined in the U.S. EPA neurotoxicity risk assessment guidelines 

as "neuroanatomical changes occurring at any level of nervous system organization" (U.S. EPA, 

1998), their biological significance is not sufficiently understood to clearly ascertain the nature 

of the adverse outcome. The effects of certain developmental insults on cerebellar structure and 

function have been well characterized (Ferguson 1996) and provide a basis with which to gauge 

the significance of the Hannah and Roth (1991) findings. Focal x-irradiation of cerebella of rats 

during the first postnatal week (before PND8 which is inclusive of the exposure period used by 

Hannah and Roth, 1991) reduces the number of granule, basket and Purkinje cells, and surviving 

Purkinje cells are misaligned and exhibit aberrant dendritic form. These animals exhibit tremor, 

ataxia, altered spontaneous activity and their performance on rotorod and cognitive tests (swim 

maze) is impaired. Similar neuroanatomical and functional alterations are observed after early 

life treatment of rats (PND 4 onwards) with antimitotic agents such as methylazoxymethanol. 

Dorman et al., (2002) did not find evidence of the types of neuroanatomical alterations or 

behavioral impairments associated with these models in their dose-response assessment of 

developmental exposure to hydrogen sulfide. While it is noteworthy that Dorman et al. (2002) 

did not did not assess Purkinje cell dendritic morphology or employ a cognitive test sensitive to 

cerebellar insult, such as conditioned eye-blink, their observations indicate that developmental 

exposure to hydrogen sulfide does not produce the substantial neuroanatomical or behavioral 

alterations associated with well-characterized models of developmental cerebellar insult. While 

a definitive study with hydrogen sulfide in which neural structure and function are intensively 

examined is not available, the currently available data do not indicate that developmental 

hydrogen sulfide exposure is associated with overt pathology or functional deficits. Furthermore, 

the currently available evidence does not provide a basis to clearly ascertain adverse 

consequences associated with the subtle alterations in Purkinje cell dendrites reported by Hannah 

and Roth (1991). Nonetheless, it should be noted that the exposure levels used by Hannah and 

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Roth (1991) are nearly the same as those used in eliciting the nasal tract effects discussed below, 

indicating that the uncertainty factors applied to the nasal effects would also provide a similar 

degree of conservatism for both of these endpoints. 

The few available occupational studies in humans are judged not to be suitable for 

derivation of an inhalation RfC because of limitations including lack of adequate exposure data, 

limited number of subjects examined, and co-exposure to other chemicals and lack of 

information on the critical effect (nasal toxicity) designated above. 

Three subchronic animal studies were considered for derivation of an inhalation RfC: 

Dorman et al. (2000), CIIT (1983a), and Brenneman et al. (2000). In comparing high-exposure 

and control male animals exposed subchronically to 80 ppm H2S Dorman et al. (2000) reported a 

few histologic testicular diagnoses in the high-exposure group (80 ppm); in none of these cases, 

however, was statistical significance achieved when compared to the control group. CIIT 

(1983a) reported that male and female mice exposed to 80 ppm (111 mg/mg3

) H2S, 6 hr/day, 5 

days/week for 90 days exhibited minimal to mild inflammation of the anterior portion of the 

nasal mucosa. Only minimal lesions were observed in two species of rats (Sprague-Dawley and 

Fischer-344) examined by the same authors (CIIT, 1983b,c) and in mice exposed to lower 

concentrations of H2S. However, in male Sprague- Dawley rats exposed to 0, 10, 30, and 80 

ppm (0, 14, 42, and 111 mg/m3) H2S for 6 hr/day, 7 days/week, for 10 weeks, Brenneman et al. 

(2000) reported nasal lesions of the olfactory mucosa in the 30 and 80 ppm (42 and 111 mg/m3) 

exposure groups. No effects were observed in the control or 10 ppm (14 mg/m3

) exposure 

animals that were considered treatment-related. Nasal lesions consisted of multifocal, bilaterally 

symmetrical olfactory neuron loss and basal cell hyperplasia affecting the lining of the dorsal 

medial meatus and dorsal and medial region of the ethmoid recess. The difference in results 

between Brenneman et al. (2000) and CIIT (1983a) may indicate a variability in animal 

response. 

The study by Brenneman et al. (2000) was considered to be the most appropriate for the 

derivation of an inhalation RfC for several reasons. First, the critical effect (nasal lesions of the 

olfactory mucosa; summarized in Table 2 in Section 4.2.2) has been reported by other 

investigators (Dorman et al., 2000; CIIT, 1983a; Lopez et al., 1988b); second, the effect is 

consistent with the irritant properties of this gas; third, along with the neurological system, the 

respiratory system has been reported be a target organ of H2S toxicity by numerous researchers; 

fourth, the LOAEL (42 mg/m3

) and NOAEL (14 mg/m3) are at lower concentrations than those 

in the other subchronic studies. 

5.2.2. Methods of Analysis—including Models (PBPK, BMD, etc.) 

The results of Brenneman et al. (2000) (Table 2, Section 4.2.2) yielded a NOAEL of 10 

ppm and a LOAEL of 30 ppm . The nature of the data (total incidence preceded by zero 

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incidence; see Table 2) was not amenable to BMD modeling, thus NOAEL and LOAELs were 

designated. Converting ppm to mg/m3

 resulted in a NOAEL of 14 mg/m3 and a LOAEL of 42 

mg/m3 (1 ppm = 1.39 mg/m3

). The LOAEL and NOAEL were then adjusted to a weekly 

continuous exposure such that (N)LOAELADJ = (N)LOAEL (mg/m3) x hrs exp/24 hours x day 

exp/week : 

NOAELADJ = 14 mg/m3

 x 6/24 hours x 7/7 days/week = 3.5 mg/m3 

LOAELADJ = 42 mg/m3

 x 6/24 hours x 7/7 days/week = 10.5 mg/m3 

Thus, a LOAELADJ of 10.5 mg/m3 and a NOAELADJ of 3.5 mg/m3 were obtained. 

Conversion of these (adjusted) effect levels to human equivalent concentrations, i.e. 

HECs, is typically accomplished through application of procedures specified in the inhalation 

RfC methodology (U.S. EPA 1994b). Species specific parameters of anatomy, physiology and 

location of the lesion are utilized to generate a dosimetric adjustment factor (DAF) that is then 

applied to the effect level of interest, a NOAEL in this instance, to generate a NOAELHEC For 

H2S, this DAF is termed as the “regional gas dose ratio” (RGDR) and is calculated as the ratio of 

rat to human ventilation rate over the surface area of the ET region (see U.S. EPA, Chapter 4). 

The ventilation rate (VE) was calculated for male Sprague-Dawley rats using the default body 

weight of 0.267 kilograms shown in Table 4-5 of U.S. EPA (1994b). The ventilation rate for 

male rats was calculated as follows: 

lnVE = b0 + b1ln(0.267kg) 

The parameters, b0 and b1, are species specific parameters listed in Table 4-6 of U.S. EPA 

(1994). This gave a VE of 0.19 liters/minute for male rats. The default ventilation rate for 

humans is 13.8 liters/minutes given on page 4-27 of U.S. EPA (1994b). The default ET surface 

areas for the rat and human are 15 and 200 cm2

, respectively. These values are given on page 

4-26 of U.S. EPA (1994b). The RGDR was calculated as follows: 

RGDRET = (VE/SAET)animal/(VE/SAET)human = (0.19/15)/(13.8/200) = 0.184 

The animal (N)LOAELADJ is then multiplied by the RGDRET to yield the (N)LOAELHEC. 

NOAELHEC = NOAELADJ x RGDRET = 3.5 mg/m3 x 0.184 = 0.64 mg/m3 

LOAELHEC = LOAELADJ x RGDRET = 10.5 mg/m3 x 0.184 = 1.9 mg/m3 

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The computational fluid dynamic (CFD) modeling information provided by Moulin et al. 

(2002) on the nasal pathology of H2S provide useful insight into the results from the current 

default RfC procedure used in constructing the HEC for H2S. From the work of Brenneman et 

al. (2000) it is clear that the lesions observed in rats from H2S exposure are restricted to the 

olfactory epithelium and do not impact the respiratory epithelium. Moulin et al. (2002) further 

refined these findings by showing that CFD simulated levels of H2S flux (and therefore H2S 

dose) correlated with the incidence of lesions within the olfactory epithelium, suggesting that the 

appropriate dose response metric for H2S would be highly specific, i.e., flux to the olfactory 

epithelium. The current RfC default procedures (used above) do not employ any kind of CFDtype air flow adjustment, nor do they use a surface area measure that discriminates between 

olfactory and respiratory epithelium. 

Thus, although the modeling information provided by Moulin et al. (2002) is not used in 

this assessment to provide quantitative assessment of species comparison of H2S exposure (i.e, 

an HEC) the results may be used to provide valuable insight into the limitations of the current 

procedures. Future work in this area may provide information to perform a more quantitative 

evaluation of the HEC for H2S. 

5.2.3. RfC Derivation—including Application of Uncertainty Factors (UFs) and Modifying 

Factors (MFs) 

The RfC for H2S is derived by dividing the NOAELHEC for nasal effects by the UF of 300. 

Uncertainty factors were applied for interspecies extrapolation from animals to humans (3) and 

intraspecies variability (10). The interspecies uncertainty factor of 3 (101/2) was used rather than 

10 because of a dosimetric adjustment from rat to human was used. The use of an additional 

uncertainty factor of 10 was employed for the use of a subchronic, rather than chronic, study. 

Although a single generation reproductive study noted testicular alterations that were 

observed only in the high dose group, the alterations were not significantly different from 

controls and had no apparent effects on reproductive performance. No other indicators of 

reproductive toxicity were observed in this study. No significant histopathology of reproductive 

organs was noted in a longer duration (subchronic) study. These results can be considered to 

lessen the concern for lack of a multi-generational reproductive study. 

As noted above, the significance and relevance of the Purkinje dendritic alterations noted 

in developing animals are indicated in the neurotoxicity risk assessment guidelines to be a 

possible indicator of a neurotoxic effect, not as a clearly adverse effect. The RfC is based on an 

effect that could be considered a neurological effect (olfactory neuronal loss) that is clearly 

adverse occurring in adult animals at a lower dose than did the altered dendritic arborization in 

developing animals. This dose-response relationship between adult and neonatal effects tends to 

ameliorate concerns that children are a susceptible population at least in the situation of long49
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term relatively low level exposures. Some circumstantial data seem to indicate that, under high 

level exposures, children may be more susceptible than adults to the acute effects of hydrogen 

sulfide. 

In consideration of the points above and the totality of information available on hydrogen 

sulfide, no data base uncertainty factor is evoked. 

Therefore, a total uncertainty factor of 300 (10 for subchronic to chronic, 10 for 

intraspecies variability, and 3 for interspecies extrapolation) is applied to the NOAELHEC of 0.64 

mg/m3

, yielding an inhalation RfC of 0.002 mg/m3. 

RfC = NOAELHEC / Uncertainty Factors

RfC = 0.64 mg/m3 / (10 x 10 x 3) = 0.002 mg/m3 = 2E-3 mg/m3

5.3. CANCER ASSESSMENT 

No data pertaining to the potential carcinogenicity of H2S were identified. 

6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION 

OF HAZARD AND DOSE RESPONSE 

6.1. HAZARD IDENTIFICATION 

Hydrogen sulfide is a colorless gas and has a strong odor of rotten eggs. Its primary uses 

include the production of elemental sulfur and sulfuric acid, the manufacture of heavy water and 

other chemicals, in metallurgy, and as an analytical reagent. Although quantitative data are 

lacking, toxicity studies suggest that H2S gas is absorbed rapidly through the lungs. Oral 

exposure is not likely to occur. In animals and humans, it distributes to the blood, brain, lung, 

heart, liver, spleen, and kidney. Oxidation is the primary metabolic pathway for H2S, with 

thiosulfate and sulfate as metabolites. Metabolism in laboratory animals and in humans appears 

to be similar. Hydrogen sulfide is excreted in the urine. 

Human data pertaining to inhalation exposure (the expected route of ambient exposure) 

consist of a plethora of case reports and a variety of occupational epidemiological studies. 

Although these studies have limitations that preclude their use for quantitative risk assessment, 

they indicate that exposure to H2S (at high concentrations) has profound effects on the 

respiratory system leading to unconsciousness with attendant neurologic sequelae and, 

sometimes, death. An increase in cardiovascular-related deaths due, in part, to H2S exposure was 

reported in one occupational study. 

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Inhalation studies in adult rodents demonstrate sensitivity of nasal olfactory epithelium to 

low concentrations of H2S. The RfC is based on these lesions. Limited evidence suggests that 

exposure of humans to low concentrations may also cause neurologic symptoms although 

quantitative exposure-response data is lacking. Because of similar access of inhaled H2S to the 

olfactory tissues of humans, these lesions are likely of relevance to humans and a reasonable 

choice as a critical effect. Relevance to olfactory lesions seen in rodents to humans is also 

suggested by Hirsch and Zavala (1999) who reported decreased persistent olfactory function in 

workers exposed to hydrogen sulfide chronically. Whereas adverse nasal effects are of relevance 

and concern in adult human exposure scenarios, inhalation studies of perinatal or neonatal 

exposure in rats demonstrates abnormal cellular development in the brain as well as significant 

alterations in neurotransmitter levels; the toxicological significance of these findings is 

uncertain. 

Relevant quantitative human oral toxicity data are not available and ingestion is not a 

likely route of exposure. An RfD based on GI disturbances in pigs consuming feed containing 

hydrogen sulfide was derived in the previous IRIS entry. A review of the RfD (see Section 

4.2.1) indicates that the effects on which that value were based are not reproducible and probably 

not related to H2S. Therefore the previous RfD will be withdrawn and a new RfD will not be 

derived based on data base deficiencies. 

The indicators of a possible effect noted in the developing brain cells of newborn rats 

indicate the possibility that the developing human fetus could also be at risk. The exposure 

levels producing these effects, however, are in the same range or somewhat higher than those 

producing the critical sentinel clearly adverse effect (nasal tract lesions) in adult animals, thereby 

ameliorating the concern that young animals (and possibly children) may be especially 

susceptible to the effects from relatively low-level chronic exposures to hydrogen sulfide. Other 

observations in the data base do indicate a possible concern regarding the susceptibility of 

children exposed to high levels of H2S, i.e., > 600 ppb. However, the relevance of this apparent 

susceptibility at environmental levels of H2S where toxicity is not likely to occur, such as the 

RfC value derived herein, is not at all clear. 

There is no evidence indicating that H2S exposure is associated with carcinogenesis. 

Under the Draft Revised Guidelines for Carcinogen Risk Assessment (U.S. EPA, 1999), data are 

inadequate for an assessment of the carcinogenic potential of hydrogen sulfide. 

6.2. DOSE RESPONSE 

The effects of H2S in humans can be acute and/or chronic. The weight-of-evidence from 

the human and animal studies suggest that the effects of H2S are dose-dependent. The exposureresponse relationship for acute effects, particularly CNS and respiratory, can be very steep. 

Levels in the range of 500 to 1,000 ppm (695 to 1,390 mg/m3) are life-threatening and can cause 

51
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immediate unconsciousness followed by serious and debilitating neurologic and respiratory 

sequelae. Lower levels have been associated with lung function deficit and eye, nose, and throat 

irritation. Precise determination of acute effect levels is precluded by the lack of accurate 

monitoring data since nearly all cases were those involving acute accidental overexposure. 

Levels dangerous to human health may not be detected by odor since high levels of H2S can 

paralyze the olfactory nerves making detection impossible. 

Little is known of the concentration-response relationship at low levels of chronic 

exposure. Occupational studies have been confounded by exposure to other substances and 

inadequate monitoring to establish cause-effect relationships. 

Controlled exposure studies in laboratory animals indicate that the portal-of-entry (the 

nasal tract) and neurologic tissues are both targets for H2S toxicity. Based on the clear nature of 

their adversity (demonstrable histopathology), information available on exposure-dose-response 

and the commonality of the underlying mechanism (irritation) between animals and humans, and 

indications that such effects also occur in humans chronically exposed to hydrogen sulfide 

(Hirsch and Zavala, 1999), nasal tract effects were chosen as the critical effect and used to 

develop an inhalation reference concentration (RfC). Available information indicates that the 

dose-response character of indicators of neurotoxicity (such as the alterations to cerebellar 

Purkinje cells reported by Hannah and Roth, 1991) and nasal tract lesions (Brenneman et al., 

2000) may be similar to one another such that consideration of one may be inclusive of the other. 

However, more extensive and definitive information on the neurologic endpoints could reveal 

that these should be the most relevant endpoint, more so than nasal tract lesions. Such 

information may provide sufficient reason to reassess H2S. 

Some data exist inferring that children/neonates may be more susceptible than adults to 

the neurological effects of H2S. In the case-study of Gaitonde et al. (1987) neurological effects 

observed in a 20-month-old that were associated with an extended exposure to high levels of H2S 

(>600 ppb) were not reported in the parents or other children in the family. It is clear that the 

findings of Hannah and Roth (1991) on developing brain cells in rats exposed both pre- and 

postnatally to high levels of H2S (20 and 50 ppm) are possible indicators of a neurotoxic effect. 

However, the toxicologic significance of the observed alterations are not yet understood 

although the effects noted in both of these reports possibly having the same underlying 

mechanism, inhibition of mitochondrial cytochrome oxidase activity. Overall, these 

observations indicate a possible concern regarding the susceptibility of children exposed to high 

levels of H2S, i.e., > 600 ppb. However, the relevance of this apparent susceptibility at 

environmental levels of H2S where toxicity is not likely to occur, such as the RfC value derived 

herein, is not at all clear. 

The daily inhalation exposure to the human population that is likely to be without an 

appreciable risk of deleterious effects during a lifetime, the RfC, has been determined to be 

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0.002 mg/m3

 or 1.4 ppb. This value was derived from a subchronic inhalation study in 

laboratory rats by Brenneman et al. (2000). This study reported critical effects, nasal tract 

lesions in olfactory epithelium, in male rats exposed to 30 or 80 ppm (42 or 111 mg/m3) but not 

at 10 ppm (14 mg/m3), the NOAEL. An overall uncertainty factor of 300 was applied to account 

for interspecies extrapolation from animal to human, intrapecies variability, and subchronic 

exposures. The use of nasal lesions permits the derivation of an RfC that is considered 

protective against any long-term CNS and respiratory effects in ambient exposure environments. 

The overall confidence in the RfC assessment is medium-high; confidence in the 

principal study is medium because it was well-designed and conducted, and examined sensitive 

endpoints. However, it was subchronic in duration, and examined only male rats. Overall 

confidence in the data base is medium-high because the endpoints are supported by other 

subchronic studies. A chronic inhalation study in rats and mice, with additional dose groups and 

a more extensive comparative examination of neurologic and nasal tract endpoints from the same 

exposures, would increase the confidence in the data base. Ongoing scientific efforts to provide 

further refinements to the assessment of H2S, particularly in the area of regional dosimetry of the 

nasal tract, are acknowledged. 

7. REFERENCES 

Allyn, LB. (1931) Notes on hydrogen sulfide poisoning. Ind. and Eng. Chem. 23:234. 

Almeida, AF; Guidotti, TL. (1999) Differential sensitivity of lung and brain to sulfide exposure: 

a peripheral mechanism for apnea. Toxicol. Sci. 50:287-293. 

Anderson, RC. (1987) 90-Day subchronic oral toxicity in rats. Test material: hydrogen sulfide 

(H2S). Arthur D. Little, Inc. Report to Dynamac Corporation. Work Assignment Nol 11148-

008. Reference 55461. 

Andrew, FD, Renne, RA; Cannon, WC. (1980) Reproductive toxicity testing for effects of H2S 

in rats. Pacific northwest laboratory annual report for 1979 to the DOE assistant secretary for 

environment: part 1 biomedical sciences. Contract Number EY-76-C-06-1830 

Arnold, IMF; Dufresne, RM; Alleyne, BC; et al. (1985) Health implications of occupational 

exposures to hydrogen sulfide. J. Occup. Med. 27:373-376. 

Baldelli, RJ; Green, FH; Auer, RN. (1993) Sulfide toxicity: mechanical ventilation and 

hypotension determine survival rate and brain necrosis. J. Appl. Physiol. 75(3):1348-1353. 

Bartholomew, TC; Powell, GM; Dodgson, KS; et al. (1980) Oxidation of sodium sulphide by rat 

liver, lungs and kidney. Biochem. Pharmacol. 29:2431-2437. 

Beauchamp, RO Jr; Bus, JS; Popp, JA; et al. (1984) A critical review of the literature on 

hydrogen sulfide toxicity. Crit. Rev. Toxicol. 13:25-97. 

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-54-

Berglin, EH; Carlsson, J. (1986) Effect of hydrogen sulfide on the mutagenicity of hydrogen 

peroxide in Salmonella typhimurium strain TA102. Mutat. Res. 175(1):5-9. 

Berry, M; Flinn, R. (1984) Vertex analysis of Purkinje cell dendritic trees in the cerebellum of 

the rat. Proc. R. Soc. Lond. B 221 (321-348). 

Borda, RP. (1997) Cognitive impairment associated with chronic low-level exposure to 

hydrogen sulfide. Presented at the American Public Health Association 125th annual meeting, 

November. 

Brenneman, KA; James, RA; Gross, EA; Dorman, DC. (2000) Olfactory neuron loss in adult 

male CD rats following subchronic inhalation exposure to hydrogen sulfide. Toxicol. Pathol. 

28(2):326-333. 

Brenneman KA; Meleason DF; Sar M; Marshall MW; James RA; Gross EA; Martin JT; Dorman 

DC.( 2002). Olfactory mucosal necrosis in male CD rats following acute inhalation exposure to 

hydrogen sulfide: reversibility and the possible role of regional metabolism. Toxicol. Pathol. 

30(2):200-208 

Breysse, PA. (1961) Hydrogen sulfide fatality in a poultry feather fertilizer plant. Industrial 

Hygiene Journal 22:220-222. 

Burnett, WW; King, EG; Grace, M.; Eng, P; Hall, WF. (1977) Hydrogen sulfide poisoning: 

review of 5 years’ experience. Canadian Med. Assn. J. 117:1277-1280. 

CIIT (Chemical Industry Institute of Toxicology). (1983a) 90-Day vapor inhalation toxicity 

study of hydrogen sulfide in B6C3F1 mice. EPA/OTS 0883-0255. 

CIIT (1983b) 90-Day vapor inhalation toxicity study of hydrogen sulfide in Fischer-344 rats. 

EPA/OTS 0883-0255. 

CIIT (1983c) 90-Day vapor inhalation toxicity study of hydrogen sulfide in Sprague-Dawley 

rats. EPA/OTS 0883-0255. 

Dorman, DC; Brenneman, KA; Struve, MF; et al. (2000) Fertility and developmental 

neurotoxicity effects of inhaled hydrogen sulfide in Sprague-Dawley rats. Neurotoxicol Teratol. 

22:71-84. 

Dorman, DC; Moulin, FJM; McManus, BE; et al. (2002) Cytochrome oxidase inhibition induced 

by acute hydrogen sulfide inhalation: correlation with tissue sulfide concentrations in the rat 

brain, liver, lung, and nasal epithelium. Toxicol. Sci. 65:18-25. 

Elovaara, E; Tossavinen, A; Savolainen, H. (1978) Effects of subclinical hydrogen sulfide 

intoxication on mouse brain protein metabolism. Exp. Neurol. 62(1):93-98. 

Ferguson, SA. (1996). Neuroanatomical and functional alterations resulting from early postnatal 

cerebellar insults in rodents. Pharmacol. Biochem. Behavior 55(4): 663-671. 

Gaitonde, UB; Sellar, RJ; O’Hare, AE. (1987) Long term exposure to hydrogen sulfide 

producing subacute encephalopathy in a child. Br. Med. J. 294(6572):614. 

54
-55-

Glass, DC. (1990) A review of the health effects of hydrogen sulphide exposure. Ann. Occup. 

Hyg. 34: 323-327. 

Green, FH; Schèurch, S; De Sanctis, GT; et al. (1991) Effects of hydrogen sulfide exposure on 

surface properties of lung surfactant. J. Appl. Physiol. 70(5):1943-1949. 

Guidotti, TL. (1996) Hydrogen sulfide. Occup. Med. 46(5): 367-371. 

Gunina, AI. (1959) Oxidation of hydrogen sulfide in the organism. U.S. Atomic Energy 

Commission, Report No. AEC tr 3661:534-541. 

Haahtela, T; Marttila, O; Vilka, V; et al. (1992) The South Karelia air pollution study: acute 

health effects of malodorous sulfur pollutants released by a pulp mill. Am. J. Public Health 

82(4): 603-605. 

Haggard, HW. (1921) The fate of sulfides in the blood. J. Biol. Chem. 49:519-529. 

Haggard, HW; Henderson, Y. (1922) The influence of hydrogen sulphide upon respiration. Am. 

J. Physiol. 41:289-297. 

Hagley, SR; South, DL. (1983) Fatal inhalation of liquid manure gas. Med. J. Aust. 2:459-460. 

Hall, AH; Rumack, BH. (1997) Hydrogen sulfide poisoning: an antidotal role for sodium nitrite? 

Vet. Human Toxicol. 39(3): 152-154. 

Hannah, RS; Roth, SH. (1991) Chronic exposure to low concentrations of hydrogen sulfide 

produces abnormal growth in developing Purkinje cells. Neurosci. Lett. 122: 225-228. 

Hannah, RS; Hayden, LJ; Roth, SH. (1989) Hydrogen sulfide exposure alters the amino acid 

content in developing rat CNS. Neurosci. Lett. 99: 323-327. 

Haskell Laboratory. (1994) The effects of chronic exposure to sulfonated hydrogen; the effect of 

chronic exposure to hydrogen sulfide (and with carbon disulfide, with cover letter dated 

05/11/94). EPA/OTS (1339). Doc. ID 86940000854. 

Hayden, LJ; Goeden, H; Roth, SH. (1990a) Exposure to low levels of hydrogen sulfide elevates 

circulating glucose in maternal rats. J. Toxicol. Environ. Health 31(1):45-52. 

Hayden, LJ; Goeden, H; Roth, SH. (1990b) Growth and development in the rat during subchronic exposure to low levels of hydrogen sulfide. Toxicol. Ind. Health 6(3):389-401. 

Hessel, PA; Herbert, FA; Melenka, LS; et al. (1997) Lung health in relation to hydrogen sulfide 

exposure in oil and gas workers in Alberta, Canada. Am. J. Ind. Med. 31(5):554-557. 

Higashi, T; Toyama, T; Sakurai, H; et al. (1983) Cross-sectional study of respiratory symptoms 

and pulmonary functions in rayon textile workers with special reference to H2S exposure. Ind. 

Health 21(4):281-292. 

Higuchi, Y; Fukamachi, M. (1977) Behavioral studies on toxicity of hydrogen sulfide by means 

of conditioned avoidance responses in rats. Folia Pharmacol. Jpn. 73:307-319. 

55
-56-

Hirsch, AR; Zavala, G. (1999) Long-term effect on the olfactory system of exposure to 

hydrogen sulphide. Occup. Environ. Med. 56: 284-287. 

Hughes, TJ; Sparacino, C; Frazier, S. (1984) Validation of chemical and biological techniques 

for evaluation of vapors in ambient air testing of twelve (12) vapor-phase compounds (project 

summary). Research Triangle Park, NC: Environmental Protection Agency, Health Effects 

Research Laboratory 3:EPA-6001. 

Ikonomidou, C; Bittigau, P; Ishimaru, MJ; et al. (2000) Ethanol-induced apoptotic 

neurodegeneration and fetal alcohol syndrome. Science 287(5455):1056-1060 

Imamura, T; Kage, S; Kudo, K; et al. (1996) A case of drowning linked to ingested sulfides: a 

report with animal experiments. Int. J. Legal Med. 109(1):42-44. 

Kage, S; Nagata, T; Kimura, K; et al. (1992) The usefulness of thiosulfate as an indicator of 

hydrogen sulfide poisoning in forensic toxicological examination: a study with animal 

experiments. Jpn. J. Forensic Toxicol. 10(3):223-227. 

Kangas, J; Savolainen, H. (1987) Urinary thiosulphate as an indicator of exposure to hydrogen 

sulphide vapour. Clinica. Chimica. Acta. 164:7-10. 

Khan, AA; Schuler, MM; Prior, MG; et al. (1990) Effects of hydrogen sulfide exposure on lung 

mitochondrial respiratory chain enzymes in rats. Toxicol. Appl. Pharmacol. 103(3):482-490. 

Khan, AA; Yong, S; Prior, MG; et al. (1991) Cytotoxic effects of hydrogen sulfide on 

pulmonary alveolar macrophages in rats. J. Toxicol. Environ. Health 33(1):57-64. 

Kilburn, KH. (1993) Case report: profound neurobehavioral deficits in an oil field worker 

overcome by hydrogen sulfide. Am. J. Med. Sci. 306(5):301-305. 

Kilburn, KH; Warshaw, RH. (1995) Hydrogen sulfide and reduced-sulfur gases adversely affect 

neurophysiological functions. Toxicol. Environ. Health 11(2):185-197. 

Kilburn, KH. (1997) Exposure to reduced sulfur gases impairs neurobehavioral function. 

Southern Med. J. 90(10): 997-1006. 

Kilburn, KH. (1999) Evaluating health effects from exposure to hydrogen sulfide: central 

nervous system dysfunction. Environ. Epidemiol. Toxicol. 1: 207-216. 

Kimbell, JS; Godo, MN; Gross, EA; Joyner, DR; Richardson, RB; Morgan, KT. (1997) 

Computer simulation of inspiratory airflow in all regions of the Fisher 344 rat nasal passages. 

Toxicol. Appl. Pharmacol. 145: 388-398. 

Kimbell, JS; Subramaniam, RP. (2001) use of computational fluid dynamics models for the 

dosimetry of inhaled gases in the nasal passages. Inhal. Toxicol. 13 (5): 325-334. 

Kimura, K; Hasegawa, M; Matsubara, K; et al. (1994) A fatal disaster case based on exposure to 

hydrogen sulfide - an estimation of the hydrogen sulfide concentration at the scene. Foren. Sci. 

Int. 66:111-116. 

56
-57-

Kleinfeld, M; Giel, C; Rosso, A. (1964) Acute hydrogen sulfide intoxication: an unusual source 

of exposure. Ind. Med. Surg. 33:656-660. 

Kohno, M; Tanaka, E; Nakamura, T; et al. (1991) Influence of the short-term inhalation of 

hydrogen sulfide in rats. Eisei. Kagaku 37(2):103-106. 

Kombian, SB; Warenycia, MW; Mele, FG; et al. (1988) Effects of acute intoxication with 

hydrogen sulfide on central amino acid transmitter systems. Neurotoxicology 9(4):587-595. 

Kosmider, S; Rogala, E; Pacholek, A. (1967) Electrocardiographic and histochemical studies of 

the heart muscle in acute experimental hydrogen sulfide poisoning. Arch. Immun. Ther. Exp. 

15:731-740. 

Laug, EP; Draize, JH. (1942) The percutaneous absorption of ammonium hydrogen sulfide and 

hydrogen sulfide. J Pharma col Exp Ther 76:179-188. 

Lefebvre, M; Yee, D; Fritz, D; et al. (1991) Objective measures of ocular irritation as a 

consequence of hydrogen sulphide exposure. Vet. Hum. Toxicol. 33(6):564-566. 

Legator, MS; Singleton, CR; Morris, DL; Philips DL. (2001) Health effects from chronic lowlevel exposure to hydrogen sulfide. Arch. Environ. Health. 56(2):123-31. 

Levitt, MD; Furne, J; Springfield, J; et al. (1999) Detoxification of hydrogen sulfide and 

methanethiol in the cecal mucosa. J. Clin. Invest. 104(8):1107-1114. 

Lopez, A; Prior, M ; Lillie, LE; et al. (1988a) Histologic and ultrastructural alterations in lungs 

of rats exposed to sub-lethal concentrations of hydrogen sulfide. Vet. Pathol. 25(5):376-384. 

Lopez, A; Prior, M; Reiffenstein, RJ; et al. (1989) Peracute toxic effects of inhaled hydrogen 

sulfide and injected sodium hydrosulfide on the lungs of rats. Fundam. Appl. Toxicol. 12(2):367-

373. 

Lopez, A; Prior, M; Yong, S; et al. (1987) Biochemical and cytologic alterations in the 

respiratory tract of rats exposed for 4 hours to hydrogen sulfide. Fundam. Appl. Toxicol. 

9(4):753-762. 

Lopez, A; Prior, M; Yong, S; et al. (1988b) Nasal lesions in rats exposed to hydrogen sulfide for 

four hours. Am. J. Vet. Res. 49(7):1107-1111. 

Mehlman, MA. (1994) Dangerous and cancer-causing properties of products and chemicals in 

the oil refining and petrochemical industry. Part VII: adverse health effects and toxic 

manifestations caused by exposure to hydrogen sulfide, a component of crude oil. In: Advances 

in Modern Environmental Toxicology, Vol XXIII. The identification and control of 

environmental and occupational diseases: hazards and risks of chemicals in the oil refining 

industry. 

Milby, TH. (1962) Hydrogen sulfide intoxication; review of the literature and report of an 

unusual accident resulting in two cases of nonfatal poisoning. J. Occup. Med. 4: 431-437. 

57
-58-

Milby, TH; Baselt, RC. (1999a) Health hazards of hydrogen sulfide: current status and future

directions. Environ. Epidemiol. Toxicol. 1: 262-269.

Milby, TH; Baselt, RC. (1999b) Hydrogen sulfide poisoning: Clarification of some

controversial issues. Am. J. Ind. Med. 35: 192-195.

Moulin, FJ; Brenneman, KA; Kimbell, JS; Dorman, DC. (2002) Predicted regional flux of

hydrogen sulfide correlates with distribution of nasal olfactory lesions in rats. Toxicol. Sci. 66:

7-15.

National Research Council (NRC). (1983) Risk assessment in the federal government: managing

the process. Committee on the Institutional means for Assessment of Risks to Public Health,

Commission on Life Sciences. Washington, DC: National Academy Press.

Osbern, LN; Crapo, RO. (1981) Dung lung: a report of toxic exposure to liquid manure. Ann.

Int. Med. 95:312-314.

Parra, O; Monso, E; Gallego, M; et al. (1991) Inhalation of hydrogen sulphide: a case of

subacute manifestations and long term sequelae. Brit. J. Ind. Med. 48:286-287.

Partlo, LA; Sainsbury, RS; Roth, SH. (2001) Effects of repeated hydrogen sulphide (H2S)

exposure on learning and memory in the adult rat. Neurotox. 22:177-189.

Prior, MG; Sharma, AK; Yong, S; et al. (1988) Concentration-time interactions in hydrogen

sulphide toxicity in rats. Can. J. Vet. Res. 52: 375-379.

Reiffenstein, RJ; Hulbert, WC; Roth, SH. (1992) Toxicology of hydrogen sulfide. Ann. Rev.

Pharmacol. Toxicol. 109-134.

Richardson, DB. (1995) Respiratory effects of chronic hydrogen sulfide exposure. Am. J. Ind.

Med. 28(1):99-108.

Robinson, AV. (1982) Effect of in vitro exposure to hydrogen sulfide on rabbit alveolar

macrophages cultured on gas-permeable membranes. Environ. Res. 27(2):491-500.

Rogers, RE; Ferin, J. (1981) Effect of hydrogen sulfide on bacterial inactivation in the rat lung.

Arch. Environ. Health 36(5):261-264.

Roth, SH; Skrajny, B; Bennington, R; et al. (1997) Neurotoxicity of hydrogen sulfide may result

from inhibition of respiratory enzymes. Proc. West Pharmacol. Soc. 40(3):41-43.

Roth, SH. (2002) Personal communication to the U.S. EPA Chemical Manager.

Saillenfait, AM; Bonnet, P; de Ceaurriz, J. (1989) Effects of inhalation exposure to carbon

disulfide and its combination with hydrogen sulfide on embryonal and fetal development in rats.

Toxicol. Lett. 48:57-66.

Shusterman, D. (1992). Critical review: the health significance of environmental odor pollution. 

Arch. Environ. Health 47:76-87.

Simson, RE; Simpson, GR. (1971) Fatal hydrogen sulphide poisoning associated with industrial

58
-59-

waste exposure. Med. J. Aust. 1;331-334 

Skrajny, B; Hannah, RS; Roth, SH. (1992) Low concentrations of hydrogen sulphide alter 

monoamine levels in the developing rat central nervous system. Can. J. Physiol. Pharmacol. 

70(11):1515-1518. 

Skrajny, B; Reiffenstein, RJ; Sainsbury, RS; et al. (1996) Effects of repeated exposures of 

hydrogen sulphide on rat hippocampal EEG. Toxicol. Lett. 84:43-53. 

Smith, RP; Abbanat, RA. (1966) Protective effect of oxidized glutathione in acute sulfide 

poisoning. Toxicol. Appl. Pharmacol. 9:209-217. 

Smith, RP; Gosselin, RE. (1964) The influence of methemoglobinemia on the lethality of some 

toxic anions. II. Sulfide. Toxicol. Appl. Pharm. 6:584-592. 

Snyder, JW; Safir, EF; Summerville, GP; et al. (1995) Occupational fatality and persistent 

neurological sequelae after mass exposure to hydrogen sulfide. Am. J. Emerg. Med. 13: 199-203 

Struve, MF; Brisbois, JN; James, RA; Marshall, MW; Dorman, DC. (2001) Neurotoxicological 

effects associated with short-term exposure of Sprague Dawley rats to hydrogen sulfide. 

Neutotoxicology 22(3):375-385. 

Subramaniam, RP; Richardson, RB; Morgan, KT; Guilmette, RA and Kimbell, JS (1998) 

Computational fluid dynamics simulations of inspiratory airflow in the human nose and 

nasopharynx. Inhal. Toxicol. 10: 91-120. 

Tvedt, B; Edland, A; Skyberg, K; et al. (1991a) Delayed neuropsychiatric sequelae after acute 

hydrogen sulfide poisoning: affection of motor function, memory, vision and hearing. Acta. 

Neurol. Scand. 84:348-351. 

Tvedt, B; Skyberg, K; Aaserud, O; et al. (1991b) Brain damage caused by hydrogen sulfide: a 

follow-up study of six patients. Am. J. Ind. Med. 20:91-101. 

U.S. EPA (U.S. Environmental Protection Agency). (1986a) Guidelines for the health risk 

assessment of chemical mixtures. Federal Register 51 (185):34014-34025. 

U.S. EPA. (1986b) Guidelines for mutagenicity risk assessment. Federal Register 

51(185):34006-34012. 

U.S. EPA. (1988) Recommendations for and documentation of biological values for use in risk 

assessment. Prepared by Environmental Criteria and Assessment Office, Office of Health and 

Environmental Assessment, Cincinnati, OH. ERPA 600/6-87/008. Available from: National 

Technical Information Service, PB88-179874/AS. 

U.S. EPA. (1991) Guidelines for developmental toxicity risk assessment. Federal Register 

56(234):63798-63826. 

U.S. EPA. (1994a) Interim policy for particle size and limit concentration issues in inhalation 

toxicity: notice of availability. Federal Register 59(206):53799. 

59
-60-

U.S. EPA. (1994b) Methods for derivation of inhalation reference concentrations and 

application of inhalation dosimetry. Prepared by Environmental Criteria and Assessment Office, 

Office of Health and Environmental Assessment, Research Triangle Park, NC. EPA/600/8-

90/066F. 

U.S. EPA. (1994c) Peer review and peer involvement at the U.S. Environmental Protection 

Agency. Signed by the U.S. EPA Administrator, Carol M. Browner, dated June 7, 1994. 

U.S. EPA. (1995) Use of the benchmark dose approach in health risk assessment. EPA/630/R94/007. 

U.S. EPA. (1996) Guidelines for reproductive toxicity risk assessment. Federal Register 

61(212):56274-56322. http://www.epa.gov/ORD/WebPubs/repro/ 

U.S. EPA. (1998) Guidelines for neurotoxicity risk assessment. Federal Register 63(93):26926-

26954. http://www.epa.gov/nceawww1/neurotox.htm 

U.S. EPA. (1999) Revised draft guidelines for carcinogen risk assessment. NCEA-F-0644, July, 

1999, Washington, DC. 

U.S. EPA. (2000a) Science policy council handbook: peer review. Prepared by the Office of 

Science Policy, Office of Research and Development, Washington, DC. EPA 100-B-00-001. 

U.S. EPA. (2000b) Science police council handbook: risk characterization. Prepared by the 

Office of Science Policy, Office of Research and Development, Washington, DC. EPA 100-B00/002 

Vanhoorne, M; de Rouck, A; de Bacquer, D. (1995) Epidemiological study of eye irritation by 

hydrogen sulphide and/or carbon disulphide exposure in viscose rayon workers. Ann. Occup. 

Hyg. 39(3):307-315. 

Warenycia, MW; Goodwin, LR; Benishin, CG; et al. (1989a) Acute hydrogen sulfide poisoning 

demonstration on selective uptake of sulfide by the brainstem by measurement of brain sulfide 

levels. Biochem. Pharmacol. 38(6):973-982. 

Warenycia, MW; Smith, KA; Blashko, CS; et al. (1989b) Monoamine oxidase inhibition as a 

sequel of hydrogen sulfide intoxication increases in brain catecholamine and 5 

hydroxytryptamine levels. Arch. Toxicol. 63(2):131-136. 

Wasch, HH; Estrin WJ; Yip, P; et al. (1989) Prolongation of the P-300 latency associated with 

hydrogen sulfide exposure. Arch. Neurol. 46:902-904. 

Watt, MM; Watt, SJ; Seaton, A. (1997) Episode of toxic gas exposure in sewer workers. Occup. 

Environ. Med. 54: 277-280. 

Weedon, FR; Hartzell, A; Setterstrom, C. (1940) Toxicity of ammonia, chlorine, hydrogen 

cyanide, hydrogen sulphide and sulphur dioxide gases V. Animals. Contributions from BoyceThompson Institute 11:365-385. 

60
-61-

Weisiger, RA; Pinkus, LM; Jakoby, WB. (1980) Thiol S-methyltransferase: suggested role in 

detoxication of intestinal hydrogen sulfide. Biochem. Pharmacol. 29:2885-2887. 

Wetterau, H; Oerkert, W; Knape, WG. (1964) Tests for the application of dried green fodder 

with higher H2S content (experiments with poultry and fattened pigs). Fetterung 5: 383-393. 

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APPENDIX A: EXTERNAL PEER REVIEW—SUMMARY OF 

COMMENTS AND DISPOSITION 

The support document and IRIS summary for hydrogen sulfide have undergone both 

internal peer review performed by scientists within EPA and a more formal external peer review 

performed by scientists in accordance with EPA guidance on peer review (U.S. EPA, 1998, 

2000a). Comments made by the internal reviewers were addressed prior to submitting the 

documents for external peer review and are not part of this appendix. Public comments were 

received and considered. The three external peer reviewers were tasked with providing written 

answers to general questions on the overall assessment and on chemical-specific questions in 

areas of scientific controversy or uncertainty. The reviewers made a number of editorial 

suggestions to clarify specific portions of the text. These changes were incorporated in the 

document as appropriate and are not discussed further. A summary of significant comments 

made by the external reviewers and EPA’s response to these comments follows: 

(1) General Questions for Peer Reviewers 

A. General Question: Are you aware of any other data/studies that are relevant (i.e., useful for 

the hazard identification or dose-response assessment) for the assessment of the adverse health 

effects, both cancer and noncancer, of this chemical? 

Comment: All three reviewers considered that the data base appears to reference/summarize the 

relevant studies although each reviewer did suggest some additional and recently-published 

studies for incorporation. 

Response: The studies identified by the reviewers were reviewed and information incorporated 

where appropriate. 

B. General Question: For the RfD and RfC, has the most appropriate critical effect been chosen 

(i.e., that adverse effect appearing first in a dose-response continuum)? Points relevant to this 

determination include whether or not the choice follows from the dose-response assessment, 

whether the effect is considered adverse, and if the effect and the species in which it is observed 

in a valid animal model for humans. 

Comment: One reviewer agreed that the principal study (Brenneman et al., 2000) was adequate 

for the determination of the RfC. However, it was suggested that, depending on the adequacy of 

the study design, the Hannah and Roth (1991) study could be considered as the principal study 

with morphological/structural changes in Purkinje cells as the critical effect. A second reviewer 

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declined to answer this and other questions related to the animal studies because of a lack of 

expertise in this area. The third reviewer did not explicitly answer this charge, but indicated that 

the “brain must be given first priority ....the nasal epithelial story in rats ....is not relevant.” This 

reviewer did not offer a suggestion for addressing CNS effects in humans from the data 

available. 

Response: The Hannah and Roth (1991) perinatal inhalation study was reconsidered for use as a 

principal study. This reconsideration proceeded along several fronts including a focused review 

by Agency scientists within the National Health and Environmental Effects Research Laboratory 

(NHEERL). 

A primary front concerns the adverse nature of the observed effect reported by Hannah 

and Roth, alterations in dendritic arborization of cerebellar Purkinje neurons in developing 

animals. The U.S. EPA neurotoxicology guidelines provide for such observations and identify 

them not as clearly adverse effects but rather as a “possible indicators of a neurotoxic effect.” 

Cerebellar-related dysfunction as a possible consequence of such a possible indicator requires 

further elucidation. It is also acknowledged that in the case of developmental scenarios such as 

those used by Hannah and Roth that predicting functional impairments from specific structural 

alterations is difficult due to the selective nature of the observed alterations and the dynamic 

self-organizing response of the developing brain to injury. 

Another aspect of the reconsideration involves the dose relationship of the candidate 

critical effects. It is clear that from the dose-response analysis accomplished in this assessment 

(Sections 4.3.3., 4.4.1, and 4.5.2), both nasal tract lesions and neurologic effects occur in the 

same concentration range at relatively low levels of H2S exposure, and both are clearly of 

relevance to humans. That the levels of H2S associated with these effects appear to be similar for 

either endpoint (e.g., the possible indicator of neurotoxic effects reported by Hannah and Roth to 

occur at 28 mg H2S /m3

 and the no-effect level of nasal tract lesions reported by Brenneman et al. 

at 14 mg H2S /m3

) is some indication that consideration for the one will also address the other. 

Based on the relative clarity of the mechanistic basis, the dose-response information at 

lower levels of H2S exposure, and adverse nature of the endpoint for nasal tract lesions as 

compared to the neurologic endpoints, as well as the recent information suggesting olfactory 

tissue to be a sensitive target tissue in rats (and therefore most likely in humans), nasal tract 

lesions are chosen as the critical effect on which to base the noncancer dose-response 

assessments. 

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C. General Question: Have the noncancer assessments been based on the most appropriate 

studies? These studies should present the critical effect/cancer (tumors or appropriate precursor) 

in the clearest dose-response relationship. If not, what other study (or studies) should be chosen 

and why? 

Comment: One reviewer felt that although the principal and supporting studies were appropriate 

for derivation of RfC a more thorough discussion of the neurotoxicity toxicity studies in both the 

Toxicological Review and IRIS Summary should be presented. Studies requiring further 

discussion were identified. It was also suggested that NOAELs and LOAELs be reported for all 

animal studies. The second reviewer declined to answer this question. The third reviewer felt that 

the rat nasal studies should be removed from consideration because they are not relevant. 

Response: The discussion of the neurotoxicity studies in both the Toxicological Review and 

IRIS summary were strengthened with additional information and discussion of the significance 

of hydrogen sulfide-induced CNS effects in humans was expanded in section 6 of the 

Toxicological Review. Additions of text analyzing the comparative dose-response between 

nasal tract effects and sensitive (potential) neurologic endpoints, in particular the anatomic 

alterations noted in cerebellar Purkinje cells by Hannah and Roth (1991), were added to Sections 

4.3.3, 4.4.1, and 4.5.2. These additions provide clarification that neurologic changes and nasal 

tract effects (olfactory epithelium) occur at about the same externally applied concentrations of 

H2S, but that the nasal tract lesions are more clearly regarded as adverse than are the most 

sensitive neurologic alterations noted (i.e., the alterations in Purkinje cell arborization). All 

animal studies were reviewed to see if it would be instructive to identify NOAELs and LOAELs 

for studies other than the principal and supporting studies. The nasal lesions caused by H2S are 

relevant to humans for a variety of reasons, to include (1) a sentinel response alerting individuals 

to the presence of H2S, (2) damage caused by H2S to the olfactory nerves causing anosmia, and 

(3) protection against nasal lesions serves to prevent effects elsewhere, such as the brain. Along 

with adverse histopathological lesions localized specifically within olfactory tissue of rats 

exposed to airborne H2S, Moulin et al. (2002) reported modeled simulations of H2S flux to this 

tissue that was highly correlated with the lesions. These simulations imply that human olfactory 

tissues could also be exposed to inhaled H2S, and, as no compelling reason exists to suspect that 

human olfactory tissue would react any differently to the irritant properties of H2S, also imply 

that similar effects would be observed in humans. 

D. General Question: Studies included in the RfD and RfC under the heading 

"Supporting/Additional studies" are meant to lend scientific justification for the designation of 

critical effect by including any relevant pathogenesis in humans, any applicable mechanistic 

information, any evidence corroborative of the critical effect, or to establish the 

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comprehensiveness of the data base with respect to various endpoints (such as 

reproductive/developmental toxicity studies). Should other studies be included under the 

"Supporting/Additional" category? Should some studies be removed? 

Comment: The only reviewer responding to this question suggested that a more comprehensive 

treatment of the neurotoxicity studies in humans and animals be included in the “Supporting or 

Additional” category. 

Response: An expanded discussion of this area was included both in the Toxicological Review 

and IRIS summary. 

E. General Question: For the noncancer assessments, are there other data that should be 

considered in developing the uncertainty factors or the modifying factor? Do you consider that 

the data support the use of different (default) values than those proposed? 

Comment: Only one reviewer responded to this question. It was suggested that a discussion of 

species differences in breathing style and nasal anatomy be included as there is evidence to 

suggest that rats are more sensitive than humans as a result of these differences. The last 

sentence in the last paragraph on page 5 of the summary, “For these reasons, an additional 

uncertainty factor of 3 was considered for incorporation ...,” should be deleted. If there is 

concern that an RfC based on olfactory lesions is not sufficiently protective of possible 

neurological effects, this point should be discussed in Section I.B.3. Also, is a factor of 10 

needed for sensitive populations and for subchronic exposure? 

Response: The reason and purpose for the dosimetric adjustment procedures applied to the effect 

levels in rats (i.e., to accommodate the differences in breathing style and anatomy) were made 

clearer to the reader in the Toxicological Review and the IRIS Summary documents. 

Based on the response to the preceding questions, the concern that an RfC based on nasal 

tract lesions may not be able to totally address all possibilities regarding neurological effects has 

been made a primary consideration in the text of the Toxicological Review and the IRIS 

Summary documents and is reflected in text additions to Sections 4.3.3, 4.4.1, and 4.5.2. 

Although the mechanism of action eliciting the nasal tract lesions (irritation) would be 

expected to vary directly with the delivery of the material to the nasal tract tissues, and that 

interindividual variability to such a direct mechanism would be minimal, the information needed 

to consider this matter (and a concomitant reduction of uncertainty) is not available to 

sufficiently compel such a reduction at this time. It is also questionable as to whether such a 

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direct mechanism would promote effects at lower concentration, even for much longer (i.e., 

lifetime) exposures, the precept on which the subchronic to chronic uncertainty factor is evoked. 

However, the same response has to be made for the interindividual UF, i.e., the information 

needed to consider this matter (and a concomitant reduction of uncertainty) is not available to 

sufficiently compel such a reduction at this time. 

The results of Moulin et al. (2002) and Brenneman et al. (2000) indicate that olfactory 

tissues in rats are sensitive to the effects of H2S, more so than respiratory epithelium. Modeling 

simulations (computational fluid dynamics) in rats of specific air flows to these tissues indicate 

olfactory tissues to be specifically sensitive to the effects of H2S, despite higher regional flow to 

the respiratory epithelium (Moulin et al., 2002). As discussed above, these simulations imply 

that human olfactory tissues could also be exposed to inhaled H2S, and, as no compelling reason 

exists to suspect that human olfactory tissue would react any differently to the irritant properties 

of H2S (the suspected mechanism of damage), it is reasonable to imply that similar effects would 

be observed in humans. 

F. General Question: Do the confidence statements and weight-of-evidence statements present 

a clear rationale and accurately reflect the utility of the studies chosen, the relevancy of effects to 

humans, and the comprehensiveness of the data base? Do these statements make sufficiently 

apparent all of the underlying assumptions and limitations of these assessments? If not, what 

needs to be added? 

Comment: Only one reviewer responded to this question. It was indicated that it was unclear 

why there was low confidence in the RfC when the confidence in the principal study and data 

base was medium. 

Response: The confidence statement was corrected such that the overall confidence in the RfC 

reflected that of the data base, i.e., medium to high. 

2) Chemical-Specific Questions for Peer Reviewers 

G. Specific Question: Should the reproductive toxicity of hydrogen sulfide, as seen in the 

Dorman et al. (2000) study be given more weight in the determination of the principal study for 

RfC determination? 

Comment: Only one reviewer responded this question, indicating that this question was 

unanswerable as the effect seen on testicular tubules in rats was not clearly adverse and not 

confirmed in another species. 

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Response: A statement to this effect was added to the text of the Toxicological Review and the 

IRIS Summary in the study description of the Dorman et al. (2000) study. 

H. Specific Question: Can you reconcile the controversy over whether hydrogen sulfide affects 

the brain directly with H2S-induced brain damage due to cytochrome oxidase enzyme inhibition 

causing morbidity and mortality (Warenycia et al., 1989a,b) versus the peripheral (lung-related) 

effect of H2S that ultimately results in hypoxia due to apnea (Baldelli et al., 1993; Almeida and 

Guidotti, 1999)? 

Comment: One reviewer indicated that the cytochrome oxidase hypothesis and the overall 

weight of scientific evidence indicate that the instantaneous collapse seen upon exposure to 750-

1,000 ppm is a result of H2S directly on the brain. There must be an exposure to H2S lasting for a 

minimum of about 5 minutes during a period of unconsciousness before hypoxic brain damage 

occurs. There is no reliable evidence that intermittent exposure to levels of H2S below 1 or 2 

ppm constitutes a danger to physical health. 

Response: This effect progression was more clearly expounded upon in the text of the 

Toxicological Review. The increased emphasis placed in the assessment on the role of 

cytochrome oxidase in the progression of toxicity, and the advancement of the concept that such 

an alteration (i.e., statistically significant inhibition of cytochrome oxidase activity) at low levels 

of H2S could be considered a sensitive indicator and harbinger of H2S toxicity should now be 

apparent to the reader of this assessment. 

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